Paul C. Grimm

Banff '05 Meeting Report: Differential Diagnosis of Chronic Allograft Injury and Elimination of Chronic Allograft Nephropathy (‘CAN’)

The 8th Banff Conference on Allograft Pathology was held in Edmonton, Canada, 15–21 July 2005. Major outcomes included the elimination of the non‐specific term ‘chronic allograft nephropathy’ (CAN) from the Banff classification for kidney allograft pathology, and the recognition of the entity of chronic antibody‐mediated rejection. Participation of B cells in allograft rejection and genomics markers of rejection were also major subjects addressed by the conference.

Neointimal and Tubulointerstitial Infiltration by Recipient Mesenchymal Cells in Chronic Renal-Allograft Rejection

BACKGROUND Tissue remodeling depends on mesenchymal cells (fibroblasts and myofibroblasts) and is a prominent feature of chronic renal-transplant rejection. It is not known whether the mesenchymal cells that participate in remodeling originate locally or from circulating precursor cells. METHODS We obtained biopsy specimens of renal allografts from six male recipients of an allograft from a female donor, four female recipients of an allograft from a male donor, two male recipients of an allograft from a male donor, and two female recipients of an allograft from a female donor. All the allografts were undergoing chronic rejection. All but two specimens were obtained within six months after transplantation. We used immunohistochemical methods to identify mesenchymal cells with smooth-muscle alpha-actin and in situ hybridization to identify mesenchymal cells with Y-chromosome DNA. RESULTS No Y-chromosome bodies were identified in the case of the two renal-allograft specimens in which both the donor and the recipient were female. In the case of the two renal-allograft specimens in which both the donor and the recipient were male, approximately 40 percent of mesenchymal cells contained a Y-chromosome body. In the case of the six specimens in which the donor was female and the recipient was male, a mean (+/-SD) of 34+/-16 percent of mesenchymal cells in the neointima, 38+/-12 percent of such cells in the adventitia, and 30+/-7 percent of such cells in the interstitium contained the Y-chromosomal marker, indicating that they originated from the recipient rather than the donor. In the case of the four renal-allograft specimens in which the donor was male and the recipient was female, the respective values were 24+/-15 percent, 33+/-9 percent, and 23+/-8 percent, indicating a persistent population of donor mesenchymal cells. CONCLUSIONS The presence of mesenchymal cells of host origin in the vascular and interstitial compartments of renal allografts undergoing chronic rejection provides evidence that a circulating mesenchymal precursor cell has the potential to migrate to areas of inflammation.

Computerized Image Analysis of Sirius Red–Stained Renal Allograft Biopsies as a Surrogate Marker to Predict Long-Term Allograft Function

Chronic allograft nephropathy (CAN) is a major problem in posttransplant management. The lack of a reliable and early surrogate marker of CAN has hampered patient care and research. In this study, the Cortical Fractional Interstitial Fibrosis Volume (V(IntFib)), quantitated with computerized image analysis of Sirius Red-stained protocol biopsies, was examined as a potential surrogate for time to graft failure (TTGF) in 68 renal allograft recipients. At 6 mo posttransplant, V(IntFib) was highly correlated with TTGF (r = 0.64, P < 0.001). Both the Banff Chronic Sum and the Acute Sum Scores were also correlated with TTGF, but less strongly (r = 0.28, P < 0.02; r = 0.35, P < 0.003, respectively). As V(IntFib) was not correlated with the Banff Chronic Score, a multivariate model was created that incorporated V(IntFib) and both Acute and Chronic Banff pathology. This model was highly correlated with TTGF (r = 0.7, P < 0.0001). These findings suggest that V(IntFib) determined by computerized image analysis of Sirius Red-stained protocol biopsies at 6 mo posttransplant, with or without incorporation of Banff acute and chronic scoring, may provide an early surrogate for time to graft failure in renal allograft recipients.

Incidence of PTLD in pediatric renal transplant recipients receiving basiliximab, calcineurin inhibitor, sirolimus and steroids

Pediatric renal transplant recipients were enrolled in a multicenter, randomized, double‐blind trial of steroid withdrawal. Subjects received basiliximab, calcineurin inhibitor, sirolimus and steroids. Of 274 subjects enrolled, 19 (6.9%) subjects developed posttransplant lymphoproliferative disorder (PTLD). The relative hazard (RH) for PTLD was 5.3‐fold higher in children aged ≤5 versus those >12 years (p = 0.0017). EBV seronegative subjects had a 4.7‐fold higher RH compared to EBV positive subjects (p = 0.02). Among EBV donor+/recipient– (D+/R–) subjects, the RH increased by 6.1‐fold (p = 0.0001). In a multivariate model, risk factors included recipient age ≤5 years (RH 3.2, 95% CI: 1.1–9.6, p = 0.034) and EBV D+/R– status (RH 7.7, 95% CI: 1.6–35.9, p = 0.010). Of 19 patients with PTLD, 17 are alive with functioning grafts and 2 lost their grafts, 1 of whom subsequently died of recurrent PTLD. This ‘robust’ immunosuppression protocol was associated with low rejection rates but an unacceptably high incidence of PTLD. The combination of basiliximab, calcineurin inhibitor, sirolimus and steroids resulted in over‐immunosuppression in a high‐risk pediatric population and we do not recommend its use. Future studies must include routine viral monitoring to permit early identification of viral activity and a protocol driven reduction of immunosuppression aimed at avoiding complications.

Melamine nephrotoxicity: an emerging epidemic in an era of globalization

Recent outbreaks of nephrolithiasis and acute kidney injury among children in China have been linked to ingestion of milk-based infant formula contaminated with melamine. These cases provide evidence in humans for the nephrotoxicity of melamine, which previously had been described only in animals. The consequences of this outbreak are already severe and will likely continue to worsen. Herein we summarize the global impact of the melamine milk contamination, the reemergence of melamine-tainted animal feed, and potential mechanisms of melamine nephrotoxicity. Large-scale epidemiologic studies are necessary to further characterize this disease and to assess its potential long-term sequelae. This epidemic of environmental kidney disease highlights the morbidity associated with adulterated food products available in todays global marketplace and reminds us of the unique vulnerability of the kidney to environmental insults. Melamine is the latest in a growing list of diverse potentially toxic compounds about which nephrologists and other health-care providers responsible for the diagnosis and management of kidney disease must now be aware.

Morphometric and Visual Evaluation of Fibrosis in Renal Biopsies

Interstitial fibrosis is an outcome measure of increasing importance in clinical trials of both renal transplantation and native disease, but data on the comparative advantages of fibrosis measurement methods are limited. We compared four morphometric techniques and contrasted these with two visual fibrosis-scoring methods on trichrome-stained slides. Two morphometric methods included whole-slide digital images: collagen III immunohistochemistry and a new technique using trichrome and periodic acid-Schiff subtraction morphometry; the other two methods included Sirius Red with and without polarization on multiple digital fields. We evaluated 10 serial sections from 15 renal biopsies with a range of fibrosis extent and diagnoses on duplicate sections with each method on separate days. Three pathologists performed visual scoring on whole-slide images. Visual and morphometric techniques had good to excellent interassay reproducibility (R(2) = 0.62 to 0.96) and interobserver reproducibility (R(2) = 0.75 to 0.99, all P < 0.001). Morphometry showed less variation between observers than visual assessment (mean of 1% to 5% versus 11% to 13%). Collagen III, Sirius Red unpolarized, and visual scores had the strongest correlations (R(2) = 0.78 to 0.89), the greatest dynamic range, and the best correlation with estimated GFR (R(2) = 0.38 to 0.50, P < 0.01 to 0.001). Considering efficiency, reproducibility, and functional correlation, two current techniques stand out as potentially the best for clinical trials: collagen III morphometry and visual assessment of trichrome-stained slides.

Steroid-Free Immunosuppression Since 1999: 129 Pediatric Renal Transplants with Sustained Graft and Patient Benefits

Despite early promising patient and graft outcomes with steroid‐free (SF) immunosuppression in pediatric kidney transplant recipients, data on long‐term safety and efficacy results are lacking. We present our single‐center experience with 129 consecutive pediatric kidney transplant recipients on SF immunosuppression, with a mean follow‐up of 5 years. Outcomes are compared against a matched cohort of 57 concurrent recipients treated with steroid‐based (SB) immunosuppression. In the SF group, 87% of kidney recipients with functioning grafts remain corticosteroid‐free. Actual intent‐to‐treat SF (ITT‐SF) and still‐on‐protocol SF patient survivals are 96% and 96%, respectively, actual graft survivals for both groups are 93% and 96%, respectively and actual death‐censored graft survivals for both groups are 97% and 99%, respectively. Unprecedented catch‐up growth is observed in SF recipients below 12 years of age. Continued low rates of acute rejection, posttransplant diabetes mellitus (PTDM), hypertension and hyperlipidemia are seen in SF patients, with sustained benefits for graft function. In conclusion, extended enrollment and longer experience with SF immunosuppression for renal transplantation in low‐risk children confirms protocol safety, continued benefits for growth and graft function, low acute rejection rates and reduced cardiovascular morbidity.

Computer-assisted quantification of fibrosis in chronic allograft nephropaty by picosirius red-staining: A new tool for predicting long-term graft function

Background. Chronic allograft nephropathy (CAN) has become the predominant limiting factor for long-term transplant survival. A cardinal histomorphologic correlate for CAN is interstitial fibrosis. Currently, no method has been established in routine use that reliably quantifies the extent of interstitial fibrosis in renal grafts. We have used staining with picrosirius red followed by computerized image analysis to study the correlation between graft fibrosis and future development of glomerular filtration rate (GFR) in a group of children with advanced CAN. Methods. Renal biopsies were performed in 56 children (mean age, 13.7±3.6 years) after a mean period of 4.6±3.1 years after transplantation because of significant increases in serum creatinine. All biopsy specimens were stained with picrosirius red. The magnitude of fibrotic tissue was calculated by computerized image analysis. Linear regression analysis was performed correlating the intensity of graft fibrosis and the changes in the GFR at the time points of renal biopsy and 2 years later. Results. There was a significant positive correlation (r =0.62, P <0.001) between the picrosirius red-stained cortical fractional interstitial fibrosis volume (VintFib) and the decrease of GFR within 2 years postrenal biopsy. When VintFib was below 5%, 82% of the patients had an increase in GFR within 2 years. Ninety-three percent of the patients with greater than 10% of fibrosis experienced a worsening renal function after 2 years. When comparing patients with stable GFR with patients having a decrease in GFR, a highly significant difference in VintFib was found (P =0.008). Conclusions. The quantitative measurement of fibrosis by picrosirius red staining appears to be a useful prognostic indicator for estimating long-term graft function in CAN and may provide an easy, fast, and inexpensive tool helpful for treatment decisions in patients developing CAN.

A Randomized Controlled Crossover Trial with Delayed-Release Cysteamine Bitartrate in Nephropathic Cystinosis: Effectiveness on White Blood Cell Cystine Levels and Comparison of Safety

BACKGROUND AND OBJECTIVES Immediate-release cysteamine bitartrate (Cystagon; Mylan Pharmaceuticals, Canonsburg, PA) may prevent or delay kidney transplantation and other serious outcomes in patients with cystinosis, but has never been subjected to a prospective clinical trial. Cystagon efficacy requires strict lifelong dosing every 6 hours. Such a dosing schedule and Cystagon-associated side effects are often cited by patients as reasons for nonadherence. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This open-label, randomized, controlled, crossover trial was powered to show that a new delayed-release formulation of cysteamine bitartrate, RP103, taken every 12 hours, was noninferior to Cystagon for maintenance of white blood cell (WBC) cystine at levels associated with optimal outcomes in the disease. RESULTS Forty-three patients were randomized. Using a mixed-effects statistical analysis model, the least-squares mean peak value of WBC cystine level was 0.62±0.05 nmol 1/2 cystine/mg protein after 12 hours under RP103 and 0.54±0.05 nmol 1/2 cystine/mg protein after 6 hours under Cystagon, a difference of 0.08±0.04 nmol 1/2 cystine/mg protein (95.8% confidence interval, 0-0.16). The average steady-state total daily dose of RP103 was 82% of the incoming steady-state total daily dose of Cystagon. There were three-fold more gastrointestinal side effects compared with using Cystagon. CONCLUSIONS A new delayed-release Q12H formulation of cysteamine bitartrate is not inferior to the Q6H formulation (Cystagon) in maintaining low WBC cystine levels in patients with cystinosis but at a lower total daily dose.

Screening for NPHS2 Mutations May Help Predict FSGS Recurrence after Transplantation

Steroid-resistant focal segmental glomerulosclerosis (FSGS) often recurs after renal transplantation. In this international survey, we sought to identify genotype-phenotype correlations of recurrent FSGS. We surveyed 83 patients with childhood-onset primary FSGS who received at least one renal allograft and analyzed 53 of these patients for NPHS2 mutations. The mean age at diagnosis was 6.7 years, and the mean age at first renal transplantation was 13 years. FSGS recurred in 30 patients (36%) after a median of 13 days (range, 1.5 to 152 days). Twenty-three patients received a second kidney transplant, and FSGS recurred in 11 (48%) after a median of 16 days (range, 2.7 to 66 days). None of the 11 patients with homozygous or compound heterozygous NPHS2 mutations developed recurrent FSGS compared with 45% of patients without mutations. These data suggest that genetic testing for pathogenic mutations may be important for prognosis and treatment of FSGS both before and after transplantation.