Nephrin : Intracellular trafficking and podocyte maturation

Sammanfattning: Congenital nephrotic syndrome of the Finnish type, CNF, is an autossomal recessive disorder caused by mutations in the NPHS1 gene and, consequently, in its product nephrin, a 180 kDa transmembrane protein belonging to the immunoglobulin superfamily of adhesion molecules. In kidneys, nephrin is located exclusively to the podocyte slit diaphragm and it has a key role in organising amd maintaining the renal ultrafiltation barrier. In Finland, 94% of the CNF phenotypes are caused by two mutations, Finmajor and Finminor. In contrast, the rest of the Finnish patients and patients outside Finland have unique set of mutations giving rise to the same CNF phenotype. Among those, missense mutations resulting in single amino acid substitutions in nephrin account for more that 50% of the disease-causing mutations. In this study, we investigated, in vitro, the fate of twenty-one nephrin missense mutants identified in CNF patients. Our studies show that 16 out of 21 mutants are retained in the ER, and fail to reach the plasma membrane, suggesting that defective trafficking of the mutated nephrin, due to misfolding may contribute to the CNF phenotype observed in patients carrying nephrin missense mutations. A tightly regulated quality control prevents about one third of all proteins produced by the cells to exit the ER, and target those for degradation. Among them, there are proteins that, despite the mutations and aberrant folding, still retain part of the functional properties of the wild-type molecule. Small compounds called chemical and pharmacological chaperones have been shown to be able to rescue protein mutants from ER quality control and redirect them to their proper location. We therefore tested the potential effect of 4-PBA, one chemical chaperone, on the nephrin missense mutants retained in the ER. We demonstrate that in presence of 4-PBA many of the mutants are rescued from the ER to the cell surface. Further studies indicate that these mutants behave functionally, as judged by their ability to interact with an extracellular partner, NEPH-1, and to become phosphorylated upon antibody clustering. Our results suggest that 4-PBA could represent a new therapy for patients with CNF. Nephrin has been shown to play important structural and signalling roles in podocytes, in vitro. In the last part of this study we investigated the impact of nephrin in late-embryonic-stage glomeuli. Our results show that the absence of nephrin, indeed, causes severe structural damage to the podocytes. However, nephrin has little impact on podocyte proliferation and apoptosis. Moreover, nephrin was able to regulate only a small set of genes, and had no impact on overall podocyte protein expression. One protein upregulated in the absence of nephrin is the tight junction component claudin 3, which is usually absent on the glomerulus. We suggest is that claudin 3 may be expressed as means for the podocyte to compensate for the loss of the proper slit diaphragm, being a component of the yet poorly understood junction-like structure observed between effaced podocyte foot processes of CNF patients. Taken together, our results suggest that nephrin has key importance in maturation of the slit diaphragm, specially the correct assembly of the filtration barrier. However, nephrin is not actively involved in podocyte development.