Cytoskeleton-associated protein 4 affects podocyte cytoskeleton dynamics in diabetic kidney disease
Roberto Boi, Emelie Lassén, Alva Johansson, Peidi Liu, Aditi Chaudhari, Ramesh Tati, Janina Müller-Deile, Mario Schiffer, Kerstin Ebefors, Jenny Nyström
Roberto Boi, Emelie Lassén, Alva Johansson, Peidi Liu, Aditi Chaudhari, Ramesh Tati, Janina Müller-Deile, Mario Schiffer, Kerstin Ebefors, Jenny Nyström
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Research Article Cell biology Nephrology

Cytoskeleton-associated protein 4 affects podocyte cytoskeleton dynamics in diabetic kidney disease

Abstract

Podocytes are kidney glomerular cells that depend on rigorously regulated cytoskeleton components and integrins to form and maintain the so-called foot processes, apparatuses that attach podocytes to the glomerular basement membrane and connect them to neighboring podocytes. In diabetic kidney disease (DKD) these foot processes are effaced as a result of cytoskeleton dysregulation, a phenomenon that gradually reduces glomerular filtration. Cytoskeleton-associated protein 4 (CKAP4) is a known linker between the endoplasmic reticulum, integrins, and microtubular cytoskeleton. Since CKAP4 gene expression is downregulated in glomeruli from patients with DKD but not in other chronic kidney diseases, we hypothesized a role for CKAP4 in the mechanisms leading to foot process effacement (FPE) in DKD. CKAP4 mRNA reduction in podocytes in DKD was demonstrated in human kidney biopsies. Knockdown of CKAP4 in vivo in zebrafish resulted in edema, proteinuria, and foot process effacement, all typical features of DKD. Knockdown of CKAP4 in vitro led to disruption of the actin cytoskeleton and of the microtubular orientation. Moreover, it caused a downregulation of several integrins. These findings indicate that CKAP4 is crucial for foot process dynamics of podocytes. Its reduction, unique to DKD, is mechanistically connected to the pathophysiological processes leading to podocyte FPE.

Authors

Roberto Boi, Emelie Lassén, Alva Johansson, Peidi Liu, Aditi Chaudhari, Ramesh Tati, Janina Müller-Deile, Mario Schiffer, Kerstin Ebefors, Jenny Nyström

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Figure 4

CKAP4 zebrafish homolog knockdown causes proteinuria and podocyte FPE.

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CKAP4 zebrafish homolog knockdown causes proteinuria and podocyte FPE. C...
CKAP4 was knocked down in zebrafish using MO in different concentrations, 30, 50, and 75 μM MO. Proteinuria was measured by reduction of eye fluorescence (A). Example of normal phenotype (P1, no edema) and fish with mild edema (P2) in CKAP4 MO; the arrow points at pericardial edema (B). Assessment of fish phenotypes in the different groups; from P1 to P4 (severe edema) was conducted with reference to Hanke et al. (25, 27) and Ursu et al. (26) (C). Representative electron microscopy pictures of the filtration barrier of zebrafish glomeruli from control MO and CKAP4 MO (75 μM). Control MO shows normal podocyte foot processes (this pattern is indicated with short white arrows) while the CKAP4 MO shows podocyte FPE (partial effacement patterns are indicated with red arrows, complete effacement with short red arrows) (D). Quantification of podocyte FPE percentages in control MO and CKAP4 MO shows increased effacement in CKAP4 MO–treated zebrafish (E). Validation of CKAP4 MO knockdown of CKAP4 as obtained via proteomics (F). A: a minimum of 32 larvae per group was used. Error bars: average ± SEM. *P < 0.05, ***P < 0.001, Mann-Whitney test. E: n = 336 (control MO), n = 319 (CKAP4 MO) foot processes were counted, from n = 14 (control MO) and n = 8 (CKAP4 MO) independent images per group. F: 3 independent experiments were performed; each treatment in each replicate is derived from lysate of a minimum of 8 pooled embryos. Error bars in both panels represent average ± SEM. ***P < 0.001, 1-way ANOVA with Tukey’s multiple-comparison test. CKAP4, cytoskeleton associated protein 4; MO, morpholino.