Nephron-specific knockin of the PIKfyve-binding-deficient Vac14^L156Rmutant results in albuminuria and mesangial expansion

Abstract

Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles - observed in many histologic analyses of renal diseases - is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remains largely unknown. Previous studies showed that in some cases vacuolization is linked to malfunction of the Vac14 complex. This complex regulates intracellular PIP levels, which in turn control the maturation of early into late endosomes as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the role of Vac14 in mice and observed that the nephron specific knockin of the PIKfyve-binding deficient Vac14 mutant led to albuminuria accompanied by mesangial expansion, increased glomerular size and an elevated expression of several kidney injury markers. Overexpression of this Vac14 variant in podocytes did not reveal a strong in vivo phenotype, indicating that Vac14-dependent trafficking processes are more important for tubular than for glomerular processes in the kidney. In vitro overexpression of Vac14 in MDCK cells had no impact on apico-basal polarity defects but resulted in a faster reassembly of junctional structures after Ca depletion and delayed endo- and transcytosis rates. Taken together, our data suggest that increased albuminuria of Vac14 overexpressing mice is a consequence of a lowered endo- and transcytosis of albumin in renal tubules.

Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy

“…22,23 Transmission Electron Microscopy (TEM) Analyses TEM microscopy analyses were performed as described previously. 24 In brief, kidneys were fixed in 2.5% glutaraldehyde with Sorensen's phosphate buffer and after that postfixed with 1% osmium tetroxide, dehydrated, and embedded in the embedding media epon. In addition, 60-nm ultrathin sections were cut by using Leica Ultracut R ultramicrotome (Vienna, Austria) and counterstained with uranyl acetate in 8% uranyl acetate in bidistilled water and incubated with lead citrate solution.…”