Aquaporin 1 is renoprotective in septic acute kidney injury by attenuating inflammation, apoptosis and fibrosis through inhibition of P53 expression

Abstract

Sepsis associated Acute kidney injury (AKI) is a common clinical syndrome characterized by suddenly decreased in renal function and urinary volume. This study was designed to investigate the role of Aquaporin 1 (AQP1) and P53 in the development of sepsis-induced AKI and their potential regulatory mechanisms. Firstly, transcriptome sequencing analysis of mice kidney showed AQP1 expression was reduced and P53 expression was elevated in Cecal ligation and puncture (CLP)-induced AKI compared with controls. Bioinformatics confirmed that AQP1 expression was remarkably decreased and P53 expression was obviously elevated in renal tissues or peripheral blood of septic AKI patients. Moreover, we found in vivo experiments that AQP1 mRNA levels were dramatically decreased and P53 mRNA significantly increased following the increased expression of inflammation, apoptosis, fibrosis, NGAL and KIM-1 at various periods in septic AKI. Meanwhile, AQP1 and P53 protein levels increased significantly first and then decreased gradually in kidney tissue and serum of rats in different stages of septic AKI. Most importantly, in vivo and vitro experiments demonstrated that silencing of AQP1 greatly exacerbates renal or cellular injury by up-regulating P53 expression promoting inflammatory response, apoptosis and fibrosis. Overexpression of AQP1 prevented the elevation of inflammation, apoptosis and fibrosis by down-regulating P53 expression in Lipopolysaccharide (LPS)-induced AKI or HK-2 cells. Therefore, our results suggested that AQP1 plays a protective role in modulating AKI and can attenuate inflammatory response, apoptosis and fibrosis via downregulating P53 in septic AKI or LPS-induced HK-2cells. The pharmacological targeting of AQP1 mediated P53 expression might be identified as potential targets for the early treatment of septic AKI.

Keywords: AKI; apoptosis; aquaporin 1; inflammation; p53; sepsis.

Figure 1

Identification and bioinformatic analysis of differentially expressed genes from GSE220812, GSE186822, GSE248078 and GSE248078. (A) Volcano plot of GSE220812. (B) Volcano plot of GSE186822. (C) Heatmap of GSE220812. (D) Heatmap of GSE186822. (E, F) Expression of AQP1 and P53 in renal tissues of patients with CI-induced AKI. (G) Heatmap of GSE248078. (H, I) Peripheral blood level of AQP1and P53 with systemic inflammatory response syndrome that had AKI or HD. (J) Heatmap of GSE67401. (K, L) Venn diagram of differentially expressed genes between GSE186822 and GSE220812. Up, up-regulated; Down, down-regulated. (M, N) Venn diagram of differentially expressed genes between GSE67401 and GSE248078. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns p >0.05.

Figure 2

Renal function was assessed for septic AKI at various times. (A, B) Serum creatinine and blood urea nitrogen levels at various times in LPS-induced AKI. (C, D) The expression of NGAL mRNA and Kim-1 mRNA in kidney of rats with septic AKI at different periods. (E)The kidney tissues of rats were prepared by HE staining to observe the renal injury and morphologic analysis at various periods after LPS treatment (200×). (F) Quantitative graph of the tubular damage score. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns p >0.05.

Figure 3

Inflammatory response, apoptosis and interstitial fibrosis during different periods of septic AKI. (A–D) The levels of TNF-α, IL-1β, IL-6 and pNF-κB in kidney from rats at different times after intraperitoneal injection of LPS. (E–H) The levels of TNF-α, IL-1β, IL-6 and pNF-κB in serum of rats during different periods of LPS-induced AKI. (I) The kidney tissues of rats were prepared by immunohistochemistry to observe the Number of inflammatory cell infiltration in renal tissue at various periods after LPS treatment. (J–L) Expression of Caspase-3 mRNA, BAX mRNA and BCL-2 mRNA of in renal tissue were assessed by RT-qPCR during different periods of septic AKI. (M) CD68 positive cells score was evaluated from immunohistochemistry staining. (N–P) Level of FN mRNA, TGF-β mRNA and α-SMA mRNA of in renal tissue were evaluated by RT-qPCR at different periods in LPS induced AKI. (Q) Renal interstitial fibrosis was evaluated from Masson staining. (R) The kidney tissues from rats were prepared by Masson staining at different times after LPS treatment for analysis of the process of renal fibrosis (200×). Blue areas indicate the abundance of fibrosis of renal tissue and red areas display the absence of fibrosis. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns p >0.05.

Figure 4

Expression of AQP1, P53 and P21 proteins and mRNAs at different times of septic AKI. (A–F) Expression of AQP1, P53 and P21 proteins in kidney tissues and serum from different groups after LPS treatment. (G–I) Expression of AQP1, P53 and P21 mRNA in renal tissues from different times of septic AKI. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns p >0.05.

Figure 5

Validation of silencing AQP1 in septic AKI rats exacerbates renal injury by upregulating P53. (A) Renal AQP1 silencing in rats caused by tail vein injection different concentrations of si-AQP1. (B) Expression of NGAL mRNA and KIM-1 mRNA in kidney at various group in septic AKI. (C) Quantitative graph of the tubular damage score. (D) Injury pathology of the renal tissue of rats with LPS-induced AKI. Photomicrographs of HE stained kidney sections (200×). (E) Expression of AQP1 mRNA, P53 mRNA and P21 mRNA in kidney were determined by RT-qPCR. (F, G) Expression of AQP1, P53 and P21 proteins in kidney and serum of rats. (H, I) Expression of inflammatory mediators in kidney and serum of rats, mainly includingIL-1β, pNF-kB, TNF-α and IL-6. (J) CD68 positive cells score was evaluated from immunohistochemistry staining at various group. (K) The kidney tissues of rats were prepared by immunohistochemistry to observe the abundance of inflammatory cell infiltration in renal tissue in LPS-induced AKI. (L, M) Kidney tissue apoptosis and fibrosis gene expression evaluated by RT-qPCR in septic AKI rats. (N) Renal interstitial fibrosis was evaluated at various group in LPS-induced AKI. (O) The kidney tissues from rats were prepared by Masson staining at various group in LPS-induced AKI (200×). *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns=not significant.

Figure 6

Silencing AQP1 exacerbates LPS-induced HK-2 cell injury by upregulating P53 expression. (A, B) HK-2 cells viability was evaluated by CCK8. (C–F) Expression of AQP1, P53 and P21 protein was detected by western blot in LPS stimulated HK-2 cells. (G) 100nM of si-AQP1 was used to silence AQP1 gene. (H–K) P53 and P21 protein expression was significantly elevated after silencing AQP1. (L) Effects of different concentrations of PIF (P53 inhibitor) on HK-2 cell viability. (M, N) PIF significantly inhibited P53 protein expression. (O–R) Expression of AQP1, P53 and P21 protein was detected by western blot at various group in LPS stimulated HK-2 cells. (S) Level of AQP1, P53 and P21 gene was detected at various groups in LPS stimulated HK-2 cells. (T–V) Level of NGAL, IL-1β, IL-6, TNF-α, Caspase-3, BAX, BCL-2, FN, TGF-β and α-SMA gene was detected by RT-qPCR at various group in LPS stimulated HK-2 cells. (W, X) Apoptosis level in LPS induced HK-2 cells was detected by flow cytometry. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns=not significant.

Figure 7

Overexpression of AQP1 was verified in septic AKI rats to alleviate renal injury by inhibiting P53. (A) Different concentrations of pcDNA3.1-AQP1 injected via tail vein lead to AQP1 overexpression in rat kidney. (B) Expression of NGAL mRNA and KIM-1 mRNA in kidney at various group in septic AKI. (C) Quantitative graph of the tubular damage score. (D) Injury pathology of the renal tissue of rats with LPS-induced AKI. Photomicrographs of HE stained kidney sections (200×). (E) Expression of AQP1 mRNA, P53 mRNA and P21 mRNA in kidney were determined by RT-qPCR. (F, G) Expression of AQP1, P53 and P21 proteins in kidney and serum of rats at various groups in Endotoxin induced AKI. (H, I) Expression of IL-1β, pNF-kB, TNF-α and IL-6 in kidney and serum of rats. (J) CD68 positive cells score was evaluated from immunohistochemistry staining at various group. (K) The kidney tissues of rats were prepared by immunohistochemistry to observe the Number of inflammatory cell infiltration in renal tissue in LPS-induced AKI. (L, M) Expression of Caspase-3 mRNA, BAX mRNA, BCL-2 mRNA, FN mRNA, TGF-βmRNA and α-SMA mRNA in renal tissue at various groups in septic AKI (N) Renal interstitial fibrosis was evaluated at various group in LPS-induced AKI. (O) The kidney tissues from rats were prepared by Masson staining at various group in LPS-induced AKI (200×). *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns=not significant.

Figure 8

Overexpression of AQP1 attenuates LPS-induced HK-2 cell injury by inhibiting P53 expression. (A) 50nM of pcDNA3.1-AQP1 was used to Transfection of HK-2 cells. (B–E) Overexpression of AQP1 significantly reduced P53 and P21 protein expression (F) Effects of different concentrations of Keve (P53 agonists) on HK-2 cell viability. (G, H) Keve successfully upregulated P53 protein expression. (I–L) Expression of AQP1, P53 and P21 protein was detected by western blot at various group in LPS stimulated HK-2 cells. (M) Level of AQP1, P53 and P21 gene was detected at various groups in LPS stimulated HK-2 cells. (N–P) Level of NGAL, IL-1β, IL-6, TNF-α, Caspase-3, BAX, BCL-2, FN, TGF-β and α-SMA genes was detected by RT-qPCR at various group in LPS stimulated HK-2 cells. (Q, R) Apoptosis level at various groups in LPS induced HK-2 cells was detected by flow cytometry. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, or ns=not significant.