Overexpression of TGF-β Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans

Abstract

Background: TGF-β is known as an important stress factor of podocytes in glomerular diseases. Apart from activation of direct pro-apoptotic pathways we wanted to analyze micro-RNA (miRs) driven regulation of components involved in the integrity of the glomerular filtration barrier induced by TGF-β. Since miR-143-3p (miR-143) is described as a TGF-β inducible miR in other cell types, we examined this specific miR and its ability to induce glomerular pathology. Methods: We analyzed miR-143 expression in cultured human podocytes after stimulation with TGF-β. We also microinjected zebrafish eggs with a miR-143 mimic or with morpholinos specific for its targets syndecan and versican and compared phenotype and proteinuria development. Results: We detected a time dependent, TGF-β inducible expression of miR-143 in human podocytes. Targets of miR-143 relevant in glomerular biology are syndecans and versican, which are known components of the glycocalyx. We found that syndecan 1 and 4 were predominantly expressed in podocytes while syndecan 3 was largely expressed in glomerular endothelial cells. Versican could be detected in both cell types. After injection of a miR-143 mimic in zebrafish larvae, syndecan 3, 4 and versican were significantly downregulated. Moreover, miR-143 overexpression or versican knockdown by morpholino caused loss of plasma proteins, edema, podocyte effacement and endothelial damage. In contrast, knockdown of syndecan 3 and syndecan 4 had no effects on glomerular filtration barrier. Conclusion: Expression of versican and syndecan isoforms is indispensable for proper barrier function. Podocyte-derived miR-143 is a mediator for paracrine and autocrine cross talk between podocytes and glomerular endothelial cells and can alter expression of glomerular glycocalyx proteins.

Endothelin receptor-A mediates degradation of the glomerular endothelial surface layer via pathologic crosstalk between activated podocytes and glomerular endothelial cells

“…These data are consistent with studies of TGF-binduced glomerulonephropathy in zebrafish. 37 Similarly, we detected a significant loss of ESL in AD-treated BALB/c mice.…”

Identification of Cell-and Disease-Specific Micro RNAs in Glomerular Pathologies

“…We have confirmed the importance of this miR for the maintenance of a functional glomerular filtration barrier in the zebrafish model [16]. MiR-30 family members were also highly expressed in our cultured human podocytes.…”

“…We performed a Q-PCR based miR-screening (TaqMan® Array Human MicroRNA Card Set v3.0) in cultured human podocytes, human glomerular endothelial cells, human mesangial cells and human proximal tubular cells in unstimulated conditions and after stimulation of miR-143-3p for podocyte function and ultrastructure [16]. Examples for a glomerular endothelial cell and a mesangial cells-specific miRs are…”

The Zebrafish Model to Study the Role of microRNAs in Glomerular Function and Disease

“…We demonstrated that miR-143-3p overexpression by injection of a specific miR-143-3p mimic in zebrafish egg stage leads to a significant reduction of vcan and sdc RNA isoforms in 120 days old zebrafish larvae. Moreover, miR-143-3p overexpression caused a nephrotic phenotype with generalized edema, loss of plasma proteins, podocyte effacement, glomerular endothelial cell swelling and loss of glomerular endothelial fenestration.Comparing the functional and ultra-structural glomerular changes of miR-143-3p overexpression to MO based knockdown of miR-143-3p target genes, vcan appeared to have the highest in vivo relevance because glomerular damage induced by miR-143-3p overexpression was comparable to that seen after vcan knockdown alone.We hypothesize that podocyte-derived miR-143-3p might function as a mediator for glomerular crosstalk between podocytes and glomerular endothelial cells and act in a paracrine as well as autocrine manner [18].Another miR that was cell-type specifically up regulated in cultured human podocytes after stimulation with TGF-beta was miR-378a-3p. miR-378a-3p was described to target nephronectin (NPNT) in osteoblasts [19].…”

“…We hypothesize that podocyte-derived miR-143-3p might function as a mediator for glomerular crosstalk between podocytes and glomerular endothelial cells and act in a paracrine as well as autocrine manner [18].…”