Progressive kidney failure is a genetically and clinically heterogeneous group of disorders. Podocyte foot processes and the interposed glomerular slit diaphragm are essential components of the permeability barrier in the kidney. Mutations in genes encoding structural proteins of the podocyte lead to the development of proteinuria, resulting in progressive kidney failure and focal segmental glomerulosclerosis. Here, we show that the canonical transient receptor potential 6 (TRPC6) ion channel is expressed in podocytes and is a component of the glomerular slit diaphragm. We identified five families with autosomal dominant focal segmental glomerulosclerosis in which disease segregated with mutations in the gene TRPC6 on chromosome 11q. Two of the TRPC6 mutants had increased current amplitudes. These data show that TRPC6 channel activity at the slit diaphragm is essential for proper regulation of podocyte structure and function.
Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur-/-) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.
The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane alpha-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the alpha-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.
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