Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A.

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-ß) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.

Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization.

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.

Orellanine specifically targets renal clear cell carcinoma.

Renal cell carcinoma (RCC), arising from the proximal tubule in the kidney, accounts for approximately 85% of kidney cancers and causes over 140,000 annual deaths worldwide. In the last decade, several new therapies have been identified for treatment of metastatic RCC. Although these therapies increase survival time compared to standard care, none of them has curative properties. The nephrotoxin orellanine specifically targets proximal tubular epithelial cells, leaving other organs unaffected. We therefore hypothesized that the selective toxicity of orellanine extends to clear cell RCC (ccRCC) cells since they emanate from proximal tubular cells. Orellanine would thus target both primary and metastatic ccRCC in vitro and in vivo. We found that orellanine induces dose-dependent cell death in proximal tubular cells and in all ccRCC cells tested, both primary and cell lines, with no toxicity detected in control cells. The toxic action of orellanine involve decreased protein synthesis, disrupted cell metabolism and induction of apoptosis. In nude rats carrying human ccRCC xenografts, brief orellanine treatment eliminated more than 90% of viable tumor mass compared to control rats. This identifies orellanine as a potential treatment concept for ccRCC patients on dialysis, due to its unique selective toxicity towards ccRCC.

Role of podocyte B7-1 in diabetic nephropathy.

Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase-dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy.

Proteasomal degradation of Nck1 but not Nck2 regulates RhoA activation and actin dynamics.

The ubiquitously expressed adapter proteins Nck1/2 interact with a multitude of effector molecules to regulate diverse cellular functions including cytoskeletal dynamics. Here we show that Nck1, but not Nck2, is a substrate of c-Cbl-mediated ubiquitination. We uncover lysine 178 in Nck1 as the evolutionarily conserved ubiquitin acceptor site. We previously reported that synaptopodin, a proline-rich actin-binding protein, induces stress fibres by blocking the Smurf1-mediated ubiquitination of RhoA. We now find that synaptopodin competes with c-Cbl for binding to Nck1, which prevents the ubiquitination of Nck1 by c-Cbl. Gene silencing of c-Cbl restores Nck1 protein abundance and stress fibres in synaptopodin knockdown cells. Similarly, expression of c-Cbl-resistant Nck1(K178R) or Nck2 containing the SH3 domain 2 of Nck1 restores stress fibres in synaptopodin-depleted podocytes through activation of RhoA signalling. These findings reveal proteasomal regulation as a key factor in the distinct and non-redundant effects of Nck on RhoA-mediated actin dynamics.