Eric J. Steenbergen

Ciliopathies with Skeletal Anomalies and Renal Insufficiency due to Mutations in the IFT-A Gene WDR19

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.

Renal Progenitor Cells Contribute to Hyperplastic Lesions of Podocytopathies and Crescentic Glomerulonephritis

Glomerular injury can involve excessive proliferation of glomerular epithelial cells, resulting in crescent formation and obliteration of Bowmans space. The origin of these hyperplastic epithelial cells in different glomerular disorders is controversial. Renal progenitors localized to the inner surface of Bowmans capsule can regenerate podocytes, but whether dysregulated proliferation of these progenitors contributes to crescent formation is unknown. In this study, we used confocal microscopy, laser capture microdissection, and real-time quantitative reverse transcriptase-PCR to demonstrate that hypercellular lesions of different podocytopathies and crescentic glomerulonephritis consist of three distinct populations: CD133(+)CD24(+)podocalyxin (PDX)(-)nestin(-) renal progenitors, CD133(+)CD24(+)PDX(+)nestin(+) transitional cells, and CD133(-)CD24(-)PDX(+)nestin(+) differentiated podocytes. In addition, TGF-beta induced CD133(+)CD24(+) progenitors to produce extracellular matrix, and these were the only cells to express the proliferation marker Ki67. Taken together, these results suggest that glomerular hyperplastic lesions derive from the proliferation of renal progenitors at different stages of their differentiation toward mature podocytes, providing an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis.

Sirolimus-associated heavy proteinuria in a renal transplant recipient: evidence for a tubular mechanism.

Sirolimus is a new and potent immunosuppressive agent. Recently, increased proteinuria has been recognized as an important complication. However, the mechanism thereof has remained unclear. We describe a patient who received sirolimus as standard therapy after living donor kidney transplantation. Within 10 days the patient developed a severe proteinuria that disappeared completely after substituting tacrolimus for sirolimus. Renal biopsy disclosed normal glomeruli even without effacement of the podocytic foot processes. Using FITC labeled anti‐albumin antibodies we noted complete absence of albumin in the proximal tubules, whereas an abundant albumin staining was observed in a control patient with a comparable level of proteinuria due to a recurrence of focal segmental glomerulosclerosis after transplantation. Our data suggest that sirolimus can induce severe proteinuria, and that reduced tubular protein reabsorption contributes to the protein loss.

Renal transplantation in patients with hemolytic uremic syndrome: high rate of recurrence and increased incidence of acute rejections.

Background. The reported outcome of renal transplantation in patients with the hemolytic uremic syndrome (HUS) varies greatly, probably related to the diverse causes of HUS. In this single-center retrospective study, we have analyzed the recurrence rate, the incidence of acute rejections, and graft survival in patients suffering from adult-onset and childhood-onset HUS. Methods. The medical records of 35 patients with end-stage renal disease caused by HUS, who received 50 renal allografts, were reviewed. A definite recurrence of HUS was diagnosed if both clinical and histologic signs of thrombotic microangiopathy (TMA) were present in the absence of any endovasculitis. If there were signs of mild endovasculitis, a probable recurrence was diagnosed. Results. After first renal transplantation, 0 definite and 1 (6%) probable recurrence occurred in 18 patients with childhood-onset HUS, as opposed to 7 (41%) definite and 3 (18%) probable recurrences in 17 adult-onset HUS patients (odds ratio [OR], 13.4; 95% confidence interval [CI], 1.7–105.7). In the latter patients, early use of cyclosporine A increased the risk for recurrence. The incidence of acute rejections was increased compared with matched controls (OR, 1.52; 95% CI, 1.05–2.19 for adult-onset HUS and OR, 1.88; 95% CI, 1.34–2.62 for childhood-onset HUS). One-year graft survival in adult-onset HUS was poor (29%), whereas 1-year graft survival in childhood-onset HUS was comparable to matched controls. Conclusions. In adult-onset HUS, the recurrence rate and the incidence of acute rejections are high, resulting in a detrimental graft survival. In childhood-onset HUS, the recurrence rate is low, but the posttransplantation course is complicated by an increased incidence of acute rejections.

Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis.

Podocyte foot process effacement is characteristic of proteinuric renal diseases. In minimal change nephrotic syndrome (MCNS) foot processes are diffusely effaced whereas the extent of effacement varies in focal segmental glomerulosclerosis (FSGS). Here we measured foot process effacement in FSGS and compared it to that in MCNS and in normal kidneys. A clinical diagnosis was used to differentiate idiopathic FSGS from secondary FSGS. Median foot process width, determined morphometrically by electron microscopy, was 3236 nm in 17 patients with idiopathic FSGS, 1098 nm in 7 patients with secondary FSGS, and 1725 nm in 15 patients with MCNS, as compared to 562 nm in 12 control patients. Multivariate analysis showed that foot process width did not correlate with proteinuria or serum albumin levels but was significantly associated as an independent factor with the type of disease. Foot process width over 1500 nm differentiated idiopathic from secondary FSGS with high sensitivity and specificity. Our results show that quantitative analysis of foot processes may offer a potential tool to distinguish idiopathic from secondary FSGS.

Site-Specific Inhibition of Glomerulonephritis Progression by Targeted Delivery of Dexamethasone to Glomerular Endothelium

Glomerulonephritis represents a group of renal diseases with glomerular inflammation as a common pathologic finding. Because of the underlying immunologic character of these disorders, they are frequently treated with glucocorticoids and cytotoxic immunosuppressive agents. Although effective, use of these compounds has limitations as a result of toxicity and systemic side effects. In the current study, we tested the hypothesis that targeted delivery of dexamethasone (dexa) by immunoliposomes to activated glomerular endothelium decreases renal injury but prevents its systemic side effects. E-selectin was chosen as a target molecule based on its disease-specific expression on activated glomerular endothelium in a mouse anti-glomerular basement membrane glomerulonephritis. Site-selective delivery of AbEsel liposome-encapsulated dexamethasone strongly reduced glomerular proinflammatory gene expression without affecting blood glucose levels, a severe side effect of administration of free dexamethasone. Dexa-AbEsel liposomes reduced renal injury as shown by a reduction of blood urea nitrogen levels, decreased glomerular crescent formation, and down-regulation of disease-associated genes. Immunoliposomal drug delivery to glomerular endothelium presents a powerful new strategy for treatment of glomerulonephritis to sustain efficacy and prevent side effects of potent anti-inflammatory drugs.

Bigenic heterozygosity and the development of steroid-resistant focal segmental glomerulosclerosis

BACKGROUND Focal segmental glomerulosclerosis (FSGS) is a major cause of steroid-resistant nephrotic syndrome in childhood with a central role for the podocytes in the pathogenesis. Mutated proteins expressed in podocytes cause proteinuria. The role of combined gene defects in the development of FSGS is less clear. METHODS We analysed seven podocyte genes known to cause proteinuria and FSGS in a group of 19 non-familial childhood-onset steroid-resistant FSGS patients. These genes include NPHS1, NPHS2, ACTN4, CD2AP, WT-1, TRPC6 and PLCE1. We also screened for the mitochondrial A3243G DNA transition associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), and occasionally FSGS. RESULTS No mutations were found in the ACTN4 and TRPC6 genes, and no mitochondrial A3243G DNA transition was found in our group of patients. Two patients showed mutations in the CD2AP gene, one combined with an NPHS2 mutation. A tri-allelic hit was found in a patient carrying compound heterozygous NPHS2 mutations and a heterozygous NPHS1 mutation. In another patient a de novo WT-1 mutation was found combined with a heterozygous NPHS1 mutation, and finally two patients showed three heterozygous PLCE1 mutations. CONCLUSIONS In our rather small group of 19 steroid-resistant FSGS patients, we found 11 mutations in podocyte genes in 6 patients. In four of them the found mutations could explain the pathology. Our data suggest that combined gene defects in podocyte genes may play a role in the development of FSGS.

The Parietal Epithelial Cell: A Key Player in the Pathogenesis of Focal Segmental Glomerulosclerosis in Thy-1.1 Transgenic Mice

Focal segmental glomerulosclerosis (FSGS) is a hallmark of progressive renal disease. Podocyte injury and loss have been proposed as the critical events that lead to FSGS. In the present study, the authors have examined the development of FSGS in Thy-1.1 transgenic (tg) mice, with emphasis on the podocyte and parietal epithelial cell (PEC). Thy-1.1 tg mice express the Thy-1.1 antigen on podocytes. Injection of anti-Thy-1.1 mAb induces an acute albuminuria and development of FSGS lesions that resemble human collapsing FSGS. The authors studied FSGS lesions at days 1, 3, 6, 7, 10, 14, and 21, in relation to changes in the expression of specific markers for normal podocytes (WT-1, synaptopodin, ASD33, and the Thy-1.1 antigen), for mouse PEC (CD10), for activated podocytes (desmin), for macrophages (CD68), and for proliferation (Ki-67). The composition of the extracellular matrix (ECM) that forms tuft adhesions or scars was studied using mAb against collagen IV alpha2 and alpha4 chains and antibodies directed against different heparan sulfate species. The first change observed was severe PEC injury at day 1, which increased in time, and resulted in denuded segments of Bowmans capsule at days 6 and 7. Podocytes showed foot process effacement and microvillous transformation. There was no evidence of podocyte loss or denudation of the GBM. Podocytes became hypertrophic at day 3, with decreased expression of ASD33 and synaptopodin and normal expression of WT-1 and Thy-1.1. Podocyte bridges were formed by attachment of hypertrophic podocytes to PEC and podocyte apposition against denuded segments of Bowmans capsule. At day 6, there was a marked proliferation of epithelial cells in Bowmans space. These proliferating cells were negative for desmin and all podocyte markers, but stained for CD10, and thus appeared to be PEC. The staining properties of the early adhesions were identical to that of Bowmans capsule, suggesting that the ECM in the adhesions was produced by PEC. In conclusion, the authors propose the following sequence of events leading to FSGS lesions in the Thy1.1 tg mice: (1) PEC damage and denudation of Bowmans capsule segments; (2) podocyte hypertrophy and bridging; and (3) PEC proliferation with ECM production.

Rituximab as induction therapy after renal transplantation: a randomized, double-blind, placebo-controlled study of efficacy and safety

We evaluated the efficacy and safety of rituximab as induction therapy in renal transplant patients. In a double‐blind, placebo‐controlled study, 280 adult renal transplant patients were randomized between a single dose of rituximab (375 mg/m2) or placebo during transplant surgery. Patients were stratified according to panel‐reactive antibody (PRA) value and rank number of transplantation. Maintenance immunosuppression consisted of tacrolimus, mycophenolate mofetil and steroids. The primary endpoint was the incidence of biopsy proven acute rejection (BPAR) within 6 months after transplantation. The incidence of BPAR was comparable between rituximab‐treated (23/138, 16.7%) and placebo‐treated patients (30/142, 21.2%, p = 0.25). Immunologically high‐risk patients (PRA >6% or re‐transplant) not receiving rituximab had a significantly higher incidence of rejection (13/34, 38.2%) compared to other treatment groups (rituximab‐treated immunologically high‐risk patients, and rituximab‐ or placebo‐treated immunologically low‐risk (PRA ≤ 6% or first transplant) patients (17.9%, 16.4% and 15.7%, p = 0.004). Neutropenia (<1.5 × 109/L) occurred more frequently in rituximab‐treated patients (24.3% vs. 2.2%, p < 0.001). After 24 months, the cumulative incidence of infections and malignancies was comparable. A single dose of rituximab as induction therapy did not reduce the overall incidence of BPAR, but might be beneficial in immunologically high‐risk patients. Treatment with rituximab was safe.

High urinary excretion of kidney injury molecule-1 is an independent predictor of end-stage renal disease in patients with IgA nephropathy

BACKGROUND The variable course of immunoglobulin A nephropathy (IgAN) warrants accurate tools for the prediction of progression. Urinary kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are markers for the detection of early tubular damage caused by various renal conditions. We evaluated the prognostic value of these markers in patients with IgAN. METHODS We included patients (n = 65, 72% male, age 43 ± 13 years) with biopsy-proven IgAN, who were evaluated for proteinuria. Urinary KIM-1 and NGAL were measured by enzyme-linked immunosorbent assay. We analysed data using Cox regression for the outcome end-stage renal disease (ESRD). RESULTS Median serum creatinine was 142 μmol/L and proteinuria 2.2 g/day. During follow-up (median 75 months), 23 patients (35%) developed ESRD. In patients with IgAN median urinary KIM-1 excretion was 1.7 ng/min and NGAL excretion was 47 ng/min, both significantly higher than in healthy controls. KIM-1 and NGAL were correlated with proteinuria (r = 0.40 and 0.34, respectively, P < 0.01) and each other (r = 0.53, P < 0.01) but not with estimated glomerular filtration rate (eGFR). Interestingly, KIM-1 was not significantly correlated with the excretion of α(1)-microglobulin (α(1)m) and β(2)-microglobulin (β(2)m), known markers of tubular injury. Univariate analysis showed that baseline serum creatinine and urinary excretion of total protein, α(1)m, β(2)m, immunoglobulin G, KIM-1 and NGAL were significantly associated with ESRD. By multivariate analysis, serum creatinine and KIM-1 excretion proved to be significant independent predictors of ESRD. CONCLUSION KIM-1 and NGAL excretion are increased in patients with IgAN and correlate with proteinuria but not with eGFR. Baseline serum creatinine and urinary KIM-1, but not proteinuria, are independent predictors of ESRD.