C3a receptor blockade protects podocytes from injury in diabetic nephropathy
Marina Morigi, Luca Perico, Daniela Corna, Monica Locatelli, Paola Cassis, Claudia Elisa Carminati, Silvia Bolognini, Carlamaria Zoja, Giuseppe Remuzzi, Ariela Benigni, Simona Buelli
Marina Morigi, Luca Perico, Daniela Corna, Monica Locatelli, Paola Cassis, Claudia Elisa Carminati, Silvia Bolognini, Carlamaria Zoja, Giuseppe Remuzzi, Ariela Benigni, Simona Buelli
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Research Article Nephrology

C3a receptor blockade protects podocytes from injury in diabetic nephropathy

Abstract

Renal activation of the complement system has been described in patients with diabetic nephropathy (DN), although its pathological relevance is still ill-defined. Here, we studied whether glomerular C3a, generated by uncontrolled complement activation, promotes podocyte damage, leading to proteinuria and renal injury in mice with type 2 diabetes. BTBR ob/ob mice exhibited podocyte loss, albuminuria, and glomerular injury accompanied by C3 deposits and increased C3a and C3a receptor (C3aR) levels. Decreased glomerular nephrin and α-actinin4 expression, coupled with integrin-linked kinase induction, were also observed. Treatment of DN mice with a C3aR antagonist enhanced podocyte density and preserved their phenotype, limiting proteinuria and glomerular injury. Mechanistically, ultrastructural and functional mitochondrial alterations, accompanied by downregulation of antioxidant superoxide dismutase 2 (SOD2) and increased protein oxidation, occurred in podocytes and were normalized by C3aR blockade. In cultured podocytes, C3a induced cAMP-dependent mitochondrial fragmentation. Alterations of mitochondrial membrane potential, SOD2 expression, and energetic metabolism were also found in response to C3a. Notably, C3a-induced podocyte motility was inhibited by SS-31, a peptide with mitochondrial protective effects. These data indicate that C3a blockade represents a potentially novel therapeutic strategy in DN for preserving podocyte integrity through the maintenance of mitochondrial functions.

Authors

Marina Morigi, Luca Perico, Daniela Corna, Monica Locatelli, Paola Cassis, Claudia Elisa Carminati, Silvia Bolognini, Carlamaria Zoja, Giuseppe Remuzzi, Ariela Benigni, Simona Buelli

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Figure 5

C3a affects mitochondrial functional integrity in cultured podocytes.

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C3a affects mitochondrial functional integrity in cultured podocytes. (A...
(A) Representative images of mitochondria labeled with MitoTracker in human podocytes exposed to control medium or C3a (1 μM) for 6 hours. Nuclei were counterstained with Hoechst (blue). Scale bars: 20 μm. The percentage of podocytes with an altered mitochondrial pattern, in terms of fragmentation and perinuclear redistribution, on total cells per field was quantified. Results are expressed as mean ± SEM (n = 4), and unpaired Student’s t test was used. **P < 0.01. (B) Quantification of mitochondrial networks, individual mitochondria, and mitochondrial area by Mitochondrial Network Analysis (MiNA) tool set in control or C3a-treated podocytes labeled with MitoTracker. Results are expressed as mean ± SEM (n = 4), and unpaired Student’s t test was used. *P < 0.05, **P < 0.01. (C) Western blot and densitometric analysis of Drp1 protein expression in purified mitochondrial fraction isolated from podocytes after incubation with control medium or C3a for 6 hours. VDAC protein expression was used as a sample loading control. Results are expressed as mean ± SEM (n = 6), and unpaired Student’s t test was used. *P < 0.05. (D) Representative images and quantification of SOD2 expression (red) in control or C3a-treated podocytes for 6 hours. Nuclei were stained with DAPI (blue) Scale bars: 20 μm. Results are expressed as mean ± SEM (n = 3), and unpaired Student’s t test was used. **P < 0.01. (E) Representative images and quantification of mitochondrial membrane potential in podocytes exposed for 6 hours to control medium or C3a. Mitochondrial potential was evaluated by staining with JC-1, a dye sensitive to mitochondrial membrane potential changes that shifts the emission spectrum from red (mitochondrial distribution, JC-1mit) to green (cytoplasmic distribution, JC-1 cyt). Nuclei were counterstained with Hoechst (blue). Mitochondrial membrane potential was evaluated as the ratio between red and green fluorescent areas and normalized for total cells/field. Scale bars: 20 μm. Results are expressed as mean ± SEM (n = 4), and unpaired Student’s t test was used. **P < 0.01.