CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD

Abstract

Scavenger receptors play a central role in atherosclerosis by processing oxidized lipoproteins and mediating their cellular effects. Recent studies suggested that the atherogenic state correlates with progression of chronic kidney disease (CKD); therefore, scavenger receptors are candidate mediators of renal fibrogenesis. Here, we investigated the role of CD36, a class B scavenger receptor, in a hypercholesterolemic model of CKD. We placed CD36-deficient mice and wild-type male mice on a high-fat Western diet for 7 to 8 wk and then performed either sham or unilateral ureteral obstruction surgery. CD36-deficient mice developed significantly less fibrosis compared with wild-type mice at days 3, 7, and 14 after obstruction. Compared with wild-type mice, CD36-deficient mice had significantly more interstitial macrophages at 7 d but not at 14 d. CD36-deficient mice exhibited reduced levels of activated NF-kappaB and oxidative stress (assessed by measuring fatty acid-derived hydroxyoctadecadienoic acid and protein carbonyl content) and decreased accumulation of interstitial myofibroblasts compared with wild-type mice. These data suggest that CD36 is a key modulator of proinflammatory and oxidative pathways that promote fibrogenesis in CKD.

Figure 1.

Total kidney collagen content determined by hydroxyproline assay is significantly decreased in obstructed kidneys from hypercholesterolemic CD36−/− mice compared with CD36+/+ mice. ▪, CD36+/+ mice (n = 6 to 7); □, CD36−/− mice (n = 8 to 10). Data are means ± SEM. ‡P < 0.05, †P < 0.01, CD36+/+ versus CD36−/−.

Figure 2.

(A) Renal E-cadherin Western blot (top) illustrates the changes in E-cadherin protein levels (120 kD) on days 7 and 14 after UUO. E-cadherin expression was normalized to β-actin. The graph summarizes the results of single-band density measurements, expressed as β-actin–normalized E-cadherin band levels. Data are means ± SEM. †P < 0.01, CD36+/+ versus CD36−/−. (B) Representative E-cadherin immunohistochemistry photomicrographs demonstrate increased tubular localization of E-cadherin in CD36−/− mice 14 d after UUO. Magnification, ×400.

Figure 3.

F4/80⁺ interstitial macrophage area is higher in CD36−/− mice 7 d after UUO despite less fibrosis. (Top) Representative F4/80 immunohistochemistry photomicrographs (400x) of CD36+/+ and CD36−/− mice at day 7 after UUO. (Bottom) Summary of the percentage F4/80⁺ interstitial area ± SEM. †P < 0.01, CD36+/+ versus CD36−/−.

Figure 4.

Renal chemokine expression in CD36−/− mice after UUO. (A) Representative RNAse protection assay blot of CD36+/+ and CD36−/− mice at day 7 after UUO. (B through E) Graphs summarize the results of band density measurements normalized against the ribosomal protein L32 band. Data are means ± SEM. ‡P < 0.05, †P < 0.01, CD36+/+ versus CD36−/−.

Figure 5.

Proinflammatory pathway attenuation in CD36−/− mice after UUO. (A) Representative Western blot of phosphorylated IκB-α and total IκB-α in CD36+/+ and CD36−/− mice at day 3 and day 7 after UUO. (B) Summary of results of phosphorylated IκB-α to total IκB-α ratios. (C) Representative EMSA illustrates translocated NF-κB heterodimer levels on pooled nuclear extracts from day 7 UUO total kidney homogenate. Arrows indicate specific NF-κB bands that were quantified for individual samples. Phorbol myristate acetate (PMA)-stimulated and unstimulated Jurkat T cells were used as positive and negative controls, respectively. The graph summarizes the results of NF-κB EMSA results performed using individual mouse samples (n = 7 to 8 per group; P = 0.02). (D) Representative supershift assay with p65 and p50 antibodies performed to identify NF-κB heterodimers in nuclear extract isolated 7 d after UUO. (E and F) Representative phosphorylated p65 immunohistochemistry photomicrographs in CD36+/+ and CD36−/− mice on day 7 after UUO. Arrows indicate p65⁺ nuclei in tubular and interstitial cells. Data are means ± SEM. ‡P < 0.05, CD36+/+ versus CD36−/−. Magnification, ×400.

Figure 6.

Kidney oxLDL levels are reduced in CD36−/− mice after UUO. (Top) Representative HOCl-LDL/oxLDL immunohistochemical photomicrographs in CD36+/+ and CD36−/− mice at day 14 after UUO. (Bottom) Summary of the quantification results obtained by computer-assisted image analysis expressed as the percentage of tubulointerstitial area staining positive for HOCl-LDL. Data are means ± SEM. †P < 0.01, CD36+/+ versus CD36−/−. Magnification, ×400.

Figure 7.

Reduction in total oxidative stress in obstructed kidneys of CD36−/− mice after UUO. Protein oxidation was measured as total protein carbonyl content by ELISA. Lipid peroxidation was measured as HODE by RP-HPLC. The levels of HODE and protein carbonyl content were reduced at day 7 after UUO in CD36+/+ and CD36−/− mice. Linear regression demonstrated a strong positive correlation between HODE levels and protein carbonyl content and indicated much lower levels in CD36−/− mice 7 d after UUO (r² = 0.8374; F = 139.1, P < 0.01). ♦, CD36+/+ sham-operated (n = 7); ▴, CD36−/− sham-operated (n = 7); ▪, CD36+/+ day 7 UUO (n = 7); •, CD36−/− day 7 UUO (n = 8).

Figure 8.

Reduction in fibrosis severity after UUO in CD36−/− mice is independent of TGF-β activation. (Top) Representative Western blot of phosphorylated Smad2 (pSmad2) and total Smad2 for days 3 and 7 after UUO in CD36+/+ and CD36−/− mice. (Bottom) Summary of the results of band density measurements for pSmad2 normalized to total Smad2. Data are means ± SEM. P > 0.05, CD36+/+ versus CD36−/−.

Figure 9.

Myofibroblast accumulation is attenuated in CD36−/− mice after UUO. (Top) Representative α-SMA immunohistochemical photomicrographs in CD36+/+ and CD36−/− mice 14 d after UUO. (Bottom) Summary of the quantification by computer-assisted image analysis, expressed as the percentage of tubulointerstitial area staining positive for α-SMA. Data are as means ± SEM. †P < 0.01, CD36+/+ versus CD36−/−. Magnification, ×400.

Figure 10.

CXCL10 levels are increased in CD36−/− mice after UUO. (Top) Representative CXCL10/IP-10 immunohistochemical photomicrographs in CD36+/+ and CD36−/− mice 7 d after UUO. (Bottom) Summary of the quantification by computer-assisted image analysis, expressed as the percentage of tubulointerstitial area staining positive for CXCL10. Data are means ± SEM. †P < 0.01, CD36+/+ versus CD36−/−. Magnification, ×400.

Figure 11.

Attenuated renal fibrosis in CD36−/− mice is independent of cholesterol levels. Total kidney collagen content determined by hydroxyproline assay is significantly decreased in obstructed kidneys from CD36−/− mice compared with CD36+/+ mice on a control diet. There was no significant difference between CD36−/− mice on a control diet and a high-fat diet after UUO. , CD36+/+ mice on control diet (n = 6 to 7); ▒, CD36−/− mice on control diet (n = 7 to 8); ▪, CD36+/+ mice on high-fat diet (n = 6 to 7); □, CD36−/− mice on high-fat diet (n = 8 to 10). Data are means ± SEM. †P < 0.01, CD36+/+ versus CD36−/−.