CA2230474A1 - Donor kidney viability test for improved preservation - Google Patents
Donor kidney viability test for improved preservation Download PDFInfo
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- CA2230474A1 CA2230474A1 CA002230474A CA2230474A CA2230474A1 CA 2230474 A1 CA2230474 A1 CA 2230474A1 CA 002230474 A CA002230474 A CA 002230474A CA 2230474 A CA2230474 A CA 2230474A CA 2230474 A1 CA2230474 A1 CA 2230474A1
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- Prior art keywords
- kidney
- gfr
- perfusion
- present
- indicative
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9493—Immunosupressants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
The present invention contemplates a methodology which employs cold perfusion of donor organs with a colloid preservation solution at a selected perfusion pressure which avoids tubular reabsorption and consequent energy loss. The present invention is also directed towards the direct continuous measurement of Glomerular Filtration Rate (GFR) through measurement of urine formation during the perfusion period. The present invention further provides a method to assess afferent arteriole and efferent arteriole vascular patency. The present invention further contemplates the maintenance of tubular patency during perfusion. The present invention is also directed to a method for monitoring cold ischemia damage as an indication for timing and dosage of nephrotoxic immunosuppressant agents, e.g., Cyclosporin A. The present invention still further contemplates a device for performing the test of the present invention.
Description
~ CA 02230474 1998-02-2~
WO 97/08949 PCT~US96/14360 -I~NOR Kl~N~Y VTAR~T-TIrY TEST FOR IMPROUnED ~K~vATIoN
The present invention relates to the ~ monitoring of donor kidneys to establish their functional state. The diagnostic method of the present invention monitors physiological events during cold storage that can be u5ed to evaluate kidney functionality at harvest, throughout the storage period and just prior to transplantation.
Today, seventy-five percent (75~) of all kidney transplants performed in the United States utilize cadaveric organs. The use of cadaveric kidneys requires cold preservation of the organ during donor-recipient preparation. The amount of storage, or cold ischemia time, influences graft survival rates for many years post transplant. Cold ischemia damage is also known to exacerbate Cyclosporin A nephrotoxicity. Thus, Cyclosporin A may cause complications that will threaten graft survival i~ administered to an is chemically damaged kidney. Current research suggests that the success rate of a transplanted organ is enhanced by a shortened ischemia time (Merkus et al. Nephrol. Dial.
Transplant, 6:881-886, 1991). The hemodynamic stability of the donor, harvest procedure, preservation procedure, 2~ and recipient integrity are other factors influencing transplant outcomes (Gnant et al. TransPl~ Proc., 25(6):3102-3103, 1993).
Perfusion of donor kidneys has been proposed to enhance kidney transplant success rates. Research 3o CA 02230474 l998-02-2~ . , W O 97/08949 PCTrUS96/14360 -l demonstrates that perfusion provides advantages in cost, microvascular preservation and graft function over cold storage (Johnson et al. Transpl. Proc. 22(2): 385-387, 1990, Belzer et al. TransPl. Proc., 21(1):1240-1241, 1989). However, due to the characteristic difficulty of perfusion and the lack of long-term data on its benefits, most kidney transplant centers currently utilize cold storage preservation.
Additional reluctance to utilize perfusion techni~ues to preserve donor kidneys i~ based on ineffective preservation solutions. Conventional perfusates are generally albumin based. Some researchers suggest that albumin may be a limiting factor in the quality and duration of kidney preservation due to substantial metabolic tubular function during cold storage (Hoffmann et al. Transpl., 47(1):32-37, 1989). Albumin also tends to denature over time, causing tissue damage (Hoffmann et al. Arch.
Surq., 118:919-921, 1983).
The presence of an impermeable colloid, e.g., albumin in a perfusate is essential however to maintain glomerular integrity, prevent cellular edema and to maintain viability of kidneys during perfusion (Hoffmann et al. supra, 1983). The colloid constituency exerts colloid osmotic pressure. Colloid osmotic pressure in the plasma opposes filtration from the blood vessels 80 that the perfusate is not forced from the blood vessels into the intercellular spaces of the kidney by the pressure needed for perfusion. Expansion of the 3o , CA 02230474 1998-02-2~
WO 97/08949 PCTnJS96~4360 -1 intercellular volume due to perfusate filtration causes compression of the kidney vasculature which causes problems in reperfusion after the kidney is transplanted.
As previously indicated, albumin has proved to be an unreliable colloid component in perfusion solutions, particularly since albumin based perfusates have been characterized by substantial metabolic tubular fun~tion during cold storage o~ rat kidney~ (Gianello et al. Transpl. Int., 7:11-16, 1994). The metabolic tubular function, documented by inulin analysis to determine filtration rate, includes concurrent reabsorption of sodium and water. Inulin analysis generally involves a multistep, labor and time intensive task of measuring inulin concentrations in blood pl~sma and urine to generate GFR. Reabsorption uses energy in the form of ATP. It is art accepted that the less energy utilization by stored kidneys, the longer the organs will maintain ~unctionality.
In contrast with albumin based perfusates, a preferable solution currently in the possession of the art is known as the "UW" (University of Wisconsin) solution. The UW solution comprises a mixture of salts, ions, osmotically active materials and colloids. The colloid in the UW solution is modified hydroxyethyl starch (HES) employed at a standard concentration of 5.0g~ (Belzer et al., Transplant, 45(4): 673-676, 1988).
The UW solution, as commonly used, is so constituted that it min;m; zes swelling of blood vessels by proper 3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 1 balance of osmotically active cell membrane permeable and impermeant salts and sugars. Several articles and patents describe using the colloid HES in kidney perfusion solutions. One such patent is U.S. Patent No.
4,879,283 to Belzer et al. Belzer et al. describe a solution for preserving and storing organs intended for transplantation containing 3.0g~ to 8.0g~ by weight of hydroxyethyl starch.
Current methods of determining the viability of donor organs both pre- and post-transplantation are described in various scientific publications. For example, kidney tissue biopsy is performed before transplantation in patients who have a history of renal disease.
Electrolytes, plasma creatinine, sodium, potassium, ATPase, kidney tissue biopsies, lysosomal enzymes and blood flow have all been used before and/or after transplantation to test organ quality. While some of these tests have been effective in limited circumstances, none has enjoyed widespread acceptance or succe~s.
Despite available techniques to measure the various related parameters, few methods are presently available to assess the continuing functional viability of a donor kidney prior to transplantation. Total - (organ) Flow (TF), for example, provides only a general measure of a kidneys functionality. TF is the total fluid flow through the kidneys, generally quantitated in ~l/min in rodents and in ml/min in hllm~n~. The only 3o CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 1 method presently available to assess the viability of a donor organ prior to transplantation employs a r determination of whether Total Flow (TF) is maintained throughout the vasculature of the kidney. Total organ ~ 5 flow can predict total organ resistance which has some use as a non-specific indicator of organ viability.
Total organ resistance provides a general indication of the patency of the kidney vasculature, e.g., as a kidney ~ails, the vascula~-ure tends to be obs~ructed as a 10 consequence of cellular swelling or edema. However, total organ flow cannot be used to assess tubule patency or to determine those segments of the kidney vasculature which are changing due to failing func~ionality. Nor can TF alone be used to evaluate the consequences of 15 those changes, e.g. TF cannot differentiate afferent or efferent arteriole or tubular resistance.
Instead, Glomerular Filtration Rate (GFR) has been used as the measure of kidney function. The consensus for using GFR as the "gold standard" for 20 evaluating the success of transplanted kidney function has long been established. However, continuous filtration testing, i.e., employing a perfusate at a defined pressure and directly measuring GFR during cold storage has never been used due to various encumbering 25 factors such as reabsorption, e.g., of water, sodium and potassium and the consequent energy loss. Such consequences do not permit measurement of GFR as a direct function of kidney viability.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -It i8 suggested in the art that assays to determine the functional viability of preserved organs should allow the surgeon to determine if an organ will be functionally viable when it is transplanted via noninvasive, nondestructive means. According to one researcher, no such assay exists (Southard et al.
CryobiologY, 26:232-238, 1989). Essentially, the prior art suggests that a need exists to develop a reliable viability assay 80 that nonviable kidneys are never transplanted (D'Alessandro et al. "Organ Preservation In: Horizons in Orqan Transplant Action, 74:1083-1095, 1994).
Increasing the number of organs for transplant therapy by increasing the number of voluntary donors has not been successful. Further complicating the situation is an increase in the incidence of renal disease in the United States and other countries around the world (Booster et al. Transpl. Proc.. 25(1):1503-1504, 1993).
An increase in organ procurement may depend on increasing recovery of available but higher risk donors and organs.
Xenografts, organs from other species, represent the potentially largest pool of donor tissue.
Xenograft survival problems are critical and, there~ore, are not a potential organ source in the near future (Lloveras et al. Transpl Proc., 25(6):3169-3170, 1993).
Patients who have sustained cardiac standstill (non-heartbeating) have become a major focus of organ procurement. However, the need for additional 3o , CA 02230474 1998-02-2~
W O 97/08949 PCTrUS96~36~ -1 precaution in preservation of organs and intraoperative procedures are required for this patient population (Rowinski et al. Transpl. Proc., 25(1~:1511-1512, 1993).
Therefore, a means for monitoring kidney functionality during cold storage in a manner which would provide an easy and direct asse~sment of kidney function rem~l n~ a critical need. The present invention provides such a methodology which employs a conventional per~usate wi~h re~uced glomerular impermeable co~loid concentration utilized at a perfusion pressure which can surprisingly avoid tubular reabsorption and consequent energy loss. Such method permits direct and continuous assessment of kidney functionality by measuring GFR by simple and convenient means.
The present invention utilizes the direct measurement of Glomerular Filtration Rate (GFR) through measurement of urine formation during the perfusion period to continuously assess the functional state of the kidney. The present invention further contemplates assessment of afferent arteriole and efferent arteriole vascular patency and the maintenance of tubular patency as well as integrity of tubule cells during perfusion.
This invention also provides a method for monitoring cold ischemia damage as an indication for timing and dosage of nephrotoxic immunosuppressant agents, e.g., Cyclosporin A. The present invention also provides a therapeutic method for maintaining tubule patency and a device for performing the tests of the present invention.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 1 The present invention is directed to a method for monitoring and determining the functional viability of a donor kidney prior to transplantation comprising perfusing said kidney with a perfusate having an impermeable colloid concentration which is correlated with the perfusion pressure in a ratio which permits glomerular filtration of said perfusate, the composition of which totally avoids kidney tubule reabsorption during said perfusion; and determining the ~FR of ~aid kidney during said perfusion as a measure of the functional viability of said kidney.
The present invention is further directed to A method for ensuring patency of a kidney tubule comprising perfusion of said kidney tubule with a perfusate having an impermeable colloid concentration which is correlated with the perfusion pressure in a ratio which permits glomerular filtration of said perfusate, the composition of which totally avoids kidney tubule reabsorption and tubule occlusion during said perfusion.
One innovation of the present invention resides in the finding that by manipulating the concentration of colloid in the st~n~rd UW solution in relation to perfusion pressure, glomerular filtration i 25 occurs in a manner which totally avoids reabsorption by the kidney tubules. The advantage provided by this discovery relative to the prior art is that GFR can be used as a direct measure of the functional viability of the kidney in the absence of other considerations.
WO 97/08949 PCT~US96/14360 -I~NOR Kl~N~Y VTAR~T-TIrY TEST FOR IMPROUnED ~K~vATIoN
The present invention relates to the ~ monitoring of donor kidneys to establish their functional state. The diagnostic method of the present invention monitors physiological events during cold storage that can be u5ed to evaluate kidney functionality at harvest, throughout the storage period and just prior to transplantation.
Today, seventy-five percent (75~) of all kidney transplants performed in the United States utilize cadaveric organs. The use of cadaveric kidneys requires cold preservation of the organ during donor-recipient preparation. The amount of storage, or cold ischemia time, influences graft survival rates for many years post transplant. Cold ischemia damage is also known to exacerbate Cyclosporin A nephrotoxicity. Thus, Cyclosporin A may cause complications that will threaten graft survival i~ administered to an is chemically damaged kidney. Current research suggests that the success rate of a transplanted organ is enhanced by a shortened ischemia time (Merkus et al. Nephrol. Dial.
Transplant, 6:881-886, 1991). The hemodynamic stability of the donor, harvest procedure, preservation procedure, 2~ and recipient integrity are other factors influencing transplant outcomes (Gnant et al. TransPl~ Proc., 25(6):3102-3103, 1993).
Perfusion of donor kidneys has been proposed to enhance kidney transplant success rates. Research 3o CA 02230474 l998-02-2~ . , W O 97/08949 PCTrUS96/14360 -l demonstrates that perfusion provides advantages in cost, microvascular preservation and graft function over cold storage (Johnson et al. Transpl. Proc. 22(2): 385-387, 1990, Belzer et al. TransPl. Proc., 21(1):1240-1241, 1989). However, due to the characteristic difficulty of perfusion and the lack of long-term data on its benefits, most kidney transplant centers currently utilize cold storage preservation.
Additional reluctance to utilize perfusion techni~ues to preserve donor kidneys i~ based on ineffective preservation solutions. Conventional perfusates are generally albumin based. Some researchers suggest that albumin may be a limiting factor in the quality and duration of kidney preservation due to substantial metabolic tubular function during cold storage (Hoffmann et al. Transpl., 47(1):32-37, 1989). Albumin also tends to denature over time, causing tissue damage (Hoffmann et al. Arch.
Surq., 118:919-921, 1983).
The presence of an impermeable colloid, e.g., albumin in a perfusate is essential however to maintain glomerular integrity, prevent cellular edema and to maintain viability of kidneys during perfusion (Hoffmann et al. supra, 1983). The colloid constituency exerts colloid osmotic pressure. Colloid osmotic pressure in the plasma opposes filtration from the blood vessels 80 that the perfusate is not forced from the blood vessels into the intercellular spaces of the kidney by the pressure needed for perfusion. Expansion of the 3o , CA 02230474 1998-02-2~
WO 97/08949 PCTnJS96~4360 -1 intercellular volume due to perfusate filtration causes compression of the kidney vasculature which causes problems in reperfusion after the kidney is transplanted.
As previously indicated, albumin has proved to be an unreliable colloid component in perfusion solutions, particularly since albumin based perfusates have been characterized by substantial metabolic tubular fun~tion during cold storage o~ rat kidney~ (Gianello et al. Transpl. Int., 7:11-16, 1994). The metabolic tubular function, documented by inulin analysis to determine filtration rate, includes concurrent reabsorption of sodium and water. Inulin analysis generally involves a multistep, labor and time intensive task of measuring inulin concentrations in blood pl~sma and urine to generate GFR. Reabsorption uses energy in the form of ATP. It is art accepted that the less energy utilization by stored kidneys, the longer the organs will maintain ~unctionality.
In contrast with albumin based perfusates, a preferable solution currently in the possession of the art is known as the "UW" (University of Wisconsin) solution. The UW solution comprises a mixture of salts, ions, osmotically active materials and colloids. The colloid in the UW solution is modified hydroxyethyl starch (HES) employed at a standard concentration of 5.0g~ (Belzer et al., Transplant, 45(4): 673-676, 1988).
The UW solution, as commonly used, is so constituted that it min;m; zes swelling of blood vessels by proper 3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 1 balance of osmotically active cell membrane permeable and impermeant salts and sugars. Several articles and patents describe using the colloid HES in kidney perfusion solutions. One such patent is U.S. Patent No.
4,879,283 to Belzer et al. Belzer et al. describe a solution for preserving and storing organs intended for transplantation containing 3.0g~ to 8.0g~ by weight of hydroxyethyl starch.
Current methods of determining the viability of donor organs both pre- and post-transplantation are described in various scientific publications. For example, kidney tissue biopsy is performed before transplantation in patients who have a history of renal disease.
Electrolytes, plasma creatinine, sodium, potassium, ATPase, kidney tissue biopsies, lysosomal enzymes and blood flow have all been used before and/or after transplantation to test organ quality. While some of these tests have been effective in limited circumstances, none has enjoyed widespread acceptance or succe~s.
Despite available techniques to measure the various related parameters, few methods are presently available to assess the continuing functional viability of a donor kidney prior to transplantation. Total - (organ) Flow (TF), for example, provides only a general measure of a kidneys functionality. TF is the total fluid flow through the kidneys, generally quantitated in ~l/min in rodents and in ml/min in hllm~n~. The only 3o CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 1 method presently available to assess the viability of a donor organ prior to transplantation employs a r determination of whether Total Flow (TF) is maintained throughout the vasculature of the kidney. Total organ ~ 5 flow can predict total organ resistance which has some use as a non-specific indicator of organ viability.
Total organ resistance provides a general indication of the patency of the kidney vasculature, e.g., as a kidney ~ails, the vascula~-ure tends to be obs~ructed as a 10 consequence of cellular swelling or edema. However, total organ flow cannot be used to assess tubule patency or to determine those segments of the kidney vasculature which are changing due to failing func~ionality. Nor can TF alone be used to evaluate the consequences of 15 those changes, e.g. TF cannot differentiate afferent or efferent arteriole or tubular resistance.
Instead, Glomerular Filtration Rate (GFR) has been used as the measure of kidney function. The consensus for using GFR as the "gold standard" for 20 evaluating the success of transplanted kidney function has long been established. However, continuous filtration testing, i.e., employing a perfusate at a defined pressure and directly measuring GFR during cold storage has never been used due to various encumbering 25 factors such as reabsorption, e.g., of water, sodium and potassium and the consequent energy loss. Such consequences do not permit measurement of GFR as a direct function of kidney viability.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -It i8 suggested in the art that assays to determine the functional viability of preserved organs should allow the surgeon to determine if an organ will be functionally viable when it is transplanted via noninvasive, nondestructive means. According to one researcher, no such assay exists (Southard et al.
CryobiologY, 26:232-238, 1989). Essentially, the prior art suggests that a need exists to develop a reliable viability assay 80 that nonviable kidneys are never transplanted (D'Alessandro et al. "Organ Preservation In: Horizons in Orqan Transplant Action, 74:1083-1095, 1994).
Increasing the number of organs for transplant therapy by increasing the number of voluntary donors has not been successful. Further complicating the situation is an increase in the incidence of renal disease in the United States and other countries around the world (Booster et al. Transpl. Proc.. 25(1):1503-1504, 1993).
An increase in organ procurement may depend on increasing recovery of available but higher risk donors and organs.
Xenografts, organs from other species, represent the potentially largest pool of donor tissue.
Xenograft survival problems are critical and, there~ore, are not a potential organ source in the near future (Lloveras et al. Transpl Proc., 25(6):3169-3170, 1993).
Patients who have sustained cardiac standstill (non-heartbeating) have become a major focus of organ procurement. However, the need for additional 3o , CA 02230474 1998-02-2~
W O 97/08949 PCTrUS96~36~ -1 precaution in preservation of organs and intraoperative procedures are required for this patient population (Rowinski et al. Transpl. Proc., 25(1~:1511-1512, 1993).
Therefore, a means for monitoring kidney functionality during cold storage in a manner which would provide an easy and direct asse~sment of kidney function rem~l n~ a critical need. The present invention provides such a methodology which employs a conventional per~usate wi~h re~uced glomerular impermeable co~loid concentration utilized at a perfusion pressure which can surprisingly avoid tubular reabsorption and consequent energy loss. Such method permits direct and continuous assessment of kidney functionality by measuring GFR by simple and convenient means.
The present invention utilizes the direct measurement of Glomerular Filtration Rate (GFR) through measurement of urine formation during the perfusion period to continuously assess the functional state of the kidney. The present invention further contemplates assessment of afferent arteriole and efferent arteriole vascular patency and the maintenance of tubular patency as well as integrity of tubule cells during perfusion.
This invention also provides a method for monitoring cold ischemia damage as an indication for timing and dosage of nephrotoxic immunosuppressant agents, e.g., Cyclosporin A. The present invention also provides a therapeutic method for maintaining tubule patency and a device for performing the tests of the present invention.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 1 The present invention is directed to a method for monitoring and determining the functional viability of a donor kidney prior to transplantation comprising perfusing said kidney with a perfusate having an impermeable colloid concentration which is correlated with the perfusion pressure in a ratio which permits glomerular filtration of said perfusate, the composition of which totally avoids kidney tubule reabsorption during said perfusion; and determining the ~FR of ~aid kidney during said perfusion as a measure of the functional viability of said kidney.
The present invention is further directed to A method for ensuring patency of a kidney tubule comprising perfusion of said kidney tubule with a perfusate having an impermeable colloid concentration which is correlated with the perfusion pressure in a ratio which permits glomerular filtration of said perfusate, the composition of which totally avoids kidney tubule reabsorption and tubule occlusion during said perfusion.
One innovation of the present invention resides in the finding that by manipulating the concentration of colloid in the st~n~rd UW solution in relation to perfusion pressure, glomerular filtration i 25 occurs in a manner which totally avoids reabsorption by the kidney tubules. The advantage provided by this discovery relative to the prior art is that GFR can be used as a direct measure of the functional viability of the kidney in the absence of other considerations.
3~
CA 02230474 1998-02-2~
W097/08949 PCT~S96/l4360-1 Previously, GFR could not be used as a direct measure of kidney functional viability during perfusion as a consequence of reabsorption of fluid by the tubules resulting from inadequate perfusates and improper perfusion pressures.
More specifically, in accordance with the present invention it has been determined that measuring Glomerular Filtration Rate and Total Flow at a constant perfusion pressure permits: (1) a determina~ion of overall kidney functionality; t2) a determination of afferent and efferent arteriole constriction; (3) correlations of functional parameters such as GFR, TF
and FF with baseline data to permit the assessment of the donor kidney for transplant; (4) an evaluation of appropriate dosages of nephrotoxic ;mmllnosuppressant agents and timing for immunosuppressant administration.
The present invention also provides a therapeutic method for ensuring the patency of the tubules.
The present invention is directed to a method for monitoring and assessing functional viability of a donor kidney comprising direct continuous measurement of glomerular filtration rate (GFR) during cold perfusion of the donor kidney employing a perfusion solution comprising a glomerular impermeable colloid at concentrations ranging from about .lg~ to about 4.0g~ as correlated with a range o~ perfusion pressure of about 5mmHg to about 6OmmHg. From these parameters a ratio can be calculated. Generally, a ratio of about lg~ to about lOmmHg can be used by the skilled artisan to 3o CA 02230474 1998-02-2~ .
W O 97/08949 PCTrUS96/14360 -1 establish appropriate colloid-pressure parameters.
Below a perfusate colloid concentration of lg~, however, it is preferred to employ a minimum of about 5mmHg to about lOmmHg of perfusion pressure. Specifically, between a colloid concentration of .lg~ to about lg~ of the perfusate the perfusion pressure is preferred to be between about 5mmHg and about lOmmHg to affect best results. These conditions permit perfusion of the tubules without causing reabsorptiorl and corlsequent utilization of metabolic resources as previously observed.
Continuous measurement of GFR is readily determined as a direct function of urine output.
The present invention is further directed to continuous monitoring of TF and calculation of FF as a fraction of GFR and TF, i.e., GFR/TF.
The present invention is also directed to in vitro methods of: assessing kidney functional viability and arteriolar status; correlating various kidney physiologic parameters, i.e., GFR, TF and FF in the determination of viability; and evaluating appropriate dosing and timing regimens for administering nephrotoxic immunosuppressant agents. These methods comprise continuous perfusion of the kidney at a conventional cold perfusion temperature of about 7~ to about 10~C
employing a perfusate having a reduced glomerular impermeable colloid concentration correlated with a particular perfusion pressure to avoid tubular reabsorption; monitoring the rate and volume of urine 3o , CA 02230474 1998-02-2~
W O 97/08949 PCT~US96114360 -1 produced by the kidney; directly measuring changes in GFR by plotting changes in urine output; and monitoring total flow.
The present invention permits continuous assessment of kidney functional viability by correlating increases in GFR, at constant TF, evidencing relative functional viability, and decreases in GFR, at constant TF, evidencing functional deterioration.
The present invention also perrnits assesRment of efferent arteriolar status by calculating filtration fraction and correlating FF spikes, relative increases in GFR and decreases in TF, with the presence of efferent arteriole constriction.
The present invention also permits assessment of afferent arteriolar status by calculating filtration fraction and correlating a relative decrease in GFR, a decrease in TF and the absence of a FF spike, with the presence of afferent arteriole constriction.
The present invention also permits as~essment of afferent and efferent arteriolar constriction by correlating a disproportionate decrease in GFR, a decrease in TF and the absence of a FF spike, with the presence of afferent and efferent arteriole constriction.
A further aspect of the present invention permits the correlation of various kidney physiologic parameters GFR, TF and FF to generate data relating to the general functional viability of a kidney comprising 3o CA 02230474 1998-02-2~ .
l direct and continuous measurement of GFR, continuous monitoring of TF and calculations of FF
A still further aspect of the present invention is directed to a method for evaluating appropriate dosing and timing regimens for administering nephrotoxic ;mmllnosuppressant agents comprising correlating lowered GFR with cold ischemia damage and the resultant need to delay administration of nephrotoxic immllnosuppressants.
Another aspect of the present invention is directed to a therapeutic method for ensuring the patency of the kidney tubules comprising continuous perfusion of the kidney at a conventional cold perfusion temperature of about 7~ to about 10~C comprising a perfusate having a selected glomerular impermeable colloid concentration and essential tubule nutrients balanced with a particular perfusion pressure wherein tubule occlusion is prevented by the absence of tubule reabsorption.
Yet another aspect of the present invention is directed to a collection device for positioning the ureter of a donor kidney during continuous cold perfusion, the device permits segregation of urine which may conveniently be collected and measured by an automatic siphon allowing for visual ~uantitation of urine output.
Figures la-lc show kidneys that have been cold perfused stored over approximately 24-48 hours. Total flow (TF), glomerular filtration rat (GFR) and 3o CA 02230474 1998-02-2~
W O 97108949 PCr~US96/14360 -1 filtration fraction (FF) are all plotted. The arrow in la indicates a large increment in GFR resulting from an increased efferent constriction. The arrows in lb and lc indicate no increment in GFR.
Figure 2 shows a biphasic response in filtration during the first ten hours of cold storage.
GFR, TF and FF are all 100~ at the time when the biphasic GFR response is at its m~; mllm, Figures 3-10 represellt indeperldent experimen~s demonstrating the biphasic response in filtration during the first ten hours of cold storage.
The present invention provides a method for assessing the functional viability of a donor kidney by monitoring one or more o~ certain kidney-specific physiologic markers. In particular, the pre~ent invention provides a method for ascer~-~;n'ng kidney function by measuring Glomerular Filtration Rate (GFR) during cold perfusion of a donor kidney employing a perfusion solution having a particular colloid concentration. Perfusion is conducted at a selected perfusion pressure which is correlated with the concentration of the colloid of the perfusate in a manner which avoids reabsorption by the kidney tubules.
It has been surprisingly discovered in accordance with the present invention that the concentration of the colloid of the perfusate and the perfusion pressure can be correlated in a ratio in such a manner as to totally avoid tubule reabsorption of e.g.
water, sodium and potassium by the kidney tubules during 3o CA 02230474 1998-02-2~ .
1 cold perfusion, thereby permitting direct assessment of the relative kidney functionality by measurement of GFR
or GFR and TF. Such measurements permit determinations of the relative functional viability of the donor kidney for transplantation. These parameters can be determined in order to further assess arteriolar status, tubule patency and appropriate dosing and timing regimens for administering nephrotoxic ;mmllnosuppressant agents post-transplant.
The present invention also permits continuous monitoring of two additional parameters Total Flow (TF) and Filtration Fraction (FF). By Total Flow i8 meant the total fluid flow through the kidneys. The Total Flow is generally quantitated in ~l/min in rodents and in ml/min in ~llm~n~ Total Flow is determined by the additive resistances of the afferent and efferent arterioles. TF can influence glomerular pressure but does not determine glomerular pressure per se. The filtration fraction (FF) is the fraction of the kidney fluid ~low that becomes glomerular filtrate. Thus the filtration fraction can be described as the ratio of GFR
to TF i.e., FF=GFR/TF. By continuously measuring GFR
and TF it is now possible in accordance with the present invention to assess the status of the kidney vasculature.
By functional viability is meant the physiological changes in the ability of the kidney to filter during cold storage at about 7~ to about 10~C
3o , CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 -1 over the period of time from harvest of the organ to transplantation.
The perfusion solution of the present invention i8 a modified Belzer-MPS~ solution the formulation of which comprise~ Sodium Gluconate, Potassium Phosphate, Monobasic NF, Magnesium Gluconate, USP (dihydrate), Glucose, Beta D(+), ~nine (free base), Ribose (D-), HEPES (free acid), Glutathione (reduced form), ~a~cium ~hloride, USP ~dihydrate), Mannitol, USP, Modified Hydroxyethyl Starch (3.75g~), Sodium Hydroxide (pH 7.4) and Water. In use, the skilled artisan may augment the solution with conventional additives such as Insulin, Dexamethasone and Penicillin.
By Glomerular Filtration Rate (GFR) is meant the rate at which fluid is filtered through the kidney.
GFR is the art established "Gold Standard" measurement of kidney function. More specifically, GFR means the quantity of filtrate formed by the kidney. GFR is also directly related to the pressure in the glomerulus.
Glomerular pressure is determined by the relative resistances between the afferent and efferent arterioles. The afferent arteriole serves as the pathway by which fluid enters the glomerulus. The efferent arteriole is the pathway by which fluid leaves the glomerulus.
The arterioles form an important part of the kidney vasculature and the assessment of their respective functional and physiological condition is 3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 extremely advantageous to the overall determination of kidney viability. The kidney vasculature supplies needed nutrients to the surrounding tissue and plays a unique function in forming an ultrafiltrate of plasma (glomerular filtration). The tubule component of the kidney is functionally interrelated with the vascular component. Once fluid is filtered through the glomerulus it enters the tubule. The tubule component generally serves to reabsorb fluids.
Previous studies attempting to evaluate tubular activity in cold stored kidneys reported substantial tubular reabsorption, energy utilization and obstruction. (Gianello et al. supra.) These studies utilized albumin-based perfusates. Thus, prior researchers found that perfusion of the tubules would necessarily result in substantial tubular reabsorption, cellular sloughing and energy utilization. Tubular reabsorption, cellular sloughing and energy utilization impeded prior efforts to directly assess kidney viability because the tubules would either be clogged with dead cells or the tubules would be actively reabsorbing fluid.
The present invention employs the above-identified composition or its equivalent, a glomerular impermeable colloid based perfusate at a concentration of about .lg~ to about 4.0g~, a perfusion pressure range of about 5mmHg to about 6OmmHg and a temperature of about 7~ to about 10~C. From these parameters a ratio can be calculated. Generally, a ratio of about lg~ to 3o . , CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 -1 about lOmmHg can be used by the skilled artisan to establish appropriate colloid-pressure parameters.
Below a perfusate colloid concentration of lg~, however, it is preferred to employ a m;n;mllm of about 5mmHg to about lOmmHg of perfusion pressure. Specifically, between a colloid concentration of .lg~ to about lg~ of the perfusate the perfusion pressure is preferred to be between about 5mmHg and about lOmmHg to affect best results. These conditions permit perfusion of the tubules without causing reabsorption and consequent utilization of metabolic resources as previously observed.
By glomerular impermeable colloid i9 meant a component of a perfusate (e.g. modified hydroxyethyl starch or albumin) which characteristically stabilizes the glomerular membrane throughout a perfusion period.
A preferred glomerular impermeable colloid based perfusate employs modified hydroxyethyl starch at the concentration, pressures and temperatures identified above.
In accordance with the present invention, a biphasic response in filtration (GFR) has been observed which can be utilized in the assessment and determination of kidney viability. This phenomenon has generally been observed during the first ten hours of cold storage by continuous monitoring of the mammalian kidneys by the method of the present invention. The biphasic response is characterized by an initial increase in filtration (GFR) as determined by measuring 3o CA 02230474 l998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 changes in urine output per unit of time. The increase in filtration is followed by a steady state at which filtration and total flow are about the same. The steady state is then followed by a decrease in filtration as determined by measuring changes in urine output per unit of time.
The rate at which increases or decreases in GFR occur serves as a direct indicator of kidney functionality. ~pecifically a steep rate of decline in GFR measured per unit of time is indicative of a deteriorating kidney. A rate of incline in GFR measured per unit time is indicative of a functionally viable kidney. A steady GFR (no slope) in conjunction with a steady Total Flow indicates that GFR has plateaued and the kidney is functionally relatively viable (but less 80 than in the inclining state). A steady GFR or one declining with a small slope relative to unit time, especially with no appreciable Total Flow, indicates that the kidney has deteriorated and is not transplantable.
Thus, another embodiment of the present invention i8 directed to mea~3uring the rate of increase or decrease in filtration, i.e. the GFR biphasic response, during cold perfusion in accordance with the present method to determine the relative viability of the kidney.
In accordance with the present invention a donor kidney is harvested and cold perfused as soon as practicable employing a perfusate comprising from about 3o . CA 02230474 1998-02-2~
W O 97/08949 PCTnUS96~436a -l .lg~ to about 4.0g~ Modified Hydroxyethyl Starch at a pressure of from about 5mmHg to about 60mmHg (as determined by the conventional ratio ~etween colloid concentration and perfusion pressure) and a temperature from about 7~ to about 10~C. For exarnple, a conventional in-line probe can be used to monitor perfusion pressure and total flow. The donor kidney is placed in a conventional cold perfusion cassette, which is removably a~tached to a coilventional recircula~ing perfusion pump (such as the Waters MOX-lOODCM cassette and perfusion pump), the renal artery is removably connected to a tube in the cassette, which is removably connected to the perfusion pump. The renal vein is not attached to the perfusion apparatus and r~m~;n~ free to drain. The ureter is positioned on an inclined trough or other collection device provided within the perfusion cassette, thus permitting gravitational urine flow from the kidney. The inclined trough is removably attached to an automatic siphon, for example, :in the perfusion cassette. The perfusate is pumped in~-o the renal artery and urine exits the kidney through the ureter, while other fluid exits the kidney through the renal vein.
Renal vein fluid is recirculated through the per~usion apparatus, while urine is collected with the aid of an automatic siphon, a drop counter or a volumetric chamber. Urine volume and urine flow rate are conveniently measured continuously in ~l or ml increments depending on the kidney species. Urine output is then correlated directly with GFR. Increasing CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 GFR per unit of time correlates generally with kidney functional viability, while decreaeing GFR per unit of time correlates generally with kidney deterioration and no GFR correlates with a non-viable kidney.
In accordance with the present invention the ureter may be placed on an inclined trough which is .e~ dbly attached to an automatic siphon within the perfusion cassette which permits urine output to be measured directly during monitoring.
In accordance with the present invention GFR
may be measured employing an art recognized balance combined with a computer software program to instantaneously and continuously measure perfusion pressure and total flow in milliliter or microliter increments of urine output in weight per unit time depending on the species of kidney.
The present invention permits GFR to be measured by conventional means known to the skilled artisan including visual counting of urine drops formed and more preferably with a volumetric chamber or an automatic siphon.
Utilizing the methodology of the present invention, the inventor has unexpectedly discovered that a sequence of changes in afferent and efferent arteriole resistance occur during cold storage monitoring. When FF spike (steeply sloped FF) is observed it generally indicates that the relationship between GFR and TF has changed due to a proportionately greater GFR. This can occur when efferent resistance preferentially increases 3o CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 causing glomerular capillary pressure to rise. This situation will cause an increase in filtration (GFR) and a decrease in TF. The inventor has discovered that this manifestation is characteristic of efferent arteriole constriction. Similarly, a relative decrease in GFR
with a concomitant decrease in TF and the absence of a FF spike indicates afferent arteriole constriction. In addition, a disproportionate decrease in GFR with a concomitant decrease in TF and the absence o~ a FF spike 1 suggests concurrent efferent and afferent arteriole constriction. Each of the three foregoing results provide useful information for the practitioner wishing to administer appropriate vasodilators or vasoconstrictors post-transplantation as well as in the assessment of the relative viability of the kidney.
In a still further embodiment, the inventors have discovered that the present methodology can assist practitioners in deciding the timing and dosing of initial post-transplant Cyclosporin A lmm~lnosuppression.
By continuously monitoring filtration (GFR) from harvest to transplant, a decreasing GFR immediately prior to transplant signals an ischemically damaged organ, which should not receive nephrotoxic immunosuppression e.g.
Cyclosporin A. Ischemia is known to enhance the nephrotoxicity of Cyclosporin A. Thus postponing immunosuppressant therapy until GFR is steadily increasing, as determined by conventional in vivo tests, may aid overall gra~t survival.
3o CA 02230474 1998-02-2~ .
In accordance with the present invention the inventor has now discovered that tubular perfusion i6 beneficial and can be used to perform the same functions as vascular perfusion by maint~;n;ng lumen (tubule) 5 patency while providing valuable nutrients. Further-more, tubular obstruction, which is a common result of tubule ischemic damage, may be mln;m;zed by tubular perfusion. Absent any reabsorption in the tubules, the inventor has, ~or the first time recognized that the 1 rate at which the fluid, e.g., urine, leaves the kidney is identical to the rate at which it was filtered through the glomerulus, i.e., GFR. The recognition that urine output and GFR can be equated provides the skilled artisan with a novel and simple diagnostic tool which can be used to continuously assess kidney function.
3o , CA 02230474 1998-02-2~
WO 97/08949 P ~ ~US96/1436a -1 E~MPL~ 1 Phy~ioloqic Marker Monitoring Lewis rat kidneys (N=20) were cold perfused at 7~C to 10~C and continuously monitored for up to 72 5 hours to ~m; ne the range of cold storage responses (see Figures la-lc). During cold storage, TF decrea~ed over time. Efferent arteriole resistance (constriction of the efferent arteriole, as measured by relative increases in ~FR and de~reases in Total Flow) increased during cold storage monitoring causing glomerular capillary pressure to rise. The increase in glomerular capillary pressure caused a relative increase in filtration (GFR) and a decrease in total flow and marked spike in filtration fraction (FF) (see Figure la). The decrease in TF and relative increase in GFR caused the spike in the FF.
Efferent constriction accompanied by afferent constriction resulted in a dramatic increase in FF.
This increase was likely due to a drop in TF, rather than an increase in GFR. (See Figures lb and lc).
The drop in TF caused an increase in the GFR/TF ratio and thus increased FF. The increase in FF may occur at different times and may be a marker of kidney viability as shown in Figures la-lc.
In Figure lb, FF dramatically increased at approximately 40 hours post-harvest in vitro but in Figure lc, the spike in FF occurred at approximately 15 hours post-harvest in vitro. The FF spike appears to be 3o W O 97/08949 PCT~US96/14360 -1 an indicator of irreversible renal tissue deterioration and a useful marker for maximum cold storage time.
These results indicated that cold storage monitoring can inferentially differentiate kidney 5 arteriolar status during cold storage.
3o . . CA 02230474 1998-02-2~
W O 97/08949 PCTrUS96/14360-1 E~U~MPLE 2 Predictive Utility of Biphasic GFR
Continuous monitoring of the kidney post-harvest i vitro uncovered a biphasic response in 5 filtration during the first ten hours of storage (Figures 2-10). The "GFR m~;mllm~ (see Figures 2-10), occurred at different times within the first ten hours following harvest (i.e., ln vitro time=X axis). The Y
axis ~ maximum~ is a percentage of an actual value determined by choosing, as a reference point, ("GFR
m~;mllm~), the time at which GFR reached a m~;mllm in the first 10 hours of cold storage. Thus, GFR, TF and FF are all standardized to 100~ at the time when the GFR
is at its m~; ml~m . This st~n~rdization allowed for more efficient comparisons of kidney values as shown in Figures 2-10.
Specifically, GFR increased an average of 34.1~ (pc.002) from 2 hours to "GFR m~; ml~m" and dropped an average of 49.2~ (pc.001) from "GFR maximum" to 10 hours. Statistical significance was evaluated using a post-hoc ANOVA comparison of means using repeated measures and the Newman-Kuels test.
Neither the initial increase in filtration nor the decrease that followed required a change in total organ flow (TF). TF at 2 hours, "GFR m~imllm~, and 10 hours, was not significantly different and averaged 4533+250~1/min, 4650+380~1/min, and 4083+460~1/min respectively.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 Data from the biphasic GFR response demonstrated marked clinical implications. Knowledge of the biphasic GFR response reflects the length of time a donor kidney can be cold stored without significant 5 damage. It has been reported that cadaveric kidneys transplanted in less than 6 hours, or living non-related donor kidney transplants (LNR), have similar outcomes as living related transplants (LR) (Teraski et al., TransPlants, Los Ar.geles, CA, UCLA Tissue Typing 10 Laboratory, 1991). Since cadaveric kidneys are usually transplanted after several hours of cold storage and have historically poorer graft outcomes than LR
transplants, knowledge that a decrease in filtration following the GFR peak correlates to a loss in post-transplant function provides a useful indicator of graftsuccess .
3o CA 02230474 1998-02-2~
W O 97/08949 PCT~US96~14360 -Kidney Harvest, Perfu~ion Storaqe and Transplant A starch based perfusate (Belzar MPS~
Perfusate, modified starch concentration 3.75g~), was 5 u~ed in twenty genetically matched Lewis rats (275-300g) (Harland Sprague-Dawley, Indianapolis, IN). Kidneys were initially cold perfused ln vivo at 7~-10~C, through a distal aortic catheter. Cold per~usate was pumped t~lrough the kidneys at physiologic pressures while simultaneously clamping the aortas above the kidneys.
Only left kidneys were harvested immediately following aortic clamping. The renal artery, vein and ureter were catheterized during cold perfusion. The renal vein catheter had to be large (.07"ID, .ll"OD) to accommodate low pressure venous flow. The kidney and all three catheters were stabilized on a small platform in a kidney transplant cassette.
Immediately, following harvest, each kidney was tested to insure proper kidney and catheter patency by cold perfusing the renal artery at 60mmHg and measuring the total kidney flow (TF). A TF of 3.6ml/min - 4.2ml/min was required to insure uniform resistance in all kidneys.
The kidney cassette was then connected to a perfusion apparatus and pump (Baxter Rotary Dialysis Pump, Chicago, Il.) using universal silastic fittings on the renal artery and vein. This system mimicked the in vlvo vasculature of the rat kidney by recirculating cold perfusate at approximately 450mls/min. The system does 3o CA 02230474 1998-02-2~ .
1 not force fluid through the kidney but allowed the actual kidney resistance, at any given moment, to determine flow. The system therefore permitted the arterioles to react to pressure changes naturally.
A catheter coming from the main recirculating line (from the pump) was connected to the kidney module, thus mimicking the way the renal artery comes off the aorta in vivo. Since, kidney flow out of the recirculating line was less (1-lOml/min) than the total flow being recirculated (approximately 450ml/min), changes in kidney flow did not significantly change pressure in the recirculating line. This system allowed kidney flow to increase or decrease as a result of kidney resistance without altering central pressure generated from the main recirculating line.
Total flow and GFR were monitored. An in-line probe (Transonic FM #T106, Ithaca, N.Y.) was used to measure TF. A 27 gauge needle was inserted into the renal artery silastic catheter to monitor perfusion pressure (Stathum Strain Gauge, Gould Windograf #40-8474, Valley View, OH). The ureter catheter (.02"ID) was extended and placed in a beaker under oil inside a Mettler PM100 balance. The Mettler balance permitted accurate measurements of microliter increments of urine.
Instantaneous microliter GFR changes in a cold perfused kidney were monitored (i.e., in weight per unit time) by combining the Mettler balance with a Mettler computer software program (Balancelink, Mettler). The Mettler 3o , , CA 02230474 1998-02-2~
l balance and accompanying software permitted continuous 2 day GFR monitoring of rat kidneys.
The perfusion pump, kidney cassette and recirculating tubing were placed in a 7~-10~~ cold room 5 to maintain tissue temperature during monitoring.
Transplantation was accomplished by matching the male universal fittings on the renal artery and vein to the female fittings on the aorta and vena cava extending from the abdomen of the reclpient ~ewis rat.
lO The transplanted kidney was insulated with Saran WrapTM
and maintained at body temperature 37~C.
Utilizing a st~n~rd in vlvo iohexal measurement of urine flow rate, the transplanted kidneys retained an average of 87~ of their overall function as compared to the untouched kidney in the genetically matched recipient rat when transplanted within two hours post-harvest. The urine concentration of iohexal multiplied by the urine volume divided by the plasma concentration of iohexal (.1 mg/ml) provided a ~ measurement GFR. The transplanted kidney GFR divided by the GFR of the untouched kidney multiplied by 100 provides a measurement of relative kidney function achieved by the transplanted kidney.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -A human kidney is cold (7~C to 10~C) perfused n vivo at physiological pressure without cessation of heart beat. A starch based perfusate (Belzar MPS~
5 Perfusate, modified colloid concentration .lg~ - 4g%), is employed throughout the perfusion period.
The kidney is harvested as soon as practicable, while continually perfusing the aorta. The renal artery is catheterized, as soon as practicable, lO and the renal artery is attached to a Waters MOX-lOODCM
Perfusion Apparatus, taking care to avoid air embolisms during transfer to the perfusion apparatus. The renal vein is left free so that fluid from the renal vein flows freely into the perfusion apparatus. The ureter is 5 positioned in an inclined trough thereby permitting gravity induced fluid flow therefrom. The trough supporting the ureter is attached to an automatic siphon capable of collecting lOml urine. The kidney is stabilized on an inclined platform in the perfusion apparatus and is not handled again until transplant.
The kidney is initially perfused at a pressure of 45mmHg to ensure proper kidney patency by measuring TF.
Cold perfusate is recirculated through the kidney at approximately 200ml/min. The system does not force fluid through the kidney but allows the actual kidney resistance, at any given moment, to determine flow. The system therefore permits the arterioles to react to pressure changes naturally.
3~
, . CA 02230474 l998-02-2~
W O 97/08949 PCTrUS96/14360-1 A catheter coming from the main recirculating line, (through the pump) is connected to the kidney , thus mimicking the way the renal artery comes off the aorta in vivo. Since, kidney flow out of the 5 recirculating line is less, i.e., about 80ml/min than the total flow being recirculated (about 200ml/min), changes in kidney flow will not significantly change pressure in the recirculating line. This system allows ki~-ley flow to increase or decrease as a result of 10 kidney resistance without altering central pressure generated from the main recirculating line.
Total flow, GFR and perfusion pressure are continuously monitored by conventional systems resident in the perfusion apparatus and determinations respecting kidney functional viability are made. Instantaneous and continuous milliliter GFR changes in a cold perfused kidney are monitored conventionally by measuring how the rate at which the automatic siphon empties. The automatic siphon will permit continuous 2 day GFR
monitoring of urine output.
The perfusion pump, kidney cassette and recirculating tubing are placed in a 7~-10~C cold room to maintain tissue temperature during monitoring.
Transplantation of the monitored kidney will greatly improve graft success rates, climinish hospital stays, eliminate the need for further dialysis and prolong the useful life of the kidney in the recipient.
3o
CA 02230474 1998-02-2~
W097/08949 PCT~S96/l4360-1 Previously, GFR could not be used as a direct measure of kidney functional viability during perfusion as a consequence of reabsorption of fluid by the tubules resulting from inadequate perfusates and improper perfusion pressures.
More specifically, in accordance with the present invention it has been determined that measuring Glomerular Filtration Rate and Total Flow at a constant perfusion pressure permits: (1) a determina~ion of overall kidney functionality; t2) a determination of afferent and efferent arteriole constriction; (3) correlations of functional parameters such as GFR, TF
and FF with baseline data to permit the assessment of the donor kidney for transplant; (4) an evaluation of appropriate dosages of nephrotoxic ;mmllnosuppressant agents and timing for immunosuppressant administration.
The present invention also provides a therapeutic method for ensuring the patency of the tubules.
The present invention is directed to a method for monitoring and assessing functional viability of a donor kidney comprising direct continuous measurement of glomerular filtration rate (GFR) during cold perfusion of the donor kidney employing a perfusion solution comprising a glomerular impermeable colloid at concentrations ranging from about .lg~ to about 4.0g~ as correlated with a range o~ perfusion pressure of about 5mmHg to about 6OmmHg. From these parameters a ratio can be calculated. Generally, a ratio of about lg~ to about lOmmHg can be used by the skilled artisan to 3o CA 02230474 1998-02-2~ .
W O 97/08949 PCTrUS96/14360 -1 establish appropriate colloid-pressure parameters.
Below a perfusate colloid concentration of lg~, however, it is preferred to employ a minimum of about 5mmHg to about lOmmHg of perfusion pressure. Specifically, between a colloid concentration of .lg~ to about lg~ of the perfusate the perfusion pressure is preferred to be between about 5mmHg and about lOmmHg to affect best results. These conditions permit perfusion of the tubules without causing reabsorptiorl and corlsequent utilization of metabolic resources as previously observed.
Continuous measurement of GFR is readily determined as a direct function of urine output.
The present invention is further directed to continuous monitoring of TF and calculation of FF as a fraction of GFR and TF, i.e., GFR/TF.
The present invention is also directed to in vitro methods of: assessing kidney functional viability and arteriolar status; correlating various kidney physiologic parameters, i.e., GFR, TF and FF in the determination of viability; and evaluating appropriate dosing and timing regimens for administering nephrotoxic immunosuppressant agents. These methods comprise continuous perfusion of the kidney at a conventional cold perfusion temperature of about 7~ to about 10~C
employing a perfusate having a reduced glomerular impermeable colloid concentration correlated with a particular perfusion pressure to avoid tubular reabsorption; monitoring the rate and volume of urine 3o , CA 02230474 1998-02-2~
W O 97/08949 PCT~US96114360 -1 produced by the kidney; directly measuring changes in GFR by plotting changes in urine output; and monitoring total flow.
The present invention permits continuous assessment of kidney functional viability by correlating increases in GFR, at constant TF, evidencing relative functional viability, and decreases in GFR, at constant TF, evidencing functional deterioration.
The present invention also perrnits assesRment of efferent arteriolar status by calculating filtration fraction and correlating FF spikes, relative increases in GFR and decreases in TF, with the presence of efferent arteriole constriction.
The present invention also permits assessment of afferent arteriolar status by calculating filtration fraction and correlating a relative decrease in GFR, a decrease in TF and the absence of a FF spike, with the presence of afferent arteriole constriction.
The present invention also permits as~essment of afferent and efferent arteriolar constriction by correlating a disproportionate decrease in GFR, a decrease in TF and the absence of a FF spike, with the presence of afferent and efferent arteriole constriction.
A further aspect of the present invention permits the correlation of various kidney physiologic parameters GFR, TF and FF to generate data relating to the general functional viability of a kidney comprising 3o CA 02230474 1998-02-2~ .
l direct and continuous measurement of GFR, continuous monitoring of TF and calculations of FF
A still further aspect of the present invention is directed to a method for evaluating appropriate dosing and timing regimens for administering nephrotoxic ;mmllnosuppressant agents comprising correlating lowered GFR with cold ischemia damage and the resultant need to delay administration of nephrotoxic immllnosuppressants.
Another aspect of the present invention is directed to a therapeutic method for ensuring the patency of the kidney tubules comprising continuous perfusion of the kidney at a conventional cold perfusion temperature of about 7~ to about 10~C comprising a perfusate having a selected glomerular impermeable colloid concentration and essential tubule nutrients balanced with a particular perfusion pressure wherein tubule occlusion is prevented by the absence of tubule reabsorption.
Yet another aspect of the present invention is directed to a collection device for positioning the ureter of a donor kidney during continuous cold perfusion, the device permits segregation of urine which may conveniently be collected and measured by an automatic siphon allowing for visual ~uantitation of urine output.
Figures la-lc show kidneys that have been cold perfused stored over approximately 24-48 hours. Total flow (TF), glomerular filtration rat (GFR) and 3o CA 02230474 1998-02-2~
W O 97108949 PCr~US96/14360 -1 filtration fraction (FF) are all plotted. The arrow in la indicates a large increment in GFR resulting from an increased efferent constriction. The arrows in lb and lc indicate no increment in GFR.
Figure 2 shows a biphasic response in filtration during the first ten hours of cold storage.
GFR, TF and FF are all 100~ at the time when the biphasic GFR response is at its m~; mllm, Figures 3-10 represellt indeperldent experimen~s demonstrating the biphasic response in filtration during the first ten hours of cold storage.
The present invention provides a method for assessing the functional viability of a donor kidney by monitoring one or more o~ certain kidney-specific physiologic markers. In particular, the pre~ent invention provides a method for ascer~-~;n'ng kidney function by measuring Glomerular Filtration Rate (GFR) during cold perfusion of a donor kidney employing a perfusion solution having a particular colloid concentration. Perfusion is conducted at a selected perfusion pressure which is correlated with the concentration of the colloid of the perfusate in a manner which avoids reabsorption by the kidney tubules.
It has been surprisingly discovered in accordance with the present invention that the concentration of the colloid of the perfusate and the perfusion pressure can be correlated in a ratio in such a manner as to totally avoid tubule reabsorption of e.g.
water, sodium and potassium by the kidney tubules during 3o CA 02230474 1998-02-2~ .
1 cold perfusion, thereby permitting direct assessment of the relative kidney functionality by measurement of GFR
or GFR and TF. Such measurements permit determinations of the relative functional viability of the donor kidney for transplantation. These parameters can be determined in order to further assess arteriolar status, tubule patency and appropriate dosing and timing regimens for administering nephrotoxic ;mmllnosuppressant agents post-transplant.
The present invention also permits continuous monitoring of two additional parameters Total Flow (TF) and Filtration Fraction (FF). By Total Flow i8 meant the total fluid flow through the kidneys. The Total Flow is generally quantitated in ~l/min in rodents and in ml/min in ~llm~n~ Total Flow is determined by the additive resistances of the afferent and efferent arterioles. TF can influence glomerular pressure but does not determine glomerular pressure per se. The filtration fraction (FF) is the fraction of the kidney fluid ~low that becomes glomerular filtrate. Thus the filtration fraction can be described as the ratio of GFR
to TF i.e., FF=GFR/TF. By continuously measuring GFR
and TF it is now possible in accordance with the present invention to assess the status of the kidney vasculature.
By functional viability is meant the physiological changes in the ability of the kidney to filter during cold storage at about 7~ to about 10~C
3o , CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 -1 over the period of time from harvest of the organ to transplantation.
The perfusion solution of the present invention i8 a modified Belzer-MPS~ solution the formulation of which comprise~ Sodium Gluconate, Potassium Phosphate, Monobasic NF, Magnesium Gluconate, USP (dihydrate), Glucose, Beta D(+), ~nine (free base), Ribose (D-), HEPES (free acid), Glutathione (reduced form), ~a~cium ~hloride, USP ~dihydrate), Mannitol, USP, Modified Hydroxyethyl Starch (3.75g~), Sodium Hydroxide (pH 7.4) and Water. In use, the skilled artisan may augment the solution with conventional additives such as Insulin, Dexamethasone and Penicillin.
By Glomerular Filtration Rate (GFR) is meant the rate at which fluid is filtered through the kidney.
GFR is the art established "Gold Standard" measurement of kidney function. More specifically, GFR means the quantity of filtrate formed by the kidney. GFR is also directly related to the pressure in the glomerulus.
Glomerular pressure is determined by the relative resistances between the afferent and efferent arterioles. The afferent arteriole serves as the pathway by which fluid enters the glomerulus. The efferent arteriole is the pathway by which fluid leaves the glomerulus.
The arterioles form an important part of the kidney vasculature and the assessment of their respective functional and physiological condition is 3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 extremely advantageous to the overall determination of kidney viability. The kidney vasculature supplies needed nutrients to the surrounding tissue and plays a unique function in forming an ultrafiltrate of plasma (glomerular filtration). The tubule component of the kidney is functionally interrelated with the vascular component. Once fluid is filtered through the glomerulus it enters the tubule. The tubule component generally serves to reabsorb fluids.
Previous studies attempting to evaluate tubular activity in cold stored kidneys reported substantial tubular reabsorption, energy utilization and obstruction. (Gianello et al. supra.) These studies utilized albumin-based perfusates. Thus, prior researchers found that perfusion of the tubules would necessarily result in substantial tubular reabsorption, cellular sloughing and energy utilization. Tubular reabsorption, cellular sloughing and energy utilization impeded prior efforts to directly assess kidney viability because the tubules would either be clogged with dead cells or the tubules would be actively reabsorbing fluid.
The present invention employs the above-identified composition or its equivalent, a glomerular impermeable colloid based perfusate at a concentration of about .lg~ to about 4.0g~, a perfusion pressure range of about 5mmHg to about 6OmmHg and a temperature of about 7~ to about 10~C. From these parameters a ratio can be calculated. Generally, a ratio of about lg~ to 3o . , CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 -1 about lOmmHg can be used by the skilled artisan to establish appropriate colloid-pressure parameters.
Below a perfusate colloid concentration of lg~, however, it is preferred to employ a m;n;mllm of about 5mmHg to about lOmmHg of perfusion pressure. Specifically, between a colloid concentration of .lg~ to about lg~ of the perfusate the perfusion pressure is preferred to be between about 5mmHg and about lOmmHg to affect best results. These conditions permit perfusion of the tubules without causing reabsorption and consequent utilization of metabolic resources as previously observed.
By glomerular impermeable colloid i9 meant a component of a perfusate (e.g. modified hydroxyethyl starch or albumin) which characteristically stabilizes the glomerular membrane throughout a perfusion period.
A preferred glomerular impermeable colloid based perfusate employs modified hydroxyethyl starch at the concentration, pressures and temperatures identified above.
In accordance with the present invention, a biphasic response in filtration (GFR) has been observed which can be utilized in the assessment and determination of kidney viability. This phenomenon has generally been observed during the first ten hours of cold storage by continuous monitoring of the mammalian kidneys by the method of the present invention. The biphasic response is characterized by an initial increase in filtration (GFR) as determined by measuring 3o CA 02230474 l998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 changes in urine output per unit of time. The increase in filtration is followed by a steady state at which filtration and total flow are about the same. The steady state is then followed by a decrease in filtration as determined by measuring changes in urine output per unit of time.
The rate at which increases or decreases in GFR occur serves as a direct indicator of kidney functionality. ~pecifically a steep rate of decline in GFR measured per unit of time is indicative of a deteriorating kidney. A rate of incline in GFR measured per unit time is indicative of a functionally viable kidney. A steady GFR (no slope) in conjunction with a steady Total Flow indicates that GFR has plateaued and the kidney is functionally relatively viable (but less 80 than in the inclining state). A steady GFR or one declining with a small slope relative to unit time, especially with no appreciable Total Flow, indicates that the kidney has deteriorated and is not transplantable.
Thus, another embodiment of the present invention i8 directed to mea~3uring the rate of increase or decrease in filtration, i.e. the GFR biphasic response, during cold perfusion in accordance with the present method to determine the relative viability of the kidney.
In accordance with the present invention a donor kidney is harvested and cold perfused as soon as practicable employing a perfusate comprising from about 3o . CA 02230474 1998-02-2~
W O 97/08949 PCTnUS96~436a -l .lg~ to about 4.0g~ Modified Hydroxyethyl Starch at a pressure of from about 5mmHg to about 60mmHg (as determined by the conventional ratio ~etween colloid concentration and perfusion pressure) and a temperature from about 7~ to about 10~C. For exarnple, a conventional in-line probe can be used to monitor perfusion pressure and total flow. The donor kidney is placed in a conventional cold perfusion cassette, which is removably a~tached to a coilventional recircula~ing perfusion pump (such as the Waters MOX-lOODCM cassette and perfusion pump), the renal artery is removably connected to a tube in the cassette, which is removably connected to the perfusion pump. The renal vein is not attached to the perfusion apparatus and r~m~;n~ free to drain. The ureter is positioned on an inclined trough or other collection device provided within the perfusion cassette, thus permitting gravitational urine flow from the kidney. The inclined trough is removably attached to an automatic siphon, for example, :in the perfusion cassette. The perfusate is pumped in~-o the renal artery and urine exits the kidney through the ureter, while other fluid exits the kidney through the renal vein.
Renal vein fluid is recirculated through the per~usion apparatus, while urine is collected with the aid of an automatic siphon, a drop counter or a volumetric chamber. Urine volume and urine flow rate are conveniently measured continuously in ~l or ml increments depending on the kidney species. Urine output is then correlated directly with GFR. Increasing CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 GFR per unit of time correlates generally with kidney functional viability, while decreaeing GFR per unit of time correlates generally with kidney deterioration and no GFR correlates with a non-viable kidney.
In accordance with the present invention the ureter may be placed on an inclined trough which is .e~ dbly attached to an automatic siphon within the perfusion cassette which permits urine output to be measured directly during monitoring.
In accordance with the present invention GFR
may be measured employing an art recognized balance combined with a computer software program to instantaneously and continuously measure perfusion pressure and total flow in milliliter or microliter increments of urine output in weight per unit time depending on the species of kidney.
The present invention permits GFR to be measured by conventional means known to the skilled artisan including visual counting of urine drops formed and more preferably with a volumetric chamber or an automatic siphon.
Utilizing the methodology of the present invention, the inventor has unexpectedly discovered that a sequence of changes in afferent and efferent arteriole resistance occur during cold storage monitoring. When FF spike (steeply sloped FF) is observed it generally indicates that the relationship between GFR and TF has changed due to a proportionately greater GFR. This can occur when efferent resistance preferentially increases 3o CA 02230474 1998-02-2~
W O 97/08949 PCT~US96/14360 causing glomerular capillary pressure to rise. This situation will cause an increase in filtration (GFR) and a decrease in TF. The inventor has discovered that this manifestation is characteristic of efferent arteriole constriction. Similarly, a relative decrease in GFR
with a concomitant decrease in TF and the absence of a FF spike indicates afferent arteriole constriction. In addition, a disproportionate decrease in GFR with a concomitant decrease in TF and the absence o~ a FF spike 1 suggests concurrent efferent and afferent arteriole constriction. Each of the three foregoing results provide useful information for the practitioner wishing to administer appropriate vasodilators or vasoconstrictors post-transplantation as well as in the assessment of the relative viability of the kidney.
In a still further embodiment, the inventors have discovered that the present methodology can assist practitioners in deciding the timing and dosing of initial post-transplant Cyclosporin A lmm~lnosuppression.
By continuously monitoring filtration (GFR) from harvest to transplant, a decreasing GFR immediately prior to transplant signals an ischemically damaged organ, which should not receive nephrotoxic immunosuppression e.g.
Cyclosporin A. Ischemia is known to enhance the nephrotoxicity of Cyclosporin A. Thus postponing immunosuppressant therapy until GFR is steadily increasing, as determined by conventional in vivo tests, may aid overall gra~t survival.
3o CA 02230474 1998-02-2~ .
In accordance with the present invention the inventor has now discovered that tubular perfusion i6 beneficial and can be used to perform the same functions as vascular perfusion by maint~;n;ng lumen (tubule) 5 patency while providing valuable nutrients. Further-more, tubular obstruction, which is a common result of tubule ischemic damage, may be mln;m;zed by tubular perfusion. Absent any reabsorption in the tubules, the inventor has, ~or the first time recognized that the 1 rate at which the fluid, e.g., urine, leaves the kidney is identical to the rate at which it was filtered through the glomerulus, i.e., GFR. The recognition that urine output and GFR can be equated provides the skilled artisan with a novel and simple diagnostic tool which can be used to continuously assess kidney function.
3o , CA 02230474 1998-02-2~
WO 97/08949 P ~ ~US96/1436a -1 E~MPL~ 1 Phy~ioloqic Marker Monitoring Lewis rat kidneys (N=20) were cold perfused at 7~C to 10~C and continuously monitored for up to 72 5 hours to ~m; ne the range of cold storage responses (see Figures la-lc). During cold storage, TF decrea~ed over time. Efferent arteriole resistance (constriction of the efferent arteriole, as measured by relative increases in ~FR and de~reases in Total Flow) increased during cold storage monitoring causing glomerular capillary pressure to rise. The increase in glomerular capillary pressure caused a relative increase in filtration (GFR) and a decrease in total flow and marked spike in filtration fraction (FF) (see Figure la). The decrease in TF and relative increase in GFR caused the spike in the FF.
Efferent constriction accompanied by afferent constriction resulted in a dramatic increase in FF.
This increase was likely due to a drop in TF, rather than an increase in GFR. (See Figures lb and lc).
The drop in TF caused an increase in the GFR/TF ratio and thus increased FF. The increase in FF may occur at different times and may be a marker of kidney viability as shown in Figures la-lc.
In Figure lb, FF dramatically increased at approximately 40 hours post-harvest in vitro but in Figure lc, the spike in FF occurred at approximately 15 hours post-harvest in vitro. The FF spike appears to be 3o W O 97/08949 PCT~US96/14360 -1 an indicator of irreversible renal tissue deterioration and a useful marker for maximum cold storage time.
These results indicated that cold storage monitoring can inferentially differentiate kidney 5 arteriolar status during cold storage.
3o . . CA 02230474 1998-02-2~
W O 97/08949 PCTrUS96/14360-1 E~U~MPLE 2 Predictive Utility of Biphasic GFR
Continuous monitoring of the kidney post-harvest i vitro uncovered a biphasic response in 5 filtration during the first ten hours of storage (Figures 2-10). The "GFR m~;mllm~ (see Figures 2-10), occurred at different times within the first ten hours following harvest (i.e., ln vitro time=X axis). The Y
axis ~ maximum~ is a percentage of an actual value determined by choosing, as a reference point, ("GFR
m~;mllm~), the time at which GFR reached a m~;mllm in the first 10 hours of cold storage. Thus, GFR, TF and FF are all standardized to 100~ at the time when the GFR
is at its m~; ml~m . This st~n~rdization allowed for more efficient comparisons of kidney values as shown in Figures 2-10.
Specifically, GFR increased an average of 34.1~ (pc.002) from 2 hours to "GFR m~; ml~m" and dropped an average of 49.2~ (pc.001) from "GFR maximum" to 10 hours. Statistical significance was evaluated using a post-hoc ANOVA comparison of means using repeated measures and the Newman-Kuels test.
Neither the initial increase in filtration nor the decrease that followed required a change in total organ flow (TF). TF at 2 hours, "GFR m~imllm~, and 10 hours, was not significantly different and averaged 4533+250~1/min, 4650+380~1/min, and 4083+460~1/min respectively.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -1 Data from the biphasic GFR response demonstrated marked clinical implications. Knowledge of the biphasic GFR response reflects the length of time a donor kidney can be cold stored without significant 5 damage. It has been reported that cadaveric kidneys transplanted in less than 6 hours, or living non-related donor kidney transplants (LNR), have similar outcomes as living related transplants (LR) (Teraski et al., TransPlants, Los Ar.geles, CA, UCLA Tissue Typing 10 Laboratory, 1991). Since cadaveric kidneys are usually transplanted after several hours of cold storage and have historically poorer graft outcomes than LR
transplants, knowledge that a decrease in filtration following the GFR peak correlates to a loss in post-transplant function provides a useful indicator of graftsuccess .
3o CA 02230474 1998-02-2~
W O 97/08949 PCT~US96~14360 -Kidney Harvest, Perfu~ion Storaqe and Transplant A starch based perfusate (Belzar MPS~
Perfusate, modified starch concentration 3.75g~), was 5 u~ed in twenty genetically matched Lewis rats (275-300g) (Harland Sprague-Dawley, Indianapolis, IN). Kidneys were initially cold perfused ln vivo at 7~-10~C, through a distal aortic catheter. Cold per~usate was pumped t~lrough the kidneys at physiologic pressures while simultaneously clamping the aortas above the kidneys.
Only left kidneys were harvested immediately following aortic clamping. The renal artery, vein and ureter were catheterized during cold perfusion. The renal vein catheter had to be large (.07"ID, .ll"OD) to accommodate low pressure venous flow. The kidney and all three catheters were stabilized on a small platform in a kidney transplant cassette.
Immediately, following harvest, each kidney was tested to insure proper kidney and catheter patency by cold perfusing the renal artery at 60mmHg and measuring the total kidney flow (TF). A TF of 3.6ml/min - 4.2ml/min was required to insure uniform resistance in all kidneys.
The kidney cassette was then connected to a perfusion apparatus and pump (Baxter Rotary Dialysis Pump, Chicago, Il.) using universal silastic fittings on the renal artery and vein. This system mimicked the in vlvo vasculature of the rat kidney by recirculating cold perfusate at approximately 450mls/min. The system does 3o CA 02230474 1998-02-2~ .
1 not force fluid through the kidney but allowed the actual kidney resistance, at any given moment, to determine flow. The system therefore permitted the arterioles to react to pressure changes naturally.
A catheter coming from the main recirculating line (from the pump) was connected to the kidney module, thus mimicking the way the renal artery comes off the aorta in vivo. Since, kidney flow out of the recirculating line was less (1-lOml/min) than the total flow being recirculated (approximately 450ml/min), changes in kidney flow did not significantly change pressure in the recirculating line. This system allowed kidney flow to increase or decrease as a result of kidney resistance without altering central pressure generated from the main recirculating line.
Total flow and GFR were monitored. An in-line probe (Transonic FM #T106, Ithaca, N.Y.) was used to measure TF. A 27 gauge needle was inserted into the renal artery silastic catheter to monitor perfusion pressure (Stathum Strain Gauge, Gould Windograf #40-8474, Valley View, OH). The ureter catheter (.02"ID) was extended and placed in a beaker under oil inside a Mettler PM100 balance. The Mettler balance permitted accurate measurements of microliter increments of urine.
Instantaneous microliter GFR changes in a cold perfused kidney were monitored (i.e., in weight per unit time) by combining the Mettler balance with a Mettler computer software program (Balancelink, Mettler). The Mettler 3o , , CA 02230474 1998-02-2~
l balance and accompanying software permitted continuous 2 day GFR monitoring of rat kidneys.
The perfusion pump, kidney cassette and recirculating tubing were placed in a 7~-10~~ cold room 5 to maintain tissue temperature during monitoring.
Transplantation was accomplished by matching the male universal fittings on the renal artery and vein to the female fittings on the aorta and vena cava extending from the abdomen of the reclpient ~ewis rat.
lO The transplanted kidney was insulated with Saran WrapTM
and maintained at body temperature 37~C.
Utilizing a st~n~rd in vlvo iohexal measurement of urine flow rate, the transplanted kidneys retained an average of 87~ of their overall function as compared to the untouched kidney in the genetically matched recipient rat when transplanted within two hours post-harvest. The urine concentration of iohexal multiplied by the urine volume divided by the plasma concentration of iohexal (.1 mg/ml) provided a ~ measurement GFR. The transplanted kidney GFR divided by the GFR of the untouched kidney multiplied by 100 provides a measurement of relative kidney function achieved by the transplanted kidney.
3o CA 02230474 1998-02-2~ .
W O 97/08949 PCT~US96/14360 -A human kidney is cold (7~C to 10~C) perfused n vivo at physiological pressure without cessation of heart beat. A starch based perfusate (Belzar MPS~
5 Perfusate, modified colloid concentration .lg~ - 4g%), is employed throughout the perfusion period.
The kidney is harvested as soon as practicable, while continually perfusing the aorta. The renal artery is catheterized, as soon as practicable, lO and the renal artery is attached to a Waters MOX-lOODCM
Perfusion Apparatus, taking care to avoid air embolisms during transfer to the perfusion apparatus. The renal vein is left free so that fluid from the renal vein flows freely into the perfusion apparatus. The ureter is 5 positioned in an inclined trough thereby permitting gravity induced fluid flow therefrom. The trough supporting the ureter is attached to an automatic siphon capable of collecting lOml urine. The kidney is stabilized on an inclined platform in the perfusion apparatus and is not handled again until transplant.
The kidney is initially perfused at a pressure of 45mmHg to ensure proper kidney patency by measuring TF.
Cold perfusate is recirculated through the kidney at approximately 200ml/min. The system does not force fluid through the kidney but allows the actual kidney resistance, at any given moment, to determine flow. The system therefore permits the arterioles to react to pressure changes naturally.
3~
, . CA 02230474 l998-02-2~
W O 97/08949 PCTrUS96/14360-1 A catheter coming from the main recirculating line, (through the pump) is connected to the kidney , thus mimicking the way the renal artery comes off the aorta in vivo. Since, kidney flow out of the 5 recirculating line is less, i.e., about 80ml/min than the total flow being recirculated (about 200ml/min), changes in kidney flow will not significantly change pressure in the recirculating line. This system allows ki~-ley flow to increase or decrease as a result of 10 kidney resistance without altering central pressure generated from the main recirculating line.
Total flow, GFR and perfusion pressure are continuously monitored by conventional systems resident in the perfusion apparatus and determinations respecting kidney functional viability are made. Instantaneous and continuous milliliter GFR changes in a cold perfused kidney are monitored conventionally by measuring how the rate at which the automatic siphon empties. The automatic siphon will permit continuous 2 day GFR
monitoring of urine output.
The perfusion pump, kidney cassette and recirculating tubing are placed in a 7~-10~C cold room to maintain tissue temperature during monitoring.
Transplantation of the monitored kidney will greatly improve graft success rates, climinish hospital stays, eliminate the need for further dialysis and prolong the useful life of the kidney in the recipient.
3o
Claims (22)
1. A method for monitoring and determining the functional viability of a donor kidney prior to a transplantation comprising perfusing said kidney with a perfusion having an impermeable colloid concentration which is correlated with the perfusion pressure in a ratio ranging from 0.00167 g%/mmHg to 0.8 g%/mmHg, and determining the GFR of said kidney during said perfusion as a measure of the functional viability of said kidney.
2. The method of Claim 1 wherein said colloid concentration is correlated with said perfusion pressure in a ratio of about 1g% to about 10mmHg.
3. The method of Claim 1 wherein said perfusion is conducted at a cold storage temperature of about 7°C to about 10°C.
4. The method of Claim 1 wherein said colloid concentration is between about .1g% to about 4g% of said perfusate.
5. The method of Claim 1 wherein said perfusion pressure is between about 5mmHg and about 60mmHg.
6. The method of Claim 1 which further comprises measuring the Total Flow (TF) of said perfusate through said donor kidney.
7. The method of Claim 6 which further comprises calculating the FF as a fraction of GFR
divided by TF.
divided by TF.
8. The method of Claim 1 wherein said GFR is measured in about 5 to about 20 minute intervals.
9, The method of Claim 6 wherein said GFR is measured in about 5 to about 20 minute intervals.
10. The method of Claim 8 wherein an increase in GFR per unit of time is indicative of relative viability.
11. The method of Claim 8 wherein a decrease in GFR per unit of time is indicative of a deteriorating kidney.
12. The method of Claim 9 wherein no change in GFR is indicative of a viable kidney when the TF is constant at the GFR maximum.
13. The method of Claim 9 wherein no change in GFR is indicative of a deteriorated kidney when TF
approaches zero.
approaches zero.
14. The method of Claim 9 wherein an increase in GFR, a decrease in TF and a resulting FF spike is indicative of efferent arteriole constriction.
15. The method of Claim 9 wherein a decrease in TF in the absence of a FF spike is indicative of afferent arteriole construction.
16. A method of Claim 9 wherein a disproportionate decrease in GFR, a decrease in TF and the absence of a FF spike is indicative of both afferent and efferent constriction.
17. The method of Claim 8 wherein a decreasing GFR is indicative of cold ischemia damage.
18. A method for ensuring patency of a kidney tubule comprising perfusion of said kidney tubule with a perfusate having an impermeable colloid concentration which is correlated with the perfusion pressure in a ratio ranging from 0.00167 g%/mmHg to 0.8 g%/mmHg.
19. The method of Claim 18 wherein said colloid concentration is correlated with said perfusion pressure in a ratio of about 1g% to about 10mmHg.
20. The method of Claim 18 wherein said perfusion is conducted at a cold storage temperature of about 7°C to about 10°C.
21. The method of Claim 18 wherein said colloid concentration is between about .1g% to about 4g%
of said perfusate
of said perfusate
22. The method of Claim 18 wherein said perfusion pressure is between about 5mmHg and about 60mmHg.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/526,121 US5712084A (en) | 1995-09-08 | 1995-09-08 | Donor kidney viability test for improved preservation |
| US08/526,121 | 1995-09-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2230474A1 true CA2230474A1 (en) | 1997-03-13 |
Family
ID=24096003
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002230474A Abandoned CA2230474A1 (en) | 1995-09-08 | 1996-09-05 | Donor kidney viability test for improved preservation |
Country Status (5)
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|---|---|
| US (1) | US5712084A (en) |
| EP (1) | EP0876096A2 (en) |
| AU (1) | AU718102B2 (en) |
| CA (1) | CA2230474A1 (en) |
| WO (1) | WO1997008949A2 (en) |
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| US7749693B2 (en) * | 1998-09-29 | 2010-07-06 | Lifeline Scientific, Inc. | Method of determining that an organ is not suitable for transplantation and using it for testing substances |
| US6977140B1 (en) * | 1998-09-29 | 2005-12-20 | Organ Recovery Systems, Inc. | Method for maintaining and/or restoring viability of organs |
| US6673594B1 (en) | 1998-09-29 | 2004-01-06 | Organ Recovery Systems | Apparatus and method for maintaining and/or restoring viability of organs |
| WO2002030452A1 (en) * | 2000-10-13 | 2002-04-18 | Pike Laboratories, Inc. | Organ and biological tissue preservation cold storage solution |
| US7014990B2 (en) * | 2000-10-13 | 2006-03-21 | Ben O'Mar Arrington | Machine perfusion solution for organ and biological tissue preservation |
| US7691622B2 (en) * | 2003-04-04 | 2010-04-06 | Lifeline Scientific, Inc. | Method and apparatus for transferring heat to or from an organ or tissue container |
| WO2004089085A2 (en) * | 2003-04-04 | 2004-10-21 | Organ Recovery Systems, Inc. | Device for separating gas from a liquid path |
| US7504201B2 (en) | 2004-04-05 | 2009-03-17 | Organ Recovery Systems | Method for perfusing an organ and for isolating cells from the organ |
| US11178866B2 (en) | 2011-03-15 | 2021-11-23 | Paragonix Technologies, Inc. | System for hypothermic transport of samples |
| US9867368B2 (en) | 2011-03-15 | 2018-01-16 | Paragonix Technologies, Inc. | System for hypothermic transport of samples |
| CA2830225C (en) | 2011-03-15 | 2020-03-24 | Paragonix Technologies, Inc. | Apparatus for oxygenation and perfusion of tissue for organ preservation |
| US8828710B2 (en) | 2011-03-15 | 2014-09-09 | Paragonix Technologies, Inc. | System for hypothermic transport of samples |
| US9253976B2 (en) | 2011-03-15 | 2016-02-09 | Paragonix Technologies, Inc. | Methods and devices for preserving tissues |
| US9426979B2 (en) | 2011-03-15 | 2016-08-30 | Paragonix Technologies, Inc. | Apparatus for oxygenation and perfusion of tissue for organ preservation |
| US9560846B2 (en) | 2012-08-10 | 2017-02-07 | Paragonix Technologies, Inc. | System for hypothermic transport of biological samples |
| US8785116B2 (en) * | 2012-08-10 | 2014-07-22 | Paragonix Technologies, Inc. | Methods for evaluating the suitability of an organ for transplant |
| JP6595984B2 (en) | 2013-05-14 | 2019-10-23 | メタボロン，インコーポレイテッド | Biomarkers associated with renal function and methods of using the same |
| US20190224275A1 (en) | 2017-05-12 | 2019-07-25 | Aurinia Pharmaceuticals Inc. | Protocol for treatment of lupus nephritis |
| US10286036B2 (en) | 2017-05-12 | 2019-05-14 | Aurinia Pharmaceuticals Inc. | Protocol for treatment of lupus nephritis |
| CA3066625A1 (en) | 2017-06-07 | 2018-12-13 | Paragonix Technologies, Inc. | Apparatus for tissue transport and preservation |
| US11632951B2 (en) | 2020-01-31 | 2023-04-25 | Paragonix Technologies, Inc. | Apparatus for tissue transport and preservation |
| KR20230170652A (en) * | 2021-02-22 | 2023-12-19 | 디나코 아게 | Local-regional perfusion of the kidney |
Family Cites Families (3)
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|---|---|---|---|---|
| SU665916A1 (en) * | 1977-12-22 | 1979-06-05 | Ивановский государственный медицинский институт | Method of determining viability of a kidney |
| US4798824A (en) * | 1985-10-03 | 1989-01-17 | Wisconsin Alumni Research Foundation | Perfusate for the preservation of organs |
| SU1496740A1 (en) * | 1987-07-13 | 1989-07-30 | Донецкий государственный медицинский институт им.М.Горького | Method of determining suitability of transplant for grafting |
-
1995
- 1995-09-08 US US08/526,121 patent/US5712084A/en not_active Expired - Fee Related
-
1996
- 1996-09-05 AU AU69692/96A patent/AU718102B2/en not_active Ceased
- 1996-09-05 EP EP96930751A patent/EP0876096A2/en not_active Ceased
- 1996-09-05 CA CA002230474A patent/CA2230474A1/en not_active Abandoned
- 1996-09-05 WO PCT/US1996/014360 patent/WO1997008949A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| US5712084A (en) | 1998-01-27 |
| AU6969296A (en) | 1997-03-27 |
| WO1997008949A2 (en) | 1997-03-13 |
| WO1997008949A3 (en) | 1997-04-03 |
| EP0876096A2 (en) | 1998-11-11 |
| AU718102B2 (en) | 2000-04-06 |
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