Protein phosphatase 2A modulates podocyte maturation and glomerular functional integrity in mice

Abstract

Background: Protein phosphorylation & dephosphorylation are ubiquitous cellular processes that allow for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a multifunction phosphatase that is well expressed in all cell types of kidney during early renal development, though its functions in kidney remains to be elucidated. Methods: PP2A conditional knock-out mice was generated with PP2A fl/fl mice that were crossed with Podocin-Cre mice. The phenotype of Pod-PP2A-KO mice (homozygous for the floxed PP2A allele with Podocin-Cre) and littermate PP2A fl/fl controls (homozygous for the PP2A allele but lacking Podocin-Cre) were further studied. Primary podocytes isolated from the Pod-PP2A-KO mice were cultured and they were then employed with sing label-free nano-LC - MS/MS technology on a Q-exactive followed by SIEVE processing to identify possible target molecular entities for the dephosphorylation effect of PP2A, in which Western blot and immunofluorescent staining were used to analyze further. Results: Pod-PP2A-KO mice were developed with weight loss, growth retardation, proteinuria, glomerulopathy and foot process effacement, together with reduced expression of some slit diaphragm molecules and cytoskeleton rearrangement of podocytes. Y box protein 1 (YB-1) was identified to be the target molecule for dephosphorylation effect of PP2A. Furthermore, YB-1 phosphorylation was up-regulated in the Pod-PP2A-KO mice in contrast to the wild type controls, while total and un-phosphorylated YB-1 both was moderately down-regulated in podocytes from the Pod-PP2A-KO mice. Conclusion: Our study revealed the important role of PP2A in regulating the development of foot processes and fully differentiated podocytes whereas fine-tuning of YB-1 via a post-translational modification by PP2A regulating its activity might be crucial for the functional integrity of podocytes and glomerular filtration barrier. Graphic abstract: [Figure not available: see fulltext.]