MicroRNAs in renal fibrosis

Renal fibrosis is the final common pathway of glomerular and tubulo-interstitial diseases. Focal segmental glomerulosclerosis (FSGS) is a devastating, relatively frequent human glomerular disease that increases in clinical incidence and is currently the most frequent cause of proteinuria. One diagnostic feature of this disease is progressive glomerular fibrosis/sclerosis that causes complete obliteration of glomeruli. We have recently developed a reproducible mouse model of FSGS by doxycycline-inducible overexpression of miR-193a 1. This system is also operative in human FSGS. Here we will identify the basic program of gene expression and regulation that causes fibrotic/sclerotic glomerular lesions in this mouse model and in human FSGS. FSGS will be induced in two month old mice, glomeruli will be isolated at different time points, mRNA will be extracted and subjected to deep RNA sequencing (RNASeq). From each time point the expression profile of normal control glomeruli will be subtracted from that of diseased glomeruli by a bio-informatic analysis. This will be followed by a proteomic analysis of laser-capture microdissected glomeruli from cryostat sections of mouse kidneys at different time points of the disease, as well as of normal kidneys that will be gently de-cellularized by an established extraction protocol of the unfixed tissue section with detergents. The laser capture micro-dissected cell-free matrix "ghosts" of the glomeruli will be subjected to tryptic digestion and the peptides will be analyzed by mass spectroscopy. We do expect from this combined approach a road map of the molecular mechanisms that cause initiation and progression of glomerular fibrosis. This includes the identification of relevant glomerular mRNAs, of the corresponding proteins in the fibrotic areas, and of chemokines and growth factors attached to the matrix. As miR-193a overexpression was also observed in a large fraction of human FSGS patients we will repeat the analysis as outlined for the mouse model on human renal biopsies. The gene expression analysis of glomeruli in developing FSGS in the mouse model in combination with the results on isolated human glomeruli of FSGS patients will reveal a unique data set that follows the development of FSGS of a details molecular level for the first time. The therapy experiments planned are the first targeted experimental intervention to prevent FSGS. We will apply tiny locked nucleic acid (LNA) specific for miR-193a (both strands) i.p. into mice during the development of FSGS by feeding doxycycline. The inhibition of FSGS will be analyzed by measuring proteinuria and by podocyte effacement, and isolated glomeruli will be subjected to RNASeq analysis. A first pilot experiment with the tiny LNA treatment was promising. Eventually we will compare the molecular mechanisms involved in glomerular and tubulo-interstitial fibrosis, as provided by our research partners, with particular emphasis of common pathways influenced by the miRNAs involved in both systems. The final goal of this concerted approach is to identify a common "Achilles` heel" in both forms of fibrosis, and to explore for the first the realistic possibility of a common targeted therapy that is based on our previous insights and results of the proposed experiments in this application. 1 Gebeshuber CA et al. Focal segmental glomerulosclerosis is induced by microRNA-193a and its downregulation of WT1. Nat Med. 19, 48187, 2013.

Renal inflammation causes fibrotic scarring and frequently loss of kidney function. In this project we have capitalized on our previous discovery of a unique mouse model of focal- segmental sclerosis in which the development of disease is started by activation of transgenic micro-RNA. We planned to analyze the biochemical alterations during initial steps of fibrosis by mass spectroscopy. The results point to an unexpected novel role of the coagulation system as initiator of the fibrotic process. This is of potential relevance as anti- coagulative therapy in early stages of disease could help to prevent or retard renal fibrosis.Out of this project described above a second program developed from a chance observation. We noted that the lymphatic vessels in inflamed and initially fibrotic kidneys are surrounded by a thick layer of exosomes, i.e. intracellular vesicles cells that are released by endothelial cells into the perivascular stroma. We discovered that these membrane vesicles carry proteins that control the attraction of inflammatory cells towards lymphatic vessels and thus amplify the immune response. Intriguingly, cancer cells use the same mechanism to set up lymph node metastases. These novel findings are a milestone in understanding the biology of inflammation in general and offer some so far unsuspected clues for the therapy of inflammatory diseases and their consequences, such as scarring and fibrosis.