Minimal Change Disease Is Associated With Endothelial Glycocalyx Degradation and Endothelial Activation.

Introduction: Minimal change disease (MCD) is considered a podocyte disorder triggered by unknown circulating factors. Here, we hypothesized that the endothelial cell (EC) is also involved in MCD. Methods: We studied 45 children with idiopathic nephrotic syndrome (44 had steroid sensitive nephrotic syndrome [SSNS], and 12 had biopsy-proven MCD), 21 adults with MCD, and 38 healthy controls (30 children, 8 adults). In circulation, we measured products of endothelial glycocalyx (EG) degradation (syndecan-1, heparan sulfate [HS] fragments), HS proteoglycan cleaving enzymes (matrix metalloprotease-2 [MMP-2], heparanase activity), and markers of endothelial activation (von Willebrand factor [vWF], thrombomodulin) by enzyme-linked immunosorbent assay (ELISA) and mass spectrometry. In human kidney tissue, we assessed glomerular EC (GEnC) activation by immunofluorescence of caveolin-1 (n = 11 MCD, n = 5 controls). In vitro, we cultured immortalized human GEnC with sera from control subjects and patients with MCD/SSNS sera in relapse (n = 5 per group) and performed Western blotting of thrombomodulin of cell lysates as surrogate marker of endothelial activation. Results: In circulation, median concentrations of all endothelial markers were higher in patients with active disease compared with controls and remained high in some patients during remission. In the MCD glomerulus, caveolin-1 expression was higher, in an endothelial-specific pattern, compared with controls. In cultured human GEnC, sera from children with MCD/SSNS in relapse increased thrombomodulin expression compared with control sera. Conclusion: Our data show that alterations involving the systemic and glomerular endothelium are nearly universal in patients with MCD and SSNS, and that GEnC can be directly activated by circulating factors present in the MCD/SSNS sera during relapse.

A Novel Treatment for Glomerular Disease: Targeting the Activated Macrophage Folate Receptor with a Trojan Horse Therapy in Rats.

Since activated macrophages express a functional folate receptor ß (FRß), targeting this macrophage population with folate-linked drugs could increase selectivity to treat inflammatory diseases. Using a macrophage-mediated anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN) in WKY rats, we investigated the effect of a novel folic acid-aminopterin (AMT) conjugate (EC2319) designed to intracellularly deliver AMT via the FR. We found that treatment with EC2319 significantly attenuated kidney injury and preserved renal function. Kidney protection with EC2319 was blocked by a folate competitor, indicating that its mechanism of action was specifically FRß-mediated. Notably, treatment with methotrexate (MTX), another folic acid antagonist related to AMT, did not protect from kidney damage. EC2319 reduced glomerular and interstitial macrophage infiltration and decreased M1 macrophage recruitment but not M2 macrophages. The expression of CCL2 and the pro-fibrotic cytokine TGF-ß were also reduced in nephritic glomeruli with EC2319 treatment. In EC2319-treated rats, there was a significant decrease in the deposition of collagens. In nephritic kidneys, FRß was expressed on periglomerular macrophages and macrophages present in the crescents, but its expression was not observed in normal kidneys. These data indicate that selectively targeting the activated macrophage population could represent a novel means for treating anti-GBM GN and other acute crescentic glomerulonephritis.

Glomerular endothelial cells and podocytes can express CD80 in patients with minimal change disease during relapse.

BACKGROUND: Urinary CD80 has emerged as potential biomarker in idiopathic nephrotic syndrome (INS). However, its cellular source remains controversial. The aim of the study was to assess whether CD80 is truly expressed by glomerular cells in INS patients during relapse and in the LPS mouse model of podocyte injury. METHODS: The presence of CD80 in glomeruli was evaluated by combining immunostaining, immunogold labeling, and in situ hybridization techniques. RESULTS: CD80 was present along the surface of glomerular endothelial cells (GEC) and rarely in podocytes in six of nine minimal change disease (MCD) patients in relapse, two of eleven patients with focal segmental glomerulosclerosis in relapse, and absent in controls. In mice, CD80 was upregulated at mRNA and protein level in GEC and podocytes, in a similar pattern to that seen in MCD patients. CONCLUSIONS: Glomerular endothelial cells and podocytes can express CD80 in patients with MCD during relapse. A better understanding of the role of CD80 in glomerular cells may provide further insights into the mechanisms of proteinuria in INS.

C4 Nephritic Factors in C3 Glomerulopathy: A Case Series.

BACKGROUND: C3 glomerulopathy (C3G) defines a group of rare complement-mediated kidney diseases with a shared underlying pathophysiology: dysregulation of complement in the fluid phase and glomerular microenvironment. Dysregulation can be driven by autoantibodies to C3 and C5 convertases. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 168 patients with C3G (dense deposit disease, 68; C3 glumerulonephritis, 100) selected from our C3G biobank. OUTCOMES: Patient-purified immunoglobulin Gs were tested for C4 nephritic factors (C4NeFs). These autoantibodies recognize C4b2a, the C3 convertase of the classical pathway of complement. MEASUREMENTS: C4NeFs were detected using a modified hemolytic assay. RESULTS: C4NeFs were identified in 5 patients, 4 of whom had C3 glomerulonephritis. C4NeFs were associated with dysregulation of C3 and C5 convertases, and they appear to stabilize these convertases in a dose-dependent manner. C4NeFs also appear to protect C4b2a from decay mediated by soluble CR1 and C4 binding protein. The stabilizing activity of the autoantibodies was further demonstrated by using heat treatment to inactivate complement. C4NeFs were not detected in 150 patients with another complement-mediated kidney disease, atypical hemolytic uremic syndrome. They were also absent in 300 apparently healthy controls. LIMITATIONS: In addition to C4NeFs, 2 patients had positive findings for other autoantibodies: one patient also had autoantibodies to factor H; the other patient also had autoantibodies to C3bBb (C3NeFs). CONCLUSIONS: The finding of C4NeFs in a small percentage of patients with C3G highlights the challenge in identifying autoantibodies that drive complement dysregulation and underscores the complexity of the autoantibody repertoire that can be identified in these patients.

Angiopoietin-like-4 and minimal change disease.

BACKGROUND: Minimal Change Disease (MCD) is the most common type of nephrotic syndrome in children. Angiopoietin-like-4 (Angplt4) has been proposed as mediator of proteinuria in MCD. The aim of this study was to evaluate the role of Angptl4 as a biomarker in MCD. METHODS: Patients with biopsy-proven primary MCD, focal segmental glomerulosclerosis, membranous nephropathy (60, 52 and 52 respectively) and 18 control subjects had urinary and serum Angptl4 measured by Elisa. Frozen kidney tissue sections were stained for Angptl4. RESULTS: Angptl4 was not identified in glomeruli of MCD patients in relapse. Urinary Angptl4 levels were elevated in MCD in relapse as well as in patients with massive proteinuria due to other glomerular diseases. CONCLUSION: Neither serum nor urine Angptl4 appear to be good biomarkers in MCD. Elevated urinary Angptl4 n glomerular disease appears to reflect the degree of proteinuria rather than any specific disease.

The RIVUR study: a review of its findings.

BACKGROUND: The conclusion drawn by the authors of the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) trial is that antimicrobial prophylaxis reduces the risk of recurrent urinary tract infection (UTI)-but not of renal scarring-in patients with vesicoureteral reflux (VUR). RESULTS: A review of the findings showed that the decreased recurrent UTI rate was only present at the end of the 2-year follow-up period and was only slightly increased (12.3%) above the 10% cutoff for statistical significance. The difference was not observed in children younger than two years of age with VUR grade III and IV. In addition, the rate of new renal scarring was not statistically different between the prophylaxis and placebo groups (8.2 vs. 8.4%, respectively). A high rate of uropathogen antibiotic resistance was observed in the prophylaxis group (68.4 vs. 24.6%, respectively). CONCLUSION: The analysis of the RIVUR findings questions the validity of its authors suggestion that the results may warrant reconsideration of the current recommendations by the American Academy of Pediatrics on obtaining a voiding cystourethrogram after the first febrile UTI and the use of urinary antibiotic prophylaxis in VUR patients.

Case series: CTLA4-IgG1 therapy in minimal change disease and focal segmental glomerulosclerosis.

BACKGROUND: Minimal Change Disease (MCD) in relapse is associated with increased podocyte CD80 expression and elevated urinary CD80 excretion, whereas focal segmental glomerulosclerosis (FSGS) has mild or absent CD80 podocyte expression and normal urinary CD80 excretion. METHODS: One patient with MCD, one patient with primary FSGS and three patients with recurrent FSGS after transplantation received CD80 blocking antibodies (abatacept or belatacept). Urinary CD80 and CTLA-4 levels were measured by ELISA. Glomeruli were stained for CD80. RESULTS: After abatacept therapy, urinary CD80 became undetectable with a concomitant transient resolution of proteinuria in the MCD patient. In contrast, proteinuria remained unchanged after abatacept or belatacept therapy in the one patient with primary FSGS and in two of the three patients with recurrent FSGS despite the presence of mild CD80 glomerular expression but normal urinary CD80 excretion. The third patient with recurrent FSGS after transplantation had elevated urinary CD80 excretion immediately after surgery which fell spontaneously before the initiation of abatacept therapy; after abatacept therapy, his proteinuria remained unchanged for 5 days despite normal urinary CD80 excretion. CONCLUSION: These observations are consistent with a role of podocyte CD80 in the development of proteinuria in MCD. In contrast, CD80 may not play a role in recurrent FSGS since the urinary CD80 of our three patients with recurrent FSGS was only increased transiently after surgery and normalization of urinary CD80 did not result in resolution of proteinuria.

Minimal change disease: a dysregulation of the podocyte CD80-CTLA-4 axis?

BACKGROUND: Minimal Change Disease (MCD) is associated with CD80 expression in podocytes and elevated urinary CD80 excretion during active renal disease. We have evaluated the urinary excretion of CTLA-4 and CD80 during different stages of the nephrotic syndrome in patients with MCD to test the hypothesis that persistent increased urinary CD80 excretion in patients with MCD in relapse is due to an ineffectual CTLA-4 response of the host to curtail the activation of CD80. METHODS: Thirty-two children with biopsy-proven MCD were studied during relapse and/or remission. Eleven healthy subjects served as controls. RESULTS: Urinary CD80 excretion was significantly increased in MCD patients in relapse relative to that in MCD patients in remission (p < 0.001) and controls (p < 0.001). Although urinary CTLA-4 excretion was higher in MCD patients in relapse than in MCD patients in remission (p = 0.01) and controls (p = 0.03), no significant correlation was observed between urinary CD80 excretion and urinary CTLA-4 level in MCD patients at the time of relapse (p = 0.06). At the time of remission, CD80 had decreased significantly in all patients, but CTLA-4 levels either decreased or remained unchanged in all but five patients, and no correlation was observed between urinary CD80 excretion and CTLA-4 level (p = 0.7). CONCLUSIONS: Urinary CTLA-4 levels do not correlate with urinary CD80 excretion, suggesting the possibility that the CTLA4 response may be suboptimal in this disease during relapse.

CD80, suPAR and nephrotic syndrome in a case of NPHS2 mutation.

BACKGROUND: Podocin mutations are characterized by progression to end stage renal disease and histologic findings of Focal Segmental Glomerulosclerosis (FSGS). CD80 is a podocytes protein that may play a role in proteinuria, particularly in Minimal Change Disease whereas the soluble urokinase receptor (suPAR) is characteristically elevated in the serum of FSGS patients. METHODS: In a patient with nephrotic syndrome and podocin mutation, urinary and serum CD80 as well as suPAR were measured using commercially available kits. Urinary CD80 molecular size was determined by western blot analysis. Glomerular staining for CD80 and podocin was performed. RESULTS: Patient displayed marked elevated CD80 and mildly increased suPAR urinary levels compared to controls. Serum CD80 level was within the range observed in normal controls. Serum suPAR level was elevated, albeit in the lower range reported for patients with primary FSGS. Immunofluorescence examination of kidney biopsy revealed glomerular CD80 expression. CONCLUSION: The combination of serum and urinary biomarkers can help differentiate various forms of FSGS. High urinary CD80 and elevated serum and urinary suPAR might represent a profile to differentiate this genetic form of FSGS from primary FSGS.

Acute renal failure due to bilateral pieloureteral stone impaction in a 10-month-old boy.

Urolithiasis (UL) can present with its classic signs and symptoms, such as flank or abdominal pain and gross hematuria. However, atypical complaints can be more common in younger children. We report here a case of bilateral ureteropelvic junction (UPJ) stones in a 10-month-old boy who only showed nonspecific symptoms at the time of presentation. The initial blood test revealed renal failure (serum creatinine 3.4 mg/dl), hyperkalemia (6.4 mEq/l), hyperphosphoremia (9.4 mEq/l) and mild metabolic acidosis. Medical treatment for electrolyte disorders was started. The ultrasonography revealed impacted stones in both ureteropelvic junctions. A pigtail catheter was placed in each ureter. High urine flow was promptly achieved after the pigtail procedure, and the serum creatinine level dropped quickly from 4.5 to 0.32 mg/dl. Quantitative determination of urinary amino acids by ion exchange chromatography showed high cystine levels of 8.43 mmol/g creatinine. Outpatient follow-up was scheduled every 3 months to monitor patient compliance with potassium citrate. In the first 6 months, the patient underwent three febrile urinary tract infections (UTIs). Since both pigtail catheters were removed, he has been free of UTIs and stones. Our case emphasizes the need for considering UL in infants who complain with unclear signs, because UL can only show nonspecific symptoms in children younger than 1 year old. Since cystinuria can cause loss of renal function due to urinary system obstruction and UTI, an early diagnosis and a close follow-up are the key to achieving the best long-term outcome.