Alteration in Rab11-mediated endocytic trafficking of LDL receptor contributes to angiotensin II-induced cholesterol accumulation and injury in podocytes

Abstract

Objectives: Exposure of podocytes to angiotensin II (Ang II) enhances the abundance of the cell surface glycoprotein, low-density lipoprotein receptor (LDLR) and promotes significant changes in the cellular cholesterol content. Recent investigation provides evidence that the small GTPase Rab11 is involved in the regulation of LDLR, but the exact mechanisms remain unknown. In this study, the role of Rab11 in post-transcriptional regulation of LDLR was evaluated to investigate potential mechanisms of podocyte cholesterol dysregulation in chronic kidney disease.

Materials and methods: Cholesterol content, LDLR and Rab11 expression were assessed in podocytes from Ang II-infused mice. In vitro, the intracellular localization of LDLR was detected under different conditions. Rab11 expression was modulated and we then explored the effect of anti-lipid cytotoxicity by detecting LDLR expression and trafficking, cholesterol content and apoptosis in podocytes.

Results: Cholesterol accumulation, upregulated expression of LDLR and Rab11 were discovered in podocytes from Ang II-infused mice. Ang II enhanced the co-precipitation of LDLR with Rab11 and accelerated the endocytic recycling of LDLR to the plasma membrane. Additionally, silencing Rab11 promoted lysosomal degradation of LDLR and alleviated Ang II-induced cholesterol accumulation and apoptosis in podocytes. Conversely, overexpression of Rab11 or inhibition of lysosomal degradation up-regulated the abundance of LDLR and aggravated podocyte cholesterol deposition.

Conclusions: Rab11 triggers the endocytic trafficking and recycling of LDLR; overactivation of this pathway contributes to Ang II-induced podocyte cholesterol accumulation and injury.

FIGURE 1

Ang II induced excessive glomerular cholesterol deposition and renal injury. Eight‐week‐old mice received a continuous infusion of Ang II for 8 weeks. Two groups of mice were analysed: Control (n = 5), Ang II (n = 5). (A) Representative pictures of H&E staining of renal cortical sections from different groups (scale bar: 20 μm), and bar graph analysis of the glomerular sclerosis index (GSI) to evaluate levels of glomerular sclerosis. Ultrastructure of the glomeruli by transmission electron microscopy (TEM) (scale bar: 1 μm), and bar graph analysis of the foot process fusion rate to evaluate levels of effacement; (B) Quantitative analysis of albumin‐to‐creatinine ratio (ACR) in different groups. ***p < 0.001, n = 5; (C) Representative microscopy images and quantification of lipid droplets by Oil red staining in kidney cortices (scale bar: 20 μm). Quantification of Filipin and WT1 (a marker of podocytes) double staining of kidney sections (original magnification ×600). ***p < 0.001, n = 30

FIGURE 2

Expression alteration of LDLR and Rab11 in the glomeruli from Ang II‐infused mice. (A) Representative immunohistochemistry label of LDLR and Rab11 in kidney sections of control and Ang II infused mice. (original magnification ×40). (B) Western blot analysis of LDLR and Rab11 from glomerular lysates of control and Ang II treated mice. GAPDH was used as an equal loading marker, and the graph indicates a statistical result of relative protein levels. *p < 0.05, n = 5. (C and D) Fluorescence staining of LDLR or Rab11 (green) with Synaptopodin (a marker of podocytes, red), DAPI (nucleus, blue) to evaluate the expression levels (scale bar: 20 μm). (E) Quantitative RT‐PCR analysis to detect relative gene expressions in glomeruli from different groups. *p < 0.05, **p < 0.01, n = 5. DAPI, 4′,6‐diamidino‐2‐phenylindole; LDLR, low‐density lipoprotein receptor

FIGURE 3

Enhanced interaction between LDLR and Rab11 in response to Ang II. (A) Western blot analysis of LDLR and Rab11 expression in HPCs exposed to Ang II, GAPDH was used as an equal loading marker, and the graph indicates a statistical result of relative protein levels. *p < 0.05, **p < 0.01, n = 5. (B) Quantitative RT‐PCR analysis to detect relative gene expressions in HPCs from different groups. *p < 0.05, ***p < 0.001, n = 5. (C) Co‐IP with anti‐Rab11 antibody or an IgG negative control. The resulting precipitates, as well as a portion of the whole cell lysate, were subjected to immunoblotting with anti‐LDLR, Anti‐Rab11, and anti‐GAPDH antibodies. HPCs, human podocytes; IP, immunoprecipitation; LDLR, low‐density lipoprotein receptor

FIGURE 4

Ang II activated LDL recycling through Rab11. (A and B) Fluorescence staining of LDLR (green) with Rab11 (red) or Lamp1 (red) in HPCs (scale bar: 5 μm), and the graph indicates a statistical result of the average immunofluorescence intensity. Pearsonʼs coefficient and Mander's overlap coefficient are considered to represent the degree of panel colocalization. ***p < 0.001, ns = no significance, n = 30. HPCs, human podocytes; LDLR, low‐density lipoprotein receptor

FIGURE 5

Rab11 knockdown reduced LDLR recycling and increased LDLR‐lysosomal degradation. HPCs were transfected with siRNAs targeting Rab11 (siRab11) or a scrambled control (Scramble) and then exposure to Ang II for 24 h. (A) Western blot analysis of total LDLR and Rab11 expression in HPCs, GAPDH was used as an equal loading marker, and the graph indicates a statistical result of relative protein levels. **p < 0.01, ***p < 0.001, n = 5. (B and C) Fluorescence staining of LDLR (green) with Rab11 (red) or Lamp1 (red) in HPCs (scale bar: 5 μm), quantification was performed as described in Figure 4. **p < 0.01, ***p < 0.001, n = 30. HPCs, human podocytes; LDLR, low‐density lipoprotein receptor

FIGURE 6

Rab11 inhibition reduced cholesterol and lipid droplets (LDs) content and protected podocytes from injury. (A) Representative confocal microscopy images and quantification of adipocyte differentiation‐related protein (Adrp, a marker of LDs) fluorescence staining and Filipin staining in each group. ***p < 0.001, n = 30. (B) Quantitative analysis of cholesterol content in each group. *p < 0.05, n = 6. (C) Flow cytometry analysis of podocytes apoptosis in different groups. *p < 0.05, **p < 0.01, n = 6