George W. Burke

Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a cause of proteinuric kidney disease, compromising both native and transplanted kidneys. Treatment is limited because of a complex pathogenesis, including unknown serum factors. Here we report that serum soluble urokinase receptor (suPAR) is elevated in two-thirds of subjects with primary FSGS, but not in people with other glomerular diseases. We further find that a higher concentration of suPAR before transplantation underlies an increased risk for recurrence of FSGS after transplantation. Using three mouse models, we explore the effects of suPAR on kidney function and morphology. We show that circulating suPAR activates podocyte β3 integrin in both native and grafted kidneys, causing foot process effacement, proteinuria and FSGS-like glomerulopathy. Our findings suggest that the renal disease only develops when suPAR sufficiently activates podocyte β3 integrin. Thus, the disease can be abrogated by lowering serum suPAR concentrations through plasmapheresis, or by interfering with the suPAR–β3 integrin interaction through antibodies and small molecules targeting either uPAR or β3 integrin. Our study identifies serum suPAR as a circulating factor that may cause FSGS.

Rituximab Targets Podocytes in Recurrent Focal Segmental Glomerulosclerosis

Rituximab treatment in high-risk patients with focal segmental glomerulosclerosis directly affects podocyte function and is linked to reduced incidence of recurrent proteinuria after kidney transplantation. Rituximab Prods Podocytes to Action Rituximab is a monoclonal antibody against CD20, a protein located on the surface of B cells. It is typically used to treat certain cancers and autoimmune disorders, but has also treated kidney conditions, including focal segmental glomerulosclerosis (FSGS)—a disorder that can affect both pediatric and adult patients. Recurrent FSGS is a problem for 30 to 40% of patients who have undergone kidney transplantation, and can be characterized by progression to end-stage renal disease and recurrence of proteinuria after transplant. Despite the ability of rituximab to treat FSGS, it has been unclear exactly how this drug achieves success in some patients, but not others. Fornoni and colleagues hypothesized that rituximab operates in a B cell–independent manner, targeting instead specific kidney cells called podocytes. To test this hypothesis, Fornoni et al. studied 41 patients at high risk for FSGS: 14 historical control patients who were not treated with rituximab and 27 patients who received rituximab at the time of kidney transplant. They found fewer podocytes with sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) protein in biopsies from patients who later developed recurrent FSGS. The authors had also collected serum from all patients before transplant and then later treated normal human podocytes in culture with the sera. Serum from patients who would later develop recurrent FSGS caused a decrease in both SMPDL-3b protein and acid sphingomyelinase activity. This phenomenon was prevented by rituximab. The FSGS serum from patients also disrupted the actin cytoskeleton of cultured podocytes, but pretreatment with rituximab, or even overexpression of SMPDL-3b protein, partially prevented this phenotype. Together, these data suggest that modulation of sphingolipid-related proteins plays a role in the pathogenesis of recurrent FSGS and, moreover, that these proteins and enzymes might be targets of rituximab treatment. With the mechanism solved, rituximab may represent a new therapeutic strategy to prevent recurrent proteinuria after kidney transplantation. Here’s to happy and healthy kidneys! Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized by proteinuria, progression to end-stage renal disease, and recurrence of proteinuria after kidney transplantation in about one-third of patients. It has been suggested that rituximab might treat recurrent FSGS through an unknown mechanism. Rituximab not only recognizes CD20 on B lymphocytes, but might also bind sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) protein and regulate acid sphingomyelinase (ASMase) activity. We hypothesized that rituximab prevents recurrent FSGS and preserves podocyte SMPDL-3b expression. We studied 41 patients at high risk for recurrent FSGS, 27 of whom were treated with rituximab at time of kidney transplant. SMPDL-3b protein, ASMase activity, and cytoskeleton remodeling were studied in cultured normal human podocytes that had been exposed to patient sera with or without rituximab. Rituximab treatment was associated with lower incidence of posttransplant proteinuria and stabilization of glomerular filtration rate. The number of SMPDL-3b+ podocytes in postreperfusion biopsies was reduced in patients who developed recurrent FSGS. Rituximab partially prevented SMPDL-3b and ASMase down-regulation that was observed in podocytes treated with the sera of patients with recurrent FSGS. Overexpression of SMPDL-3b or treatment with rituximab was able to prevent disruption of the actin cytoskeleton and podocyte apoptosis induced by patient sera. This effect was diminished in cultured podocytes where SMPDL-3b was silenced. Our study suggests that treatment of high-risk patients with rituximab at time of kidney transplant might prevent recurrent FSGS by modulating podocyte function in an SMPDL-3b–dependent manner.

Unexpected augmentation of mycophenolic acid pharmacokinetics in renal transplant patients receiving tacrolimus and mycophenolate mofetil in combination therapy, and analogous in vitro findings

Mycophenolate mofetil (MMF) a potent immunosuppressive agent, has recently been approved for clinical use (CellCept) in renal transplant patients in combination with cyclosporine (CsA). With the expanded use of tacrolimus (Prograf) as well in renal transplant patients, there is a lack of pharmacokinetic studies clarifying drug interactions between the three agents. A pharmacokinetic study was performed on 18 stable renal transplant patients receiving MMF and tacrolimus together, and four control groups, one receiving tacrolimus alone, two receiving CsA, in combination with MMF (1.0 or 1.5 g bid), and one receiving CsA microemulsion (Neoral). Area-under-the-curve values were calculated for each drug to assess if there was a reciprocal effect on the respective bioavailability of each. In vitro, the immunosuppressive effect of trough level plasma from each patient group was studied using mixed lymphocyte culture (MLC), as well as MLC reactions spiked with various combinations of each drug. There was a minimal effect of MMF on tacrolimus pharmacokinetics. However, patients receiving tacrolimus and MMF displayed significantly higher levels (Cmin and area under the curve) of mycophenolic acid (MPA) than those receiving CsA (Sandimmune or Neoral) and the same dose of MMF (50.2 +/- 16.5 vs 32.1 +/- 16.7 micrograms h/ml AUC, p < 0.02). Equivalent MPA levels could be attained in patients receiving CsA if the MMF dose was increased by 50% (1.5 g bid). There were also significantly lower levels of the glucuronide metabolite of MPA (MPAG) (755 +/- 280 vs 1230 +/- 250 micrograms h/ml AUC, p = 0.02), suggesting a specific inhibition (either direct or indirect) of the conversion of MPA to MPAG in tacrolimus patients, as opposed to those receiving CsA. For each drug combination, there was a positive correlation between the plasma immunosuppressive effect seen in MLC assays and the MMF dose. In addition, trough plasma from patients receiving tacrolimus and MMF was significantly more MLC inhibitory than from those receiving CsA or CsA microemulsion and equivalent-dose MMF. Culture media containing MPA and tacrolimus equal to clinical therapeutic trough concentrations (10 ng/ml) were significantly more MLC inhibitory than CsA at equivalent clinical therapeutic trough concentrations (200 ng/ml) with equivalent MPA levels. These studies in renal transplant patients suggest that tacrolimus in combination with MMF may result in a greater degree of immunosuppression than may be anticipated.

Abatacept in B7-1–Positive Proteinuric Kidney Disease

Abatacept (cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein [CTLA-4-Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize β1-integrin activation in podocytes and reduce proteinuria in patients with B7-1-positive glomerular disease.

A randomized trial of three renal transplant induction antibodies: early comparison of tacrolimus, mycophenolate mofetil, and steroid dosing, and newer immune-monitoring.

Background. New trends in immunosuppression in clinical transplantation include the use of antibody induction agents in protocols that emphasize reduction or avoidance of steroids and calcineurin inhibitors. Methods. In a randomized trial using three different antibody induction agents in 90 first renal transplant recipients from cadaver donors, group A received Thymoglobulin, group B received Alemtuzumab, and group C received Daclizumab. Maintenance immunosuppression included tacrolimus and mycophenolate in all three arms, and methylprednisolone in groups A and C only (standard clinical institutional practice). The targeted trough level of tacrolimus was between 8 and 10 ng/mL for groups A and C, respectively, with a targeted mycophenolate dose of 1 g twice daily. However, in group B, the target tacrolimus trough level was 4 to 7 ng/mL to reduce long-term nephrotoxicity, with 500 mg twice-daily doses of mycophenolate, without steroid maintenance. Results. In this 15-month median postoperative interval report, there were no notable differences in demographics and patient and graft survivals. Acute rejection rates at 1 year were equivalent, that is, 5 of 30 in all three groups (16.6%). In group B, there was slightly lower renal function at 1 month, but no difference at 1 year. There was also significantly more leukopenia, but a greater percentage of T-regulatory cells and number of Fox-P3 mRNA copies by flow cytometry and semiquantitative polymerase chain reaction analysis, respectively, in group B. Conclusions. This preliminary analysis indicates that 80% of the patients in group B remained steroid-free 1 year postoperatively, with lower tacrolimus trough levels and no difference in other adverse events.

Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T-cells

OBJECTIVE To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients. RESEARCH DESIGN AND METHODS We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays. RESULTS Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells. CONCLUSIONS We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

Dissociation of depletional induction and posttransplant lymphoproliferative disease in kidney recipients treated with alemtuzumab.

Transplant patients are at the risk for posttransplant lymphoproliferative disease (PTLD), a virally‐driven malignancy. Induction with the depleting antibody preparations Thymoglobulin and OKT3 is associated with PTLD suggesting that the T‐cell depletion increases PTLD risk. We therefore studied 59 560 kidney recipients from the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) database for a relationship between induction agent use and PTLD. Two agents with comparable T‐cell depletional effects, alemtuzumab and Thymoglobulin, were compared to nondepletional induction agents or no induction. The overall incidence of PTLD was 0.46% and differed significantly by induction strategy (p < 0.01): without induction (0.43%), basiliximab (0.38%), daclizumab (0.33%), Thymoglobulin (0.67%) and alemtuzumab (0.37%). Thymoglobulin was associated with significantly increased PTLD risk (p = 0.0025), but alemtuzumab (p = 0.74), basiliximab (p = 0.33) and daclizumab, which trended toward a protective effect (p = 0.06), were not. Alemtuzumab and Thymoglobulin treated patients did not differ in any established parameter affecting PTLD risk although alemtuzumab is known to have a more pronounced B‐cell depleting effect. Interestingly, maintenance therapy with an mTOR inhibitor was strongly associated with PTLD (0.71%, p < 0.0001). Thus, depletional induction is not an independent risk factor for PTLD. Rather, maintenance drug selection or perhaps the balance between B‐ and T‐cell depletion may be more relevant determinants of PTLD risk.

Posttransplant hyperglycemia. Increased incidence in cyclosporine-treated renal allograft recipients

The incidence of posttransplant diabetes mellitus (PTDM) was compared in two groups of renal allograft recipients. These were all nondiabetic patients who had been transplanted between 1979 and 1987 and received either azathioprine-methylprednisolone (group 1) or cyclosporine-methylprednisolone (group 2) therapy as maintenance immunosuppression. The incidence of PTDM in group 1 was 9.1% vs. 18.6% in group 2 (P less than .05). The mean daily dose of methylprednisolone during the initial 2 months posttransplant was not greater among the PTDM patients of groups 1 or 2. Cyclosporine levels and mean daily CsA doses during the initial 2 posttransplant months were also not different among the CsA-PTDM and euglycemic CsA patients. Posttransplant diabetes mellitus occurred rapidly (less than 2 months) and required insulin therapy in the majority of cases. Increased age (greater than 40 years) was associated with a higher risk for PTDM, however, the greater incidence accompanying increased body weight only approached significance. Patient gender and donor source were not associated with significant risk for PTDM. The development of PTDM was accompanied by a significant decrease in graft survival at 3 years in the entire PTDM population and at 4 years in the CsA-PTDM subgroup. Actuarial patient survival was not adversely affected. The current study suggests that CsA may be diabetogenic when administered with methylprednisolone to renal allograft recipients. The adverse effect on allograft survival requires further investigation. These results may also have important implications for pancreatic and islet cell transplantation.

A randomized long-term trial of tacrolimus/sirolimus versus tacrolimus/mycophenolate mofetil versus cyclosporine (neoral)/sirolimus in renal transplantation. II. Survival, function, and protocol compliance at 1 year

Background. In an attempt to reduce chronic calcineurin inhibitor induced allograft nephropathy in first cadaver and human leukocyte antigen non-identical living-donor renal transplantation, sirolimus (Siro) or mycophenolate mofetil (MMF) was tested as adjunctive therapy, with planned dose reductions of tacrolimus (Tacro) over the first year postoperatively. Adjunctive Siro therapy with a similar dose reduction algorithm for Neoral (Neo) was included for comparison. Methods. The detailed dose reduction plan (Tacro and Siro, group A; Tacro and MMF, group B; Neo and Siro, group C) is described in our companion report in this issue of Transplantation. The present report documents function, patient and graft survival, protocol compliance, and adverse events. Results. As mentioned (in companion report), group demographics were similar. The present study shows no significant differences in 1-year patient and graft survival but does show a trend that points to more difficulties in group C by way of a rising slope of serum creatinine concentration (P =0.02) and decreasing creatinine clearance (P =0.04). There were more patients who discontinued the protocol plan in group C. Thus far, no posttransplant lymphomas have appeared, and infectious complications have not differed among the groups. However, a greater percentage of patients in group C were placed on antihyperlipidemia therapy, with an (unexpected) trend toward a higher incidence of posttransplant diabetes mellitus in this group. Group A required fewer, and group B the fewest, antihyperlipidemia therapeutic interventions (P <0.00001). Conclusions. This 1-year interim analysis of a long-term, prospective, randomized renal-transplant study indicates that decreasing maintenance dosage of Tacro with adjunctive Siro or MMF appears to point to improved long-term function, with reasonably few adverse events.

De novo membranoproliferative glomerulonephritis in hepatitis C virus-infected renal allograft recipients.

Hepatitis C virus (HCV) is the leading cause of non-A, non-B hepatitis among renal allograft recipients. We sought to identify and describe a proteinuric renal disease occurring in our HCV-infected renal transplant patients. Patients with proteinuria exceeding 1 g/day were identified from a cohort of 98 HCV-infected kidney recipients. Qualitative and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and restriction fragment-length polymorphism of the amplified RT-PCR product was performed to detect circulating HCV RNA, viral titer, and strain type, respectively. An immune complex nephritis (ICN) of the membranoproliferative pattern (MPGN) was found on five of eight biopsies. Two patients infected with the Hutch strain-type developed nephrotic-range proteinuria within three months posttransplant while the remaining three MPGN patients had been transplanted greater than 5 years prior to the onset of proteinuria. Testing for rheumatoid factors, cryoglobulins, hypocomplementemia, and circulating immune complexes failed to show a consistent pattern. Sucrose density gradient (SDG) equilibrium centrifugation was used to determine the buoyant-density of HCV virions from control (HCV-infected nonproteinuric recipients; n = 5) and nephrotic patients (n = 5). Whereas HCV virions from the control patients had a low buoyant density on sucrose gradients, a substantial percentage of the circulating HCV RNA from the MPGN patients was present in the high-density fractions in association with IgM and IgG. Treatment of the pooled high-density layers with NP40 followed by recentrifugation resulted in a shift of the HCV RNA to the medium-density layers. In conclusion, MPGN developed in five HCV-infected kidney recipients despite pharmacologic immunosuppression. Both the physicochemical properties of the HCV virions on SDG and their association with IgG and IgM in the high-density layers provide indirect evidence for the presence of circulating complexes of anti-HCV antibody and HCV antigen(s).