The Epidermal Growth Factor System in Rare Glomerular Disease

In our preliminary work, we have identified a potential novel, microRNA (miR) controlled pathway for the pathogenesis of primary focal segmental glomerulosclerosis (FSGS). MiRs are short nucleotides, which suppress expression of genes in a cell specific fashion. Several studies have shown that deletion of miR function in podocytes (knock out of Dicer or Drosha) cause a special form of FSGS, but so far no single pathogenic miR was found. In our preliminary work we have identified the first miR (miR193) that controls the complex structure of podocytes. The experimental evidence includes: a. Transgenic doxycycline inducible over-expression of miR193 in mice causes nephrotic range proteinuria and primary FSGS. No abnormalities were observed in other tissues. b. In silico analysis of the genes regulated by miR193 (using the Ingenuity program and transcriptomic comparison of miR193 over-expressing mice to control mice) identified --among other regulated genes-- WT-1, uPA, TGFb1 and 2 (known to be involved in FSGS). In addition, multiple genes of the EGF signaling pathway were identified, including specific ADAM and matrix metalloproteases and components of the EGF down-stream signaling pathways (PI3K pathway, and SOS2 (Grb/Ras)). c. In microdissected glomeruli from human FSGS patients, miR193 was differentially regulated. Interestingly, this was also the case for patients affected by the Denys-Drash syndrome - where a mutation of transcription factor WT-1 causes FSGS. Doxycyclin inducible over-expression of miR193 in mice under the ubiquitous CMV promoter is already established. Glomeruli will be isolated by perfusion with Dynabeads and total RNA will be extracted for downstream analysis of regulated transcripts in the glomerulus in vivo. Alternatively, genetically tagged primary podocytes transgenically expressing beta-galactosidase or eGFP will be isolated by FACsorting. Total miR will be isolated, analyzed and quantified for miR193 as described above. mRNA will be extracted from the isolated glomeruli and subjected to gene expression arraying, using Affymetrix GeneChip Mouse Chip 1.0 ST Array. These studies will be performed in normal and diseased mice, in which FSGS has been induced using the 5/6 nephrectomy + DOCA salt model. The gene expression pattern will be bioinformatically analyzed by GeneSpring and Ingenuity software. To identify miR193 targets, the stably transfected line of conditionally immortalized mouse podocytes (kindly provided by Mundel), or freshly transfected primary podocytes will be used. Lentiviral transduction of miR193 will be performed, mRNA will be extracted and subjected to gene expression analysis, using Affymetrix mouse Gene Chips (Mouse Chip 1.0 ST Array). Components of the EGF system, as well as genes regulated by the EGF system will be of particular interest in this analysis. miR193 over-expressing cultured primary podocytes or podocyte cell lines will be tested for their cell turn-over (mitotic and apoptotic rates), their expression (by qPCR) and localization (by immunofluorescence) of key podocyte proteins of the cytoskeleton (actin, actinins, tubulin), the nephrin-associated slit diaphragm complex (nephrin, CD2ap), matrix adhesion (a3b1 integrin), and of the cell surface (podocalyxin). An archive of renal biopsies from thousands of patients in combination with clinical data will allow us to translate the basic findings to the clinic. The most important pathological entities will be FSGS and as controls minimal change disease and other glomerular diseases and normal glomeruli. Glomeruli will be isolated by laser capture microdissection from archival paraffin sections and extraction and quantification of miR193 will be performed as described above. Using genetic mouse models of FSGS this program deals with the first identification of a novel miR-based regulatory mechanism of podocyte stability that involves several regulated cellular pathways, including that of the EGF system. The results obtained already in preliminary studies will provide the basis of a targeted therapy of the frequent sporadic form of human FSGS.

We analysed the role of miR-193a in Focal segmental glomerulosclerosis (FSGS). FSGS is one of the most frequent glomerular diseases in adults. It is characterised by focal and segmental sclerosis of the kidney glomeruli and profuse podocyte (visceral epithelial cells) foot process effacement. The resulting kidney damage is progressive and irresponsive to treatment and leads to the nephrotic syndrome with proteinuria, hypoalbuminaemia and oedema, followed by kidney failure. While for a limited number of cases genetic mutations in podocyte-specific genes, drugs, viruses or a circulating factor are responsible, for the majority of cases the cause is not understood in detail. We have identified miR-193a as a main candidate for podocyte damage in human FSGS patients. miR-193a suppressed its target Wilms Tumor 1 (WT1), a master regulator of kidney development and podocyte homeostasis. This led to loss of podocyte proteins and induced FSGS. By specific blocking of miR-193a we could provide a proof of principle for a therapeutic approach. These findings represent a big step towards the elucidation of so far idiopathic FSGS cases and the first efficient and specific therapy and require a more thorough analysis of the miR-193a pathway in podocytes.In cooperation with other research groups we analysed signal transduction pathways promoting FSGS. Our focus was on the newly identified pathway regulated by miR-193a.