A slit-diaphragm-associated protein network for dynamic control of renal filtration

Abstract

The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm, a cell-cell junction between the foot processes of renal podocytes. The slit-diaphragm is thought to operate as final barrier or as molecular sensor of renal filtration. Using high-resolution proteomic analysis of slit-diaphragms affinity-isolated from rodent kidney, we show that the native slit-diaphragm is built from the junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling-receptors, kinases/phosphatases, transporters and scaffolds. Knockout or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide-receptor C (ANPRC), integral membrane protein 2B (ITM2B), membrane-associated guanylate-kinase, WW and PDZ-domain-containing protein1 (MAGI1) and amyloid protein A4 resulted in target-specific impairment or disruption of the filtration process. Our results identify the slit-diaphragm as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.

The molecular architecture of the kidney slit diaphragm

“…Our model suggests that the center of the SD is composed of Nephrin molecules alone, which explains the results of studies in several animal models using either siRNA-mediated knock-down or inducible deletion of the Neph1 gene showing that the amount of Nephrin incorporated into the structured SD is maintained for a long time before albuminuria occurs 9,23 . Furthermore, our model is consistent both with the compression model 15 11 , in which the SD is required for a stable podocyte network that compresses the GBM to potentially reduce permeation speed, and with the electrokinetic model 14 , in which negatively charged molecules are moved back to the capillaries by electrophoresis, which actively prevents the clogging of the glomerular filtration barrier.…”

Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process

“…Additionally, we did not identify a significant enrichment of solute transporters as in a recent MS analysis of the podocin-associated proteome. 23 This may represent a more restricted localization of the modified podocin-BioID protein along the foot process, the inability to biotinylate these proteins, or the lack of enrichment in our studies due to their normal biotinylation without the addition of excess biotin. Together, these findings highlight the efficacy of using or combining multiple approaches to obtain a more complete understanding of the localized proteome.…”

“…Our proteomics profiling identified more than 50 candidate molecules with significant enrichment in the podocyte foot process, Log2 > 1.2. Within this dataset we were able to identify novel candidates not found in previous investigations of the slit diaphragm proteome 22,23 . Additionally, we were able to identify changes to the localized proteome that occur with aging.…”

“…The identification of slit diaphragm protein complexes with immunoprecipitation followed by mass spectrometry (MS) has uncovered important localized interactions 22, 23 . However, the efficiency of these immunoprecipitations is often hindered by harsh conditions required to extract membrane proteins, which can eliminate weaker binding interactions, or by antibodies that may disrupt interactions.…”

Transcriptional regulation by the NSL complex enables diversification of IFT functions in ciliated versus nonciliated cells

“…A typical podocyte consists of a cell body, primary processes that consist primarily of microtubules, and branching secondary processes (also known as foot processes) that are enriched in actin (30,31). Slit diaphragms, specialized cell-cell junctions found between two interdigitating podocyte foot processes, function together with the glomerular basement membrane to form the glomerular filtration barrier (32)(33)(34). Therefore, the proper maintenance of cytoskeletal structures in podocytes is crucial for their function and mutations in genes involved in cytoskeletal functions are often implicated in glomerular diseases (29,35).…”