Podocyte OTUD5 alleviates diabetic kidney disease through deubiquitinating TAK1 and reducing podocyte inflammation and injury

Abstract

Recent studies have shown the crucial role of podocyte injury in the development of diabetic kidney disease (DKD). Deubiquitinating modification of proteins is widely involved in the occurrence and development of diseases. Here, we explore the role and regulating mechanism of a deubiquitinating enzyme, OTUD5, in podocyte injury and DKD. RNA-seq analysis indicates a significantly decreased expression of OTUD5 in HG/PA-stimulated podocytes. Podocyte-specific Otud5 knockout exacerbates podocyte injury and DKD in both type 1 and type 2 diabetic mice. Furthermore, AVV9-mediated OTUD5 overexpression in podocytes shows a therapeutic effect against DKD. Mass spectrometry and co-immunoprecipitation experiments reveal an inflammation-regulating protein, TAK1, as the substrate of OTUD5 in podocytes. Mechanistically, OTUD5 deubiquitinates K63-linked TAK1 at the K158 site through its active site C224, which subsequently prevents the phosphorylation of TAK1 and reduces downstream inflammatory responses in podocytes. Our findings show an OTUD5-TAK1 axis in podocyte inflammation and injury and highlight the potential of OTUD5 as a promising therapeutic target for DKD.

Fig. 1. Identification of OTUD5 as a regulator of podocyte inflammation and injury.

a A volcano plot analysis illustrating the differential expression of DUBs induced by HG/PA in podocytes. (n = 3 samples for each Ctrl group and HG/PA group; P values were determined by Wald test from DESeq2 software with Benjamini-Hochberg’s correction). b A table shows DUBs with significant differences in MPC5 cells treated with HG/PA. c The mRNA level of Otud5 in HG/PA-induced MPC5 cell lines. (n = 3 independent experiments; P values were determined by two-tailed unpaired t-test and data are presented as mean ± SD). d Representative western blot of OTUD5 expression in MPC5 cell lines after stimulation with HG/PA for different durations. (n = 3 independent experiments). e Representative immunofluorescence (IF) images of OTUD5 expression in human renal tissue from normal subjects (n = 3 samples) and patients with DKD (n = 3 samples). Scale bar, 50 μm. Representative western blot of OTUD5 expression in renal cortex of T2DM (f) mice and T1DM mice (g). (n = 6 samples). Representative western blot of OTUD5 expression in renal cortex of NOD (h) mice and db/db mice (i). (n = 6 samples). j, k Representative western blot of OTUD5 expression in primary podocytes of T2DM (j) and T1DM (k) mice. (n = 6 samples). l MPC5 cells transfected with Flag-OTUD5 were stimulated with HG/PA for 8 h. Real-time qPCR showed the mRNA levels of Il6 and Tnfα. (n = 3 independent experiments; P values were determined by one-way ANOVA with Bonferroni’s correction and data are presented as mean ± SD). Representative western blot of Cleaved Caspase3 (m) and Nephrin (n) expression in OTUD5-overexpression podocytes stimulated by HG/PA for 24 h. (n = 3 independent experiments).

Fig. 2. Podocyte-specific Otud5 knockout aggravates podocyte injury and DKD in T2DM mice.

a Schematic diagram of the strategy for the generation of podocyte-specific Otud5 knockout mice (OTUD5CKO). b Schematic diagram depicting the procedure of STZ/HFD-induced T2DM mice. c Weekly monitoring of blood glucose levels in mice. Data are presented as mean ± SD. The levels of serum creatinine (d), urea nitrogen (e), and urine albumin to creatinine ratio (f) were analyzed in mice. g, h Representative images of hematoxylin and eosin staining (H&E), periodic acid-Schiff (PAS), and transmission electron microscopy (TEM) in mice. Scale bar: black 20 μm, red 1 μm. (n = 6 samples). Quantification of glomerular basement membrane (GBM) thickness (i) and podocyte foot process numbers (j, k) in the glomeruli. l Representative immunofluorescence (IF) images of Nephrin expression in glomeruli from mice. Scale bar, 20 μm. (n = 6 samples). Real-time qPCR showing mRNA levels of Il6 (m) and Tnfα (n) in kidney tissues of each group. n = 6 for each group. For d–f, i–k, m, and n, P values were determined by one-way ANOVA with Bonferroni’s correction, and data are presented as mean ± SD.

Fig. 3. OTUD5CKO exacerbates podocyte injury and DKD in T1DM mice.

a Weekly monitoring of blood glucose levels in mice. Data are presented as mean ± SD. The levels of serum creatinine (b), urea nitrogen (c), and urine albumin to creatinine ratio (d) were analyzed in mice. e, f Representative images of H&E, PAS, and TEM in mice. Scale bar: black 20 μm, red 1 μm. (n = 6 samples). Quantification of GBM thickness (g) and podocyte foot process numbers (h, i) in the glomeruli. j Representative IF images of Nephrin expression in glomeruli from mice. Scale bar, 20 μm. (n = 6 samples). Real-time qPCR showing mRNA levels of Il6 (k) and Tnfα (l) in the kidney tissues of each group. n =6 for each group. For b–d, g–i, k, and l, P values were determined by one-way ANOVA with Bonferroni’s correction, and data are presented as mean ± SD.

Fig. 4. Identification of TAK1 as a potential substrate protein of OTUD5.

a Schematic illustration of a quantitative proteomic screen to identify proteins binding to OTUD5. b Mass spectrometry/mass spectrometry (MS/MS) spectrum of the peptide MITTSGPTSEK from TAK1. Co-immunoprecipitation (Co-IP) of OTUD5 and TAK1 in MPC5 cells (c) and kidney tissues (d). Endogenous OTUD5 was immunoprecipitated. (n = 3 independent experiments). e Co-IP of OTUD5 in NIH/3T3 co-transfected with Flag-OTUD5 and His-TAK1 plasmids. Exogenous OTUD5 was immunoprecipitated using an anti-Flag antibody. (n = 3 independent experiments). f His-TAK1 and Flag-OTUD5 were transfected into MPC5 cells with or without HG/PA treatment and then subjected to 10 μM MG132 for 6 h. Ubiquitinated TAK1 was detected by immunoblotting using an anti-ubiquitin antibody. (n = 3 independent experiments). g His-TAK1, HA-WT Ub, HA-K48 Ub, and HA-K63 Ub were transfected into NIH/3T3 together with Flag-OTUD5 and then subjected to 10 μM MG132 for 6 h. Ubiquitinated TAK1 was detected by immunoblotting using an anti-HA antibody. (n = 3 independent experiments). h His-TAK1 and HA-K63 Ub were transfected into NIH/3T3 together with Flag-OTUD5 (WT or C224S) and then subjected to 10 μM MG132 for 6 h. Ubiquitinated TAK1 was detected by immunoblotting using an anti-HA antibody. (n = 3 independent experiments). i Schematic illustration of the construct for mutating the ubiquitinated lysine residue of TAK1. j His-TAK1 (WT, K34R, K158R, K209R or K562R) and HA-WT Ub were transfected into NIH/3T3 together with Flag-OTUD5 and then subjected to 10 μM MG132 for 6 h. Ubiquitinated TAK1 was detected by immunoblotting using an anti-HA antibody. (n = 3 independent experiments).

Fig. 5. OTUD5 negatively regulates TAK1 activation and inflammation in podocytes.

MPC5 cells transfected with Flag-OTUD5 (a) or si-OTUD5 (b) were stimulated with HG/PA for 30 min. Representative western blot analysis of P-TAK1. (n = 3 independent experiments). c, d Representative western blot analysis of P-TAK1 in kidney tissues of each group. (n = 6 samples). MPC5 cells transfected with Flag-OTUD5 (e) or si-OTUD5 (f) were stimulated with HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of ERK, P38, and JNK. (n = 3 independent experiments). MPC5 cells transfected with si-OTUD5 were pretreated with 10 μM Takinib (TAK1 inhibitor) for 1 h before exposure to HG/PA. g Levels of P-TAK1, P-ERK, P-P38, and P-JNK were detected by western blot. h Real-time qPCR showing mRNA levels of Il6 and Tnfα. (n = 3 independent experiments; P values were determined by one-way ANOVA with Bonferroni’s correction and data are presented as mean ± SD). i His-TAK1 was transfected into NIH/3T3 with or without Flag-OTUD5 (WT or C224A). Co-IP was performed with an anti-His antibody, followed by a western blot of TAK1 and TAB2. (n = 3 independent experiments). j MPC5 cells transfected with Flag-OTUD5 (WT or C224A) were stimulated with HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of TAK1, ERK, P38, and JNK. (n = 3 independent experiments). k His-TAK1(WT or K158R) was transfected into NIH/3T3 with or without Flag-OTUD5. Co-IP was performed with an anti-His antibody, followed by a western blot of TAK1 and TAB2. (n = 3 independent experiments). l His-TAK1 (WT or K158R) and Flag-OTUD5 were transfected into MPC5 cells for 24 h and then stimulated by HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of TAK1, ERK, P38, and JNK. (n = 3 independent experiments).

Fig. 6. Inhibition of TAK1 eliminates the aggravated podocyte injury and DKD in OTUD5CKO-T2DM mice.

a A schematic diagram illustrating the procedure of T2DM-induced OTUD5CKO and Otud5^fl/fl mice, with or without Takinib administration. b Weekly measurements of blood glucose levels of the mice in the indicated groups. Data are presented as mean ± SD. c Representative western blot images showing phosphorylated and total protein levels of TAK1, ERK, P38, and JNK in the kidneys of indicated groups. (n = 6 samples). Serum creatinine (d), urea nitrogen (e), and urine albumin to creatinine ratio (f) levels of the mice in the indicated groups. g Representative H&E and PAS staining images of kidney sections. Scale bar: 20 μm. (n = 6 samples). h–k Representative TEM images of kidney sections and corresponding quantitative analysis. Scale bar: 1 μm. l Representative IF images of Nephrin expression in the glomeruli of the indicated groups. Scale bar, 20 μm. n = 6 for each group. For d–f, i, j, and k, P values were determined by one-way ANOVA with Bonferroni’s correction, and data are presented as mean ± SD.

Fig. 7. Podocyte-specific overexpression of OTUD5 alleviates podocyte injury and DKD in T2DM mice.

a A schematic diagram illustrating the procedure of T2DM-induced Otud5^fl/fl mice injected with AAV-EV or AAV-OTUD5. b The levels of blood glucose. Data are presented as mean ± SD. c Ubiquitinated TAK1 was detected by immunoblotting using anti-ubiquitin antibodies in the kidneys of indicated groups. (n = 6 samples). d Representative western blot images showing phosphorylated and total protein levels of TAK1 in the kidneys of indicated groups. (n = 6 samples). The levels of serum creatinine (e), urea nitrogen (f), and urine albumin to creatinine ratio (g). h Representative H&E and PAS staining of kidney sections. (n =6 samples). i–l Representative TEM images of kidney sections and corresponding quantitative analysis. Scale bar: 1 μm. m Representative IF images of Nephrin expression. Scale bar, 20 μm. (n = 6 samples). n = 6 for each group. For e–g and j–l, P values were determined by a two-tailed unpaired t-test, and data are presented as mean ± SD.