SARS-CoV-2 N protein induces acute kidney injury in diabetic mice via the Smad3-Ripk3/MLKL necroptosis pathway

Abstract

Kidney is one of major organs attacked by SARS-CoV-2, resulting in acute kidney injury (AKI) in critically ill COVID-19 patients, especially in the elderly and diabetic patients with diabetic kidney disease (DKD). 1 , 2 Among SARS-CoV-2 proteins, the N protein can be detectable in damaged tubules in COVID-19 patients with AKI. 2 , 3 However, the role and mechanisms of N protein-induced AKI in diabetes remain unclear. By using ultrasound-microbubble-mediated kidney-specific gene transfer technique, we found that SARS-CoV-2 N protein overexpression could dose-and time-dependently induce AKI in non-diabetic mice (db/m), showing tubular necrosis with renal dysfunction including a marked increase in blood urea nitrogen (BUN) and creatinine, which was further increased in diabetic db/db mice at age of 8 weeks and became much more severe in those with older age at 16 and 32 weeks with the development of DKD (Fig. 1a–d , and supplementary Figs. S1 and S2 ). In addition, kidney overexpression of the N protein also upregulated kidney injury molecule 1 (Kim1), a biomarker for AKI, in db/db mice, especially in those with DKD over 16–32 weeks (Fig. 1e, f , and supplementary Fig. S3a ), although there was no difference in expression of renal SARS-CoV-2 N mRNA in db/m and db/db mice at different groups (Fig. 1g ). These findings reveal that the SARS-CoV-2 N protein is pathogenic in AKI and is capable of promoting more severe AKI in db/db mice, especially in aged db/db with underlying DKD. Fig. 1 SARS-CoV-2 N Protein induces AKI under diabetic conditions via the Smad3-Ripk3/MLKL necroptosis pathway. a Ultrasound-microbubble-mediated kidney-specifically overexpressing SARS-CoV-2 N protein can induce severe tubular necrosis (*) in db/db mice, particularly in those with older age db/db mice (HE-staining, scale bar = 50 μm). b Semi-quantitative analysis of tubular necrosis in db/m and db/db mice with or without overexpressing SARS-CoV-2 N protein at the age of 8, 16 and 32 weeks. c , d Serum levels of creatinine and BUN in db/m and db/db mice with or without overexpressing SARS-CoV-2 N protein at the age of 8, 16, 32 weeks. e Quantitative analysis of renal Kim1 protein expression in db/m and db/db mice with or without overexpressing SARS-CoV-2 N protein at the age of 8, 16, 32 weeks. f , g Quantitative real-time PCR for Kim1 mRNA and SARS-CoV-2 N mRNA expression in db/m and db/db mice with or without overexpressing SARS-CoV-2 N protein at the age of 8, 16, 32 weeks. h Quantitative real-time PCR for Ripk3 and MLKL mRNA expression in 16-week-old db/m and db/db mice with or without overexpressing SARS-CoV-2 N protein. i Western blot analysis of Flag-NP, p-Smad3, Ripk3, p-MLKL and MLKL expression in the kidneys of 16-week-old db/m and db/db mice with or without overexpressing SARS-CoV-2 N protein. j Co-localization between SARS-CoV-2 N protein (green) and p-Smad3 (red) in the AKI kidney (scale bar = 50 μm, and scale bar = 25 μm in amplified images). k Co-localization between p-MLKL (green) and p-Smad3 (red) in the AKI kidney (scale bar = 50 μm). l Interaction between Flag-SARS-CoV-2 N protein and Smad3 in HK-2 cells by Co-IP. m Western blot analysis shows that overexpression of SARS-CoV-2 N protein induces and promotes Smad3 phosphorylation and nuclear translocation in HK-2 cells under high AGE conditions (50 μg/ml for 30 mins). n , o ChIP assay for detecting the binding of Smad3 to Ripk3 and MLKL promoter region in response to AGE (50 μg/ml). p Quantitative real-time PCR for Ripk3 and MLKL mRNA expression in HK-2 cells. q Western blot analysis of Ripk3, p-MLKL and MLKL protein expression in HK-2 cells with or without SARS-CoV-2 N protein overexpression and AGE stimulation (50 μg/ml for 6 h). r Treatment with SIS3 (10 μM) blocks SARS-CoV-2 N protein-induced activation of Smad3 (p-Smad3) and Ripk3/MLKL signaling in HK-2 cells under high AGE stimulation (50 μg/ml) conditions. s Renal pathological changes at the age of 16-week-old Smad3 KO-db/m, Smad3 WT-db/m, Smad3 KO-db/db, and Smad3WT-db/db mice with overexpression of SARS-CoV-2 N protein (HE-staining, scale bar = 50 μm). t , u Semi-quantitative analysis of tubular necrosis (*) and serum creatinine in Smad3 KO/WT-db/m or Smad3 KO/WT-db/db mice with SARS-CoV-2 N protein overexpression. v Western blot analysis of p-Smad3, Ripk3, p-MLKL and MLKL expression in Smad3 KO/WT-db/m or Smad3 KO/WT-db/db mice with overexpressing SARS-CoV-2 N protein. w Therapeutic effect of SIS3 (10 mg/kg, daily) on SARS-CoV-2 N-induced renal pathology in db/m and db/db mice (HE-staining, * tubular necrosis, scale bar = 50 μm). x , y Therapeutic effect of SIS3 on SARS-CoV-2 N-induced tubular necrosis and serum creatinine in db/m and db/db. z Western blot analysis shows the inhibitory effect of SIS3 on SARS-CoV-2 N protein-induced p-Smad3, p-MLKL, and expression of Ripk3 and MLKL in the kidney of db/m and db/db mice. The data represents as the mean ± SEM for at least 3 independent experiments in vitro or for groups of 6 mice in vivo. G Glomerulus, S3KO Smad3 knockout mice, S3WT Smad3 wild-type mice, 8w 8 weeks, 16w 16 weeks, 32w 32 weeks; *, necrotic tubules. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. VC group; # P < 0.05, ## P < 0.01, ### P < 0.001 as indicated; † P < 0.05, †† P < 0.01, ††† P < 0.001 vs. AGE + VC, S3WT + NP, or NP + DMSO group Full size image

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