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Use of two biomarkers of renal ischemia to assess machine-perfused non-heart-beating donor kidneys.

Renal transplantation is a recognized treatment for end-stage renal failure, offering a better quality of life, independence from dialysis, better survival rates, and decreased cardiovascular complications compared with renal replacement therapy (1). Recently, continued increases in the prevalence of end-stage renal failure and the length of transplantation waiting lists have not been matched by an increased supply of donors from the traditional brainstem dead donors and living donors. Non-heart-beating donor (NHBD) kidneys are regaining importance to substantially increase the kidney donor pool, with estimates indicating a potential increase of 20-40% (2, 3).

The use of NHBD kidneys is associated with the development of two adverse conditions, primary nonfunction (PNF) and delayed graft function (DGF), which are caused by the ischemic injury that occurs after cardiorespiratory arrest. Therefore, in centers with NHBD programs, it has become necessary to screen-out damaged kidneys that will never work.

Machine preservation using continuous hypothermic pulsatile perfusion has been adopted in NHBD kidney screening initiatives, and perfusion characteristics (flow, pressure, resistance, temperature, weight gain) with enzyme analysis of kidney effluents are used to assess viability. Since its isolation and identification as ligandin, glutathione S-transferase (GST) has evolved as a suitable biomarker in the pretransplantation assessment of machine-perfused NHBD kidneys. Different isoforms have been identified from the distal and proximal renal tubules, and the commonest isoform, [alpha]-GST, has previously been used as a biomarker of renal ischemia as assessed by ELISA (4). However, we have previously demonstrated that a spectrophotometric assay for total GST activity (tGST) could reliably be substituted for [alpha]-GST (5).

Original work by the Maastricht NHBD group has established threshold limits for tGST activity in kidney perfusates for the selection of "viable" kidneys for transplantation (6). This criterion has been introduced in Newcastle, England, where 69 NHBD renal transplants have been performed since 1998 with a first-year graft survival rate of 91%.

Fatty acid-binding protein (FABP), isolated in the cytosol, is involved in the transportation of free fatty acids into the mitochondria for [beta]-oxidation, with heart (H)- and liver (L)-FABP isotypes being restricted to the distal and proximal renal tubules, respectively. H-FABP, which is released more readily during renal ischemia, has been used as a biomarker in small animal studies (7).

The aim of the present study was to compare the validity of the HFABP and tGST determined during machine perfusion. The use of controlled and uncontrolled NHBD donors provided a differential ischemia model. Controlled donors represented donors from intensive therapy/high-dependency unit referrals (withdrawal of treatment); these donors had reduced primary warm ischemic times (WITs). Uncontrolled donors represented donors from Accident and Emergency Department referrals who had suffered cardio-respiratory arrest and had generally undergone a period of resuscitation; these donors had longer primary WITs.

From the initial procurement of 59 NHBDs, 69 renal transplants were performed, of which 44.9% (31 of 69) involved uncontrolled donor kidneys and 55.1% (38 of 69) involved controlled donor kidneys. After donor family consent was obtained, the kidneys were removed and machine-perfused for 4-8 h, using the Newcastle Hypothermic Preservation System (8, 9). Decisions for implantation were based on a combination of procurement and preservation characteristics, including tGST concentrations in kidney perfusates (10).

tGST activity in kidney perfusate samples, collected hourly during machine perfusion and stored at -20 [degree]C, was measured with an automated (Roche[R] Cobas Mira) spectrophotometric method, described by Habig and Jakoby (11), based on the conjugation of chlorodinitrobenzene at 25 [degree]C with glutathione to form glutathione Sdinitrobenzene, with the absorbance of the product measured at 340 nm. Calibration was performed with a series of calibrators prepared from Maastricht standard renal GST, diluted in 0.1 mol/L phosphate buffer (pH 5.9). The intra- and interassay CVs were <4% and <7% at tGST activities of 65.3 and 229 U/L.

HFABP in the kidney perfusates was measured with a sandwich ELISA using a calibrator composed of recombinant human HFABP (12). Briefly, the procedure involved coating a microtiter ELISA plate with monoclonal detector antibody 67D3 (gift from HyCult Biotechnology, Uden, The Netherlands) and incubating for 1 h with recombinant calibrator or perfusate before the addition of a second, horseradish peroxidase-labeled detector antibody, 66E2-HRP (HyCult Biotechnology). After the plate was washed, tetramethylbenzidine (Lucron Bioproduct) was added, the reaction was terminated with 2 mol/L sulfuric acid, and the resulting absorbance was measured at 450 nm. All incubations were performed at room temperature. The intra- and interassay CVs were <5% and <11% at HFABP concentrations of 1 and 12 [micro]g/L, respectively. The assays for tGST activity and HFABP were performed in separate laboratories, without previous knowledge of the procurement and perfusion characteristics.

There were 31 (of a total of 70 NHBD kidneys procured) renal transplants using uncontrolled donors and 38 (of a total of 48 NHBD kidneys procured) renal transplants using controlled donors, with lower discard rates perceived with the controlled donors (Table 1). The two groups of donors were matched for donor and recipient factors. The mean ([+ or -] SE) WITs were 24.9 [+ or -] 1.5 min and 20.5 [+ or -] 1.5 min for uncontrolled and controlled donors, respectively (Table 1). The first-year graft survival rates were 83% and 97% in recipients of uncontrolled and controlled NHBD kidneys, whereas the first-year patient survival rates were 82% and 94% in recipients of uncontrolled and controlled NHBD kidneys, respectively.

In terms of graft function, renal transplants of uncontrolled donors produced a PNF rate of 13% and DGF rate of 87%, whereas renal transplants of controlled donors produced an immediate function (IF) rate of 32% and DGF of 68%. The mean tGST activity and HFABP concentration (per 100 g of kidney tissue) at [t.sub.4] (4th hour of machine perfusion) for uncontrolled donors were 143 [+ or -] 18 U/L and 184 [+ or -] 13 [micro]gL, respectively. The mean tGST activity and HFABP concentration (per 100 g of kidney tissue) at t4 for controlled donors were 96 [+ or -] 14 U/L and 80 [+ or -] 10 [micro]g/L, respectively. Using a scatterplot of tGST vs HFABP, we obtained a Pearson correlation coefficient (r) of 0.8 (P <0.001; Fig. 1). Grafts that subsequently failed (n [+ or -] 5) had a mean tGST activity (128 [+ or -] 34 U/L per 100 g of kidney tissue) and HFABP concentration (89 [+ or-] 14 [micro]gL per 100 g of kidney tissue) at t4 similar to those of functioning grafts. tGST activity did not exceed a critical value of 200 U/L per 100 g of kidney tissue.

The differential renal ischemic clinical model was illustrated by the use of controlled and uncontrolled donors. Uncontrolled donors suffered worse ischemic insult with longer primary WITs (P <0.05, Mann-Whitney U-test). Clinically, the use of uncontrolled donors was associated with a higher discard rate (39 of 70 vs 10 of 48; P <0.001, [chi square] -test), poorer graft survival rate (83% vs 97%), higher PNF (12.9% vs 0%; P <0.05, [chi square]-test) and DGF rates (87% vs 68%), and lower IF rates (0% vs 32%, P <0.001, [chi square]-test) compared with use of controlled donors. This was reflected by higher biomarker concentrations detected in the kidney perfusate during machine perfusion of uncontrolled donors (P <0.001, Mann-Whitney U-test). Both biomarkers showed paralleled changes in concentration detected during machine perfusion, as illustrated in the scatterplot, with a Pearson correlation coefficient of 0.8 (P <0.001; Fig. 1).

[FIGURE 1 OMITTED]

Previously, viability assessment of NHBD kidneys has involved the use of many markers, such as lactate dehydrogenase, lactate, urea, pH, electrolyte changes (13), adenine nucleotides (14), macromolecules of cell structure (e.g., [beta].sub.2]-microglobulin and N-acetyl-[beta]-d-glucosaminidase) (15), lysosomal enzymes (16), metabolic enzymes (GST and alanine aminopeptidase) (17), cytosolic proteins (HFABP) (18), and the use of pretransplantation biopsies (19). These have had limited success in assessing mild ischemic injury. The use of proteomics with gel electrophoresis to identify newer markers by isolation and identification of protein fractions appears interesting but not practical (20). GST has remained the most suitable marker when used in conjunction with machine preservation. Unfortunately, the sensitivity of GST is questionable in borderline cases. The routine use of other biomarker assays, such as HFABP in conjunction with tGST activity, could provide complementary information on the potential allograft "viability". This is illustrated by the fact that there was better segregation of donor categories (controlled vs uncontrolled) with HFABP than with tGST activity. This finding therefore warrants continued evaluation.

This work was supported by the Northern Counties Kidney Research Fund and the Freeman Hospital.

References

(1.) Schnuelle P, Lorenz D, Trede M, Van Der Woude F. Impact of renal cadaveric transplantation on survival in end-stage renal failure: evidence for reduced mortality risk compared with hemodialysis during long-term follow-up. J Am Soc Nephrol 1998;9:2135-41.

(2.) Daemen J, Oomen A, Kelders W, Kootstra G. The potential pool of non-heart-beating kidney donors. Clin Transplant 1997;11:149-54.

(3.) Weber M, Dindo D, Demartines N, Ambuhl P, Clavien P. Kidney transplantation from donors without a heartbeat. N Engl J Med 2002;347:248-55.

(4.) Sundberg A, Nilsson R, Appelkvist E, Dallner G. ELISA procedures for the quantitation of glutathione transferases in the urine. Kidney Int 1995;48: 570-5.

(5.) Balupuri S, Buckley P, Mohamed M, Cornell C, Mantle D, Kirby J, et al. Assessment of non-heart-beating donor (NHBD) kidneys for viability on machine perfusion. Clin Chem Lab Med 2000;38:1103-6.

(6.) Daemen J, Oomen A, Janssen M, van de Schoot L, van Kreel B, Heineman E, et al. Glutathione S-transferase as predictor of functional outcome in transplantation of machine-preserved non-heart-beating donor kidneys. Transplantation 1997;63:89-93.

(7.) Lam K, Borkan S, Claffey K, Schwartz J, Chobanian A, Brecher P. Properties and differential regulation of two fatty acid binding proteins in the rat kidney. J Biol Chem 1988;263:15762-8.

(8.) Balupuri S, Strong A, Hoernich N, Snowden C, Mohamed M, Manas D, et al. Machine perfusion for kidneys: how to do it at minimal cost. Transplant Int 2001;14:103-7.

(9.) Gok M, Shenton B, Strong A, Buckley P, Mohamed M, Talbot D. Pump upgrade for machine perfusion at the Freeman Hospital in Newcastle. Transplant Int 2001;14:207.

(10.) Balupuri S, Buckley P, Snowden C, Mustafa M, Sen B, Griffiths P, et al. The trouble with kidneys derived from the non heart-beating donor: a single center 10-year experience. Transplantation 2000;15:842-6.

(11.) Habig W, Jakoby W. Assays for differentiation of glutathione S-transferases. Methods Enzymol 1981;77:398-405.

(12.) Wodzig K, Pelsers M, van der Vusse G, Roos W, Glatz J. One-step enzyme-linked immunosorbent assay (ELISA) for plasma fatty acid-binding protein. Ann Clin Biochem 1997;34:263-8.

(13.) Newman C, Shenton B. Re-evaluation of viability testing of cadaveric kidneys for transplantation. Br J Urol 1981;53:95-8.

(14.) Kahng M, Trifillis A, Hall-Craggs M, Regec A, Trump B. Biochemical and morphological studies on human kidneys preserved for transplantation. Am J Clin Pathol 1983;80:779-85.

(15.) Sezai A, Shiono M, Orime Y, Nakata K, Hata M, Yamada H, et al. Renal circulation and cellular metabolism during left ventricular assisted circulation: comparison study of pulsatile and nonpulsatile assists. Artif Organs 1997;21:830-5.

(16.) Pavlock G, Southard J, Starling J, Belzer F. Lysosomal enzyme release in hypothermically perfused dog kidneys. Cryobiology 1984;21:521-8.

(17.) Matteucci E, Carmellini M, Bertoni C, Boldrini E, Mosca F, Giampietro O. Urinary excretion rates of multiple renal indicators after kidney transplantation: clinical significance for early graft outcome. Renal Fail 1998;20:32530.

(18.) Nayashida N, Chihara S, Tayama E, Akasu K, Kai E, Kawara T, et al. Influence of renal function on serum and urinary heart fatty acid-binding protein levels. J Cardiovasc Surg (Torino) 2001;42:735-40.

(19.) Muruve N, Steinbecker K, Luger A. Are wedge biopsies of cadaveric kidneys obtained at procurement reliable? Transplantation 2000;69:2384-8.

(20.) Obelchuk L, Tsomartova D, Kovalyov L, Khasigov P, Rubachev P, Grachev S, et al. Standardized and extended catalog of major proteins of the human kidney. Biochemistry (Mosc) 1997;62:191-9.

Muhammed A. Gok, [1] * Maurice Pelsers, [2] Jan F.C. Glatz, [2] Brian K. Shenton, [1] Robert Peaston, [3] Chris Cornell, [3] and David Talbot [1]

([1] Department of Surgery, The Medical School, University of Newcastle, Newcastle Upon Tyne NE7 7DN, England; [2] Department of Physiology, University of Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands; [3] Department of Surgery, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4HH, England;

* address correspondence to this author at: Renal/ Liver Transplant Unit, The Freeman Hospital, Newcastle Upon Tyne NE7 7DN, England; e-mail M.A.Gok@ncl.ac.uk)
Table 1. Descriptive demographics and results for controlled and
uncontrolled donors.

 Uncontrolled Controlled
 (n = 31) (n = 38)

NHBD kidneys procured
 Transplanted 31 38
 Discarded 39 10
Age, (b) years
 Donors 50.3 [+ or -] 1.6 43.1 [+ or -] 2.8
 Recipients 49.1 [+ or -] 2.2 52.1 [+ or -] 2.4
Sex, M/F
 Donor kidneys 21/10 25/13
 Recipients 22/9 22/16
Ischernic times, (b) min
 Primary WIT 24.9 [+ or -] 1.5 20.5 [+ or -] 1.5
 Secondary WIT 38.9 [+ or -] 2.0 36.2 [+ or -] 1.2
 Cold ischemic time 1512.5 [+ or -] 51.7 1467.8 [+ or -] 53.8
First-year survival
rate, %
 Kidneys 83 97
 Patients 82 94
Function, %
 PNF 13 0
 IF 0 32
 DGF 87 68
[t.sub.4] biomarker
concentrations, (b) per
100 g of kidney tissue
 Transplanted kidneys
 tGST activity, U/L 143 [+ or -] 18 96 [+ or -] 14
 HFABP, [micro]g/L 184 [+ or -] 13 80 [+ or -] 10
 Discarded kidneys
 tGST activity, U/L 176 [+ or -] 24 84 [+ or -] 22
 HFABP, [micro]g/L 209 [+ or -] 25 83 [+ or -] 16

 Mann-Whitney
 U-test, P

NHBD kidneys procured
 Transplanted <0.001 (a)
 Discarded
Age, (b) years
 Donors 0.07 (a)
 Recipients 0.4 (a)
Sex, M/F
 Donor kidneys 0.9 (a)
 Recipients 0.4 (a)
Ischernic times, (b) min
 Primary WIT <0.05 (a)
 Secondary WIT 0.4 (a)
 Cold ischemic time 0.4 (a)
First-year survival
rate, %
 Kidneys <0.051
 Patients 0.2 (c)
Function, %
 PNF <0.05 (a)
 IF <0.001 (a)
 DGF 0.07 (a)
[t.sub.4] biomarker
concentrations, (b) per
100 g of kidney tissue
 Transplanted kidneys
 tGST activity, U/L <0.001 (a)
 HFABP, [micro]g/L <0.001 (a)
 Discarded kidneys
 tGST activity, U/L <0.05 (a)
 HFABP, wg/L <0.05 (a)

(a) [chi square] test.

(b) Mean [+ or -] SE.

(c) Log-rank P value.
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Title Annotation:Technical Briefs
Author:Gok, Muhammed A.; Pelsers, Maurice; Glatz, Jan F.C.; Shenton, Brian K.; Peaston, Robert; Cornell, Ch
Publication:Clinical Chemistry
Date:Jan 1, 2003
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