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, 2 (2), e45

Multiple Metabolic Hits Converge on CD36 as Novel Mediator of Tubular Epithelial Apoptosis in Diabetic Nephropathy

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Multiple Metabolic Hits Converge on CD36 as Novel Mediator of Tubular Epithelial Apoptosis in Diabetic Nephropathy

Katalin Susztak et al. PLoS Med.

Abstract

Background: Diabetic nephropathy (DNP) is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED) and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood.

Methods and findings: We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC) apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced by glycated albumins (AGE-BSA and CML-BSA) and free fatty acid palmitate through sequential activation of src kinase, and proapoptotic p38 MAPK and caspase 3. In contrast, paucity of expression of CD36 in PTECs in diabetic mice with diabetic glomerulopathy was associated with normal tubular epithelium and the absence of tubular apoptosis. Mouse PTECs lacked CD36 and were resistant to AGE-BSA-induced apoptosis. Recombinant expression of CD36 in mouse PTECs conferred susceptibility to AGE-BSA-induced apoptosis.

Conclusion: Our findings suggest a novel role for CD36 as an essential mediator of proximal tubular apoptosis in human DNP. Because CD36 expression was induced by glucose in PTECs, and because increased CD36 mediated AGE-BSA-, CML-BSA-, and palmitate-induced PTEC apoptosis, we propose a two-step metabolic hit model for TED, a hallmark of progression in DNP.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Differential Localization and Expression of CD36 Protein in Kidneys of Diabetic Mice with Glomerulopathy and of Humans with DNP
(A and B) Indirect double-immunofluorescence labeling of kidney sections from non-diabetic control (A) and diabetic (B) mice with anti-CD36 (green) and proximal tubular marker anti-aquaporin1 (red). (C and D) Double labeling of non-diabetic control mice with anti-CD36 (green) and loop-of-Henle marker sodium potassium chloride cotransporter anti-NKCC (red) (C) and collecting duct marker aquaporin2 (red) (D) (arrow depicts colocalization of anti-CD36 and anti-aquaporin2 staining). (E and F) Double labeling of human kidney sections from control individuals (E) and individuals with diabetes with DNP (F) using anti-CD36 (green) and anti-aquaporin1 (red). (G) Higher-magnification image of (F) with arrows depicting colocalization of anti-CD36 and anti-aquaporin1. (Note that anti-CD36 labeling is heterogeneous: staining is isolated proximal tubular cells.) (H–J) Representative images of anti-CD36 immunoperoxidase staining of sections of normal human kidney (H), human kidney with DNP (I), and human kidney with FSGS (J). Arrow in (I) depicts proximal tubular epithelial staining. (K) CD36 PTEC expression score derived from blinded, semi-quantitative analysis of distribution and intensity of proximal tubular CD36 staining of human biopsy samples from ten normal control, ten DNP, and ten FSGS kidneys and the result shown on a dot plot. Significance was calculated by Wilcoxon Rank Sum Test, and PTEC scores for DNP kidneys were significantly different from those of FSGS kidneys and normal human kidneys.
Figure 2
Figure 2. TED and IF Coincide with Proximal Tubular Apoptosis and CD36 Expression in Human DNP
(A and B) Periodic Acid–Schiff staining of diabetic mouse kidney (28-wk-old C57BLKS/J-leprdb/db) (A) and human DNP kidney (B). Arrowheads denote glomeruli with advanced mesangial expansion and glomerulosclerosis; arrows depict normal proximal tubule in diabetic mouse (A) and TED in human with DNP (B). (C) TUNEL assay (green) and anti-CD36 (red) double labeling of human DNP. Arrows indicate apoptotic, CD36-positive tubular epithelial cells. (D) TUNEL assay (green) and anti-aquaporin1 (red) double labeling of human DNP. Arrows depict TUNEL-positive and aquaporin1-positive PTECs. (E) Dot plot indicates the number of TUNEL-positive tubular cells per 100 total tubular cells in kidneys of control (CTL) and diabetic (DM) mice and humans, as indicated.
Figure 3
Figure 3. CD36 mRNA and Protein Synthesis Is Stimulated in Human, but Not in Murine, PTECs, and Is Suppressed in Murine Collecting Duct Cells by High Ambient Glucose
(A) Relative CD36 mRNA abundance determined by quantitative real-time PCR in human PTEC line HK-2 treated with 30 mM D-glucose (open bars) or control L-glucose (black bars) for 4 and 24 h following maintenance of cells in 5 mM D-glucose medium. Bars represent mean ± SEM of three to five repeat experiments. Numbers on top of bars indicate significant p-values of experimental groups relative to 0 h. (B) Bar graphs show experiment as described under (A), using mouse collecting duct cell line M1 instead of human HK-2 PTECs. The relative expression of CD36 was normalized to internal control housekeeping genes HPRT and beta actin, and to baseline controls (untreated cells). (C) Relative cell surface expression of CD36 protein determined by FACS in M1 cells (open bars) and HK-2 cells (black bars) maintained in 5 mM D-glucose medium (CTL), or in medium containing 30 mM D-glucose (D-gluc) or L-glucose (L-gluc) for 72 h. (Original FACS histograms are provided in Figure S1.) Bars represent mean ± SEM of three to five repeat experiments. Numbers indicate significant p-values of experimental groups relative to control. (D) Immunoblot showing CD36 protein levels in human HK-2 PTECs maintained in control 5 mM D-glucose (CTL), or after stimulation for 72 h with 30 mM L-glucose (L-gluc) or D-glucose (D-gluc), as indicated. Tubulin is shown for loading control. All data represent at least four independent repeat experiments.
Figure 4
Figure 4. AGE-BSA, CML-BSA, and FFA PA Induce Apoptosis in Human PTECs through CD36 Signaling
Bar graphs show mean ± SEM of apoptotic nuclei, visualized by DAPI staining and normalized to 100 total cells, in human HK-2 PTECs. Data are derived from three independent repeat experiments. Numbers on top of bars indicate significant p-values of experimental groups relative to control, or as indicated by bracket. (A) Cells were treated for 48 h with control BSA (40 μM), TSP-1 (1 μg/ml), and AGE-BSA modified for 2, 5, or 10 weeks (AGE-BSA2, AGE-BSA5, and AGE-BSA10, respectively) in the absence or presence of control IgG (10 μg/ml) or anti-CD36 neutralizing antibody (10 μg/ml), as indicated. (B) Cells were treated with control BSA (40 μM), or CMLmin-BSA at 0.5, 1, 2, 5, and 10 μM, in the absence or presence of anti-CD36 neutralizing antibody, as indicated. (C) Cells were treated with monounsaturated FFA oleic acid (OA) or PA at increasing concentrations, in the absence or presence of control IgG (10 μg/ml) or anti-CD36 neutralizing antibody (10 μg/ml), as indicated.
Figure 5
Figure 5. Activation of Intracellular Pathways following AGE-BSA and PA Treatment of Human HK-2 PTECs
(A and C) Immunoblots show levels of (A) phosphorylated (Y418) src kinase and tubulin or (C) phosphorylated p38 MAPK (pp38) and total p38 MAPK (p38) in HK-2 cells treated with AGE-BSA5 (40 μM) in the absence or presence of control IgG or anti-CD36 neutralizing antibody (10 μg/ml) for different time periods, as indicated. (B and D) As shown in (A) and (C), except HK-2 cells were treated with PA (150 μM) instead of AGE-BSA5. (E and F) Bar graphs demonstrate mean ± SEM of caspase 3 activity in three independent repeat experiments. Caspase 3 activity was measured by quantitative ELISA in HK-2 cells after 18 h of stimulation with AGE-BSA5 and PA, as per manufacturer's protocol. Numbers on top of bars indicate significant p-values of experimental groups relative to control, or as indicated by brackets. (G) Bar graphs demonstrate number of apoptotic nuclei of HK-2 cells, normalized to 100 total cells, treated with AGE-BSA5 (40 μM) or PA (150 μM) in the absence (black bars) or presence of pan-caspase inhibitor (z-VAD-fmk [100 μM]; open bars), caspase 3 inhibitor (z-DEVD-fmk [20 μM]; first striped bars), caspase 9 inhibitor (z-LEHD-fmk (20 μM); gray bars), or chemical inhibitors of p38 MAPK (SB203580 [10 μM]; second striped bars). Mean ± SEM of three independent repeat experiments is presented. Numbers on top of bars indicate the significant p-values for comparison relative to control (no inhibitor).
Figure 6
Figure 6. Expression of CD36 Transgene Confers Susceptibility to AGE-BSA-Induced Apoptosis
(A–D) Representative images show DAPI (A and C) and FITC (B and D) labeling of CD36-negative MCT cells treated with 40 μM AGE-BSA5 for 24 h after co-transfection with green fluorescent protein plasmid pEGFP and pcDNA3.1 empty control vector (A and B), or pEGFP and CD36 expression plasmid pcDNA3.1/CD36 (C and D). (E) The dot plot shows results of four independent experiments where apoptotic nuclei per 100 total cells were quantitated in transfected cell cultures with or without treatments as indicated.

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