Significance Statement: MicroRNA-192-5p (miR-192-5p) is upregulated in the glomeruli and urine of patients with idiopathic membranous glomerulonephritis (iMGN). It derives from glomerular endothelial cells, is packed into exosomes, and decreases podocyte nephronectin (NPNT) in the glomerular basement membrane (GBM) by paracrine signaling. Patients with iMGN have reduced glomerular NPNT expression. Whole-body knockdown of npnt in zebrafish and podocyte-specific knockout of Npnt in mice damage the GBM, increasing lucidity of the lamina rara interna, which admits high molecular weight proteins. Reduced NPNT leading to GBM leakiness might be an important part of iMGN pathophysiology, initiating podocyte antigen presentation, and admitting autoantibodies into the subepithelial space. NPNT might be a prognostic parameter and noninvasive marker for iMGN. Monitoring and targeting this miR could be a promising diagnostic and therapeutic approach for iMGN. Background: Autoantibodies binding to podocyte antigens cause idiopathic membranous glomerulonephritis (iMGN). However, it remains elusive how autoantibodies reach the subepithelial space because the glomerular filtration barrier (GFB) is size selective and almost impermeable for antibodies. Methods: Kidney biopsies from patients with iMGN, cell culture, zebrafish, and mouse models were used to investigate the role of nephronectin (NPNT) regulating microRNAs (miRs) for the GFB. Results: Glomerular endothelial cell (GEC)-derived miR-192-5p and podocyte-derived miR-378a-3p are upregulated in urine and glomeruli of patients with iMGN, whereas glomerular NPNT is reduced. Overexpression of miR-192-5p and morpholino-mediated npnt knockdown induced edema, proteinuria, and podocyte effacement similar to podocyte-derived miR-378a-3p in zebrafish. Structural changes of the glomerular basement membrane (GBM) with increased lucidity, splitting, and lamellation, especially of the lamina rara interna, similar to ultrastructural findings seen in advanced stages of iMGN, were found. IgG-size nanoparticles accumulated in lucidity areas of the lamina rara interna and lamina densa of the GBM in npnt-knockdown zebrafish models. Loss of slit diaphragm proteins and severe structural impairment of the GBM were further confirmed in podocyte-specific Npnt knockout mice. GECs downregulate podocyte NPNT by transfer of miR-192-5p–containing exosomes in a paracrine manner. Conclusions: Podocyte NPNT is important for proper glomerular filter function and GBM structure and is regulated by GEC-derived miR-192-5p and podocyte-derived miR-378a-3p. We hypothesize that loss of NPNT in the GBM is an important part of the initial pathophysiology of iMGN and enables autoantigenicity of podocyte antigens and subepithelial immune complex deposition in iMGN.

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3′ of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2^ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2^ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.

MicroRNAs (miRs) are small non‐coding RNAs that regulate gene expression in physiological processes as well as in diseases. Currently miRs are already used to find novel mechanisms involved in diseases and in the future, they might serve as diagnostic markers. To identify miRs that play a role in glomerular diseases urinary miR‐screenings are a frequently used tool. However, miRs that are detected in the urine might simply be filtered from the blood stream and could have been produced anywhere in the body, so they might be completely unrelated to the diseases. We performed a combined miR‐screening in pooled urine samples from patients with different glomerular diseases as well as in cultured human podocytes, human mesangial cells, human glomerular endothelial cells and human tubular cells. The miR‐screening in renal cells was done in untreated conditions and after stimulation with TGF‐β. A merge of the detected regulated miRs led us to identify disease‐specific, cell type‐specific and cell stress‐induced miRs. Most miRs were down‐regulated following the stimulation with TGF‐β in all cell types. Up‐regulation of miRs after TGF‐β was cell type‐specific for most miRs. Furthermore, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Most miRs were specifically regulated in one disease. Only miR‐155 was up‐regulated in all disease urines compared to control and therefore seems to be rather unspecific. In conclusion, a combined urinary and cell miR‐screening can improve the interpretation of screening results. These data are useful to identify novel miRs potentially involved in glomerular diseases.

Background: ANCA-associated vasculitis (AAV) is a rare small vessel disease characterized by multi-organ involvement. Biomarkers that can measure specific organ involvement are missing. Here, we ask whether certain circulating cytokines and chemokines correlate with renal involvement and if distinct cytokine/chemokine patterns can differentiate between renal, ear/nose/throat, joints, and lung involvement of AAV. Methods: Thirty-two sets of Birmingham vasculitis activity score (BVAS), PR3-ANCA titers, laboratory marker, and different cytokines were obtained from 17 different patients with AAV. BVAS, PR3-ANCA titers, laboratory marker, and cytokine concentrations were correlated to different organ involvements in active AAV. Results: Among patients with active PR3-AAV (BVAS > 0) and kidney involvement we found significant higher concentrations of chemokine ligand (CCL)-1, interleukin (IL)-6, IL21, IL23, IL-28A, IL33, monocyte chemoattractant protein 2 (MCP2), stem cell factor (SCF), thymic stromal lymphopoietin (TSLP), and thrombopoietin (TPO) compared to patients without PR3-ANCA-associated glomerulonephritis. Patients with ear, nose, and throat involvement expressed higher concentrations of MCP2 and of the (C-X-C motif) ligand-12 (CXCL-12) compared to patients with active AAV and no involvement of these organs. Conclusion: We identified distinct cytokine patterns for renal manifestation and for ear, nose and throat involvement of PR3-AAV. Distinct plasma cytokines might be used as non-invasive biomarkers of organ involvement in AAV.