Mitochondrial Dysfunction in Podocytes Caused by CRIF1 Deficiency Leads to Progressive Albuminuria and Glomerular Sclerosis in Mice

Abstract

Recent studies have implicated mitochondrial disruption in podocyte dysfunction, which is a characteristic feature of primary and diabetic glomerular diseases. However, the mechanisms by which primary mitochondrial dysfunction in podocytes affects glomerular renal diseases are currently unknown. To investigate the role of mitochondrial oxidative phosphorylation (OxPhos) in podocyte dysfunction, glomerular function was examined in mice carrying a loss of function mutation of the gene encoding CR6-interacting factor-1 (CRIF1), which is essential for intramitochondrial production and the subsequent insertion of OxPhos polypeptides into the inner mitochondrial membrane. Homozygotic deficiency of CRIF1 in podocytes resulted in profound and progressive albuminuria from 3 weeks of age; the CRIF1-deficient mice also developed glomerular and tubulointerstitial lesions by 10 weeks of age. Furthermore, marked glomerular sclerosis and interstitial fibrosis were observed in homozygous CRIF1-deficient mice at 20 weeks of age. In cultured mouse podocytes, loss of CRIF1 resulted in OxPhos dysfunction and marked loss or abnormal aggregation of F-actin. These findings indicate that the OxPhos status determines the integrity of podocytes and their ability to maintain a tight barrier and control albuminuria. Analyses of the glomerular function of the podocyte-specific primary OxPhos dysfunction model mice demonstrate a link between podocyte mitochondrial dysfunction, progressive glomerular sclerosis, and tubulointerstitial diseases.

Keywords: CRIF1; albuminuria; glomerular sclerosis; mitochondrial oxidative phosphorylation; podocyte.

Figure 1

Podocyte-specific CRIF1 deficiency leads to massive albuminuria and azotemia in mice. (A) Podocyte-specific CRIF1 knockout mice were generated by crossing CRIF1^flox/flox mice with podocin-Cre mice. PCR analyses were performed to genotype the CRIF1-loxP alleles and Cre transgene. (B) Body weights of the male (M; n = 10) and female (F; n = 10) CRIF1^pdKO and CRIF1^ctrl mice, respectively. (C) Survival rates of the CRIF1^pdKO and CRIF1^ctrl mice (n = 20 per group). (D,E) Blood urea nitrogen (D) and serum creatinine (E) levels in the CRIF1^pdKO and CRIF1^ctrl mice (F) albuminuria levels in the CRIF1^pdKO and CRIF1^ctrl mice (B,D–F) Data are represented as the mean ± SD. (D–F) * p < 0.05 by Student’s t-tests.

Figure 2

Podocyte-specific CRIF1 deficiency leads to glomerular sclerosis and tubulointerstitial fibrosis in mice. Hematoxylin and eosin staining (upper panels) and Masson’s Trichrome staining (lower panels) of the renal cortex in CRIF1^pdKO and CRIF1^ctrl mice at 3, 5, 10, and 20 weeks of age. Large black boxes show higher magnifications of the small black boxed areas in the larger panels. Scale bars, 20 μm (large panels) and 7 μm (small panels). The yellow and black arrows in the panels showing sections from 10- and 20-week-old CRIF1^pdKO mice indicate the occurrence of glomerular sclerosis and proteinaceous casts, respectively. The glomerular sclerosis scores and interstitial fibrosis areas in kidneys from 10- and 20-week-old CRIF1^ctrl and CRIF1^pdKO mice. Data are represented as the mean ± SD. * p < 0.05 by Student’s t-tests.

Figure 3

Transmission electron micrographs of renal sections from CRIF1^ctrl and CRIF1^pdKO mice at 3, 5, 10, and 20 weeks of age, and the foot process width of mice kidney at 5 weeks of age. White arrows in the panels showing 3-, 5-, 10-, and 20-week-old mice indicate foot process with normal structure. White boxes in the panel showing section from 3-week-old mice indicate mitochondria. The yellow arrows in the panel showing section from 5-week-old and 10-week-old CRIF1^pdKO mice indicate foot process fusion and effacement, and microvillous transformation. The red and black arrows in the panel showing a section from a 20-week-old CRIF1^pdKO mouse indicate extensive effacement and microvillous transformation of the foot processes, and podocyte degeneration and vacuolization, respectively. Yellow scale bar, 2 μm. White scale bar, 1 μm.

Figure 4

Podocyte-specific CRIF1 deficiency causes mitochondrial structural abnormalities. Transmission electron micrographs of renal cortex sections from 10-week-old CRIF1^ctrl mice (A) and CRIF1^pdKO (B) mice. The smaller panels show higher magnifications of the boxed areas in the larger panels. The white arrows indicate mitochondria. The yellow arrows indicate cristae of mitochondria Scale bars, 2 μm (large panels) and 0.5 μm (small panels).

Figure 5

Deletion of CRIF1 causes mitochondrial dysfunction in podocytes. (A) Immunofluorescent staining of endogenous CRIF1 in an immortalized mouse podocyte. MitoTracker Red and DAPI were used to stain the mitochondria and nucleus, respectively. Scale bar, 20 μm. (B) Immunoblot analysis (left and middle panels) of CRIF1 protein expression and qRT-PCR analysis of Crif1 mRNA expression in podocytes transfected with a scrambled control siRNA (scCONT) or a CRIF1-specific siRNA (siCRIF1). The expression levels of CRIF1 protein and Crif1 mRNA were normalized to those of actin and GAPDH, respectively. Data are represented as the mean ± SD of n = triple replicates. (C) Immunoblot analyses of mitochondrial protein complexes I–IV. The expression levels of the complexes were normalized to those of actin. Data are represented as the mean ± SD of n = triple replicates. (D) The OCRs in non-transfected (CONT), control siRNA-transfected (scCONT), and CRIF1-specific siRNA-transfected podocytes after the addition of oligomycin (Oligo; 2 μg/mL), CCCP (10 μM) and rotenone (Rote; 1 μM). * p < 0.05 (B,C) by Student’s t-tests.

Figure 6

Deletion of CRIF1 causes loss and aggregation of F-actin in podocytes. (A) Immunoblot analyses of F-actin, α–actinin-4, synaptopodin, cofilin, ZO-1, and nephrin in control siRNA- and CIRF1-specific siRNA-transfected podocytes. Data are represented as the mean ± SD of n = triple replicates. (B) Immunofluorescent staining of phalloidin (F-actin) in control siRNA- and CIRF1-specific siRNA-transfected podocytes. Mitochondria were stained using MitoTracker Red. (C) Immunofluorescent staining of phalloidin (F-actin) and nephrin in kidneys from 10-week-old CRIF1^ctrl and CRIF1^pdKO mice. * p < 0.05, ** p < 0.01 by Student’s t-tests.