Sandip Kapur

The influence of pulsatile preservation on renal transplantation in the 1990s.

BACKGROUND Unlike simple cold storage (CS), pulsatile machine preservation (MP) of kidneys for transplantation permits pharmacologic manipulation of the perfusate and aids in the pretransplant assessment of the kidney graft. These characteristics of MP may have importance in the era of increasing use of extended criteria donor kidneys. The overall aim of this article is to critically assess practices at our preservation unit with respect to graft function. Specific aims are to (1) compare the influence of MP versus CS on graft function, (2) determine which pretransplant variables have significance in pretransplant assessment, and (3) determine whether pharmacologic manipulation during MP is advantageous. METHODS There were 650 consecutive kidneys preserved in our laboratory between January 1, 1993 and March 1, 999, by either MP or CS. All MP kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer-MPS or Belzer II solution. Perfusion parameters and electrolytes were measured serially during pulsatile perfusion. All CS kidneys were stored in University of Wisconsin solution. All kidneys obtained from donors exhibiting extended criteria features underwent pretransplant frozen section biopsies. Transmission electron microscopy (EM) was performed on a subset of kidneys undergoing pharmacologic manipulation. Four agents were assessed prospectively for their ability to influence MP characteristics when added to perfusate: PGE1, trifluoperazine, verapamil, and papaverine. RESULTS MP was associated with improved immediate, 1-, and 2-year graft function and reduced length of initial hospital stay when compared with CS grafts. Changes in the machine perfusion variables flow and resistance, and the [Ca++] in perfusate, were significantly associated with delayed graft function (DGF) after the transplant. Biopsy information was not predictive of DGF. The addition of PGE1 to perfusate improved MP characteristics, reduced the release of [Ca++] into perfusate, and ameliorated mitochondrial ischemic injury in transmission EM images. Early graft function was improved in the presence of PGE1+MP, compared with function in the presence of other pharmacologic agents or CS alone. CONCLUSIONS MP is associated with improved early and long term renal function. Moreover, PGE1 augments MP in improving graft function. The combination of MP+PGE1 may be important in optimizing the ability to use extended donor criteria kidneys and, thereby, improve the overall efficiency of cadaveric renal transplantation.

In vivo hyperexpression of transforming growth factor-beta1 in mice: stimulation by cyclosporine.

BACKGROUND We have demonstrated that cyclosporine (CsA) stimulates transforming growth factor (TGF) beta1 expression in vitro and that growth of mammalian cells can be arrested by CsA via a TGF-beta1-dependent mechanism. Herein, we have explored whether CsA stimulates TGF-beta1 hyperexpression in vivo. METHODS Four groups of B6AF1 mice were studied: group 1, control; group 2, CsA pretreatment; group 3, anti-CD3 monoclonal antibody pretreatment; and group 4, CsA plus anti-CD3 pretreatment. RESULTS CsA pretreatment augmented TGF-beta1 protein expression and increased intrarenal display of TGF-beta1 mRNA. This heightened TGF-beta1 expression was associated with an impaired T cell proliferative response. CONCLUSIONS Our observations, together, advance the hypothesis that CsA might function in vivo as an immunosuppressant not only by inhibiting the expression of proinflammatory cytokines (e.g., interleukin 2), but also by stimulating the expression of TGF-beta1, a potent immunosuppressive cytokine. Moreover, prevention of TGF-beta1 hyperexpression might prevent CsA-associated renal fibrosis, as TGF-beta1 is a fibrogenic cytokine.

CD103 mRNA levels in urinary cells predict acute rejection of renal allografts1

Background. CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. Methods. We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. Results. CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). Conclusion. CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.

Survival Benefit with Kidney Transplants from HLA-Incompatible Live Donors

BACKGROUND A report from a high-volume single center indicated a survival benefit of receiving a kidney transplant from an HLA-incompatible live donor as compared with remaining on the waiting list, whether or not a kidney from a deceased donor was received. The generalizability of that finding is unclear. METHODS In a 22-center study, we estimated the survival benefit for 1025 recipients of kidney transplants from HLA-incompatible live donors who were matched with controls who remained on the waiting list or received a transplant from a deceased donor (waiting-list-or-transplant control group) and controls who remained on the waiting list but did not receive a transplant (waiting-list-only control group). We analyzed the data with and without patients from the highest-volume center in the study. RESULTS Recipients of kidney transplants from incompatible live donors had a higher survival rate than either control group at 1 year (95.0%, vs. 94.0% for the waiting-list-or-transplant control group and 89.6% for the waiting-list-only control group), 3 years (91.7% vs. 83.6% and 72.7%, respectively), 5 years (86.0% vs. 74.4% and 59.2%), and 8 years (76.5% vs. 62.9% and 43.9%) (P<0.001 for all comparisons with the two control groups). The survival benefit was significant at 8 years across all levels of donor-specific antibody: 89.2% for recipients of kidney transplants from incompatible live donors who had a positive Luminex assay for anti-HLA antibody but a negative flow-cytometric cross-match versus 65.0% for the waiting-list-or-transplant control group and 47.1% for the waiting-list-only control group; 76.3% for recipients with a positive flow-cytometric cross-match but a negative cytotoxic cross-match versus 63.3% and 43.0% in the two control groups, respectively; and 71.0% for recipients with a positive cytotoxic cross-match versus 61.5% and 43.7%, respectively. The findings did not change when patients from the highest-volume center were excluded. CONCLUSIONS This multicenter study validated single-center evidence that patients who received kidney transplants from HLA-incompatible live donors had a substantial survival benefit as compared with patients who did not undergo transplantation and those who waited for transplants from deceased donors. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.).

Quantifying the Risk of Incompatible Kidney Transplantation: A Multicenter Study

Incompatible live donor kidney transplantation (ILDKT) offers a survival advantage over dialysis to patients with anti‐HLA donor‐specific antibody (DSA). Program‐specific reports (PSRs) fail to account for ILDKT, placing this practice at regulatory risk. We collected DSA data, categorized as positive Luminex, negative flow crossmatch (PLNF) (n = 185), positive flow, negative cytotoxic crossmatch (PFNC) (n = 536) or positive cytotoxic crossmatch (PCC) (n = 304), from 22 centers. We tested associations between DSA, graft loss and mortality after adjusting for PSR model factors, using 9669 compatible patients as a comparison. PLNF patients had similar graft loss; however, PFNC (adjusted hazard ratio [aHR] = 1.64, 95% confidence interval [CI]: 1.15–2.23, p = 0.007) and PCC (aHR = 5.01, 95% CI: 3.71–6.77, p < 0.001) were associated with increased graft loss in the first year. PLNF patients had similar mortality; however, PFNC (aHR = 2.04; 95% CI: 1.28–3.26; p = 0.003) and PCC (aHR = 4.59; 95% CI: 2.98–7.07; p < 0.001) were associated with increased mortality. We simulated Centers for Medicare & Medicaid Services flagging to examine ILDKTs effect on the risk of being flagged. Compared to equal‐quality centers performing no ILDKT, centers performing 5%, 10% or 20% PFNC had a 1.19‐, 1.33‐ and 1.73‐fold higher odds of being flagged. Centers performing 5%, 10% or 20% PCC had a 2.22‐, 4.09‐ and 10.72‐fold higher odds. Failure to account for ILDKTs increased risk places centers providing this life‐saving treatment in jeopardy of regulatory intervention.

SERINE PROTEINASE INHIBITOR-9, AN ENDOGENOUS BLOCKER OF GRANZYME B/PERFORIN LYTIC PATHWAY, IS HYPEREXPRESSED DURING ACUTE REJECTION OF RENAL ALLOGRAFTS

Background. Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1′ is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. Methods. We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. Results. The levels of PI-9 (P =0.001), granzyme B (P <0.0001), and perforin mRNAs (P <0.0001), but not the levels of 18S rRNA (P =0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P =0.01). Furthermore, PI-9 levels predicted subsequent graft function (r =0.43, P =0.01). Conclusions. PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.

Epidemiology of BK virus in renal allograft recipients: independent risk factors for BK virus replication.

Background. Identification of risk factors for BK virus (BKV) replication may improve transplant outcome. We investigated the impact of immunosuppressive drugs on the prevalence of BKV replication in recipients of human renal allografts. Methods. One hundred twenty renal allograft recipients were studied prospectively at 1, 3, and 6 months posttransplantation to identify risk factors for BKV replication. BKV replication was quantified by measurement of urinary cell BKV VP1 mRNA levels using BKV specific primers and TaqMan probe in a real-time quantitative polymerase chain reaction assay. Levels of urinary cell mRNA for granzyme B, CD103, and transforming growth factor-β1 were measured to ascertain whether BKV replication is associated with an inflammatory signature. Results. The prevalence of BKV replication increased over time and was highest at 6 months compared with 1 or 3 months posttransplantation (P<0.001). A logistic regression model analysis demonstrated that steroid maintenance therapy (odds ratio: 8.3, P=0.003) and induction with rabbit anti-human thymocyte globulin (odds ratio: 5.8, P=0.008) were independent risk factors for BKV replication. Neither mycophenolate mofetil dose nor tacrolimus dose or trough levels were different between those with or without BKV replication. The development of acute rejection or antirejection treatment with methylprednisolone did not increase the risk of BKV replication. BKV replication was associated with heightened levels of urinary cell mRNA for granzyme B (P<0.002), CD103 (P<0.005) but not for transforming growth factor-β1 (P>0.05). Conclusions. Steroid maintenance therapy and induction with antithymocyte globulin are independent risk factors for BKV replication in renal allograft recipients treated with tacrolimus and mycophenolate mofetil.

Analysis of failure in living donor liver transplantation: Differential outcomes in children and adults

Over the past decade we have reported excellent outcomes in pediatric living-donor liver transplantation (LDLT) with recipient survival exceeding 90%. Principles established in these patients were extended to LDLT in adults. To compare outcomes in donors and recipients between adult and pediatric LDLT in a single center, we reviewed patient records of 45 LDLT performed between 1/98 and 2/01: 23 adult LDLT (54 ± 6.5 yr) and 22 pediatric LDLT (33.7 ± 53.5 months). Preoperative liver function was worse in adults (International Normalized Ratio [INR] 1.5 ± 0.4 vs. INR 1.2 ± 0.5; p = 0.032). 4 adults (17%) met criteria for status 1 or 2A. Only 1 child was transplanted urgently. Analysis included descriptive statistics and Kaplan-Meier estimation. Donor mortality was 0% with 1 re-exploration, 2.4%. Median hospital stay (LOS) was 6.0 days (range, 4–12 days). Donor morbidity and LOS did not differ by sex, extent of hepatectomy, or adult and pediatric LDLT (p = 0.49). In contrast, recipient outcomes were worse for adults. Adult 1 year graft survival was 65% (3 retransplants [ReTx], 5 deaths) vs. 91% for children (1 ReTx, 1 death) p = 0.02. Graft losses in adults were due to sepsis (n = 3), small for size (n = 2), suicide, and hepatic artery thrombosis (HAT), whereas in children graft losses were due to portal thrombosis and total parenteral nutrition (TPN) liver failure. Biliary leaks occurred in 22% of adults and 9% of children. Hepatic vein obstruction occurred in 17% of adults and in none of the children. Median LOS was comparable (adult, 16.5 days (range, 7–149 days); child, 17 days (range, 10–56 days), p = 0.2). Graft function (total bilirubin (TBili) < 5mg/dl, INR < 1.2, aspartate aminotransferase (AST) < 100 U/l) normalizing by day 4 in children and by day 14 in adults. Adults fared worse, with an array of problems not seen in children, in particular, hepatic vein obstruction and small-for-size syndrome. Biliary leaks were diagnosed later in adults and were lethal in 3 cases; this was later avoided with biliary drainage in adult recipients. Finally, use of LDLT in decompensated adults led to death in 3 of 4 patients, and should be restricted to elective use.

Molecular signatures of urinary cells distinguish acute rejection of renal allografts from urinary tract infection

Acute rejection (AR) and urinary tract infection (UTI) continue to plague renal transplantation. We tested the hypotheses that UTI does not increase granzyme B mRNA levels in urinary cells, and that the levels distinguish AR from UTI. We measured the levels of granzyme B mRNA in 15 urine specimens from renal allograft recipients with UTI, 29 specimens from patients with AR but without UTI, and 14 specimens from patients without AR and without UTI. We also measured transcript levels in urine specimens from 41 nontransplant individuals, 11 with UTI and 30 without UTI. UTI did not increase granzyme B mRNA levels. Granzyme B mRNA levels were lower in renal allograft recipients with UTI compared with those with AR (P<0.0001). We conclude that bacterial UTI is unlikely to confound AR diagnosis made by measurement of granzyme B mRNA levels in urinary cells.

Validation of noninvasive diagnosis of BK virus nephropathy and identification of prognostic biomarkers.

Background. BK virus nephropathy (BKVN) may cause renal allograft dysfunction and failure. The gold standard test is kidney biopsy, which is invasive and costly. A noninvasive, accurate biomarker for diagnosis of BKVN and prognostication of allograft function after BKVN infection may improve allograft survival. Methods. We tested the diagnostic accuracy of our previously reported cutoff value of 6.5×105 BKV viral capsid protein 1 (VP-1) mRNA/ng RNA in urinary cells (Ding et al., Transplantation 2002; 74: 987) using an independent cohort (n=89). We also examined whether urinary cell mRNA profiles obtained at the time of BKVN diagnosis identified patients at risk of subsequent decline in graft function. Results. BKVN was accurately diagnosed (sensitivity of 100% and specificity of 97%) using our previously reported cutoff value. Levels of granzyme B (GB) mRNA (P=0.002) and proteinase inhibitor (PI)-9 mRNA (P=0.01) in urinary cells were higher in BKVN patients with a subsequent decline in renal function (n=8) compared with patients with stable function (n=10), and were positively associated (GB, P=0.01; PI-9, P=0.04) with rise in serum creatinine from the time of BKVN diagnosis to 12 months after diagnosis. GB levels in the BKVN patients with a decline in renal function were similar to those in the acute rejection group (n=11, P>0.05), but higher than the normal biopsy group (n=36, P<0.001); levels in BKVN patients with stable function were lower than those in the acute rejection group (P<0.01) and not significantly different from the normal biopsy group (P>0.05). Conclusions. Noninvasive diagnosis of BKVN and prognostication of renal allograft function after BKVN diagnosis are feasible by measurement of transcripts for BKV viral capsid protein 1 (VP-1), GB, and PI-9 in urine.