#737 Activated HIF-1a accelerates tubular repair in acute kidney injury via cell proliferation

Field: Medicine (Health Sciences)

Abstract Background and Aims Acute kidney injury (AKI) is a common syndrome in hospitalized patients worldwide and is associated with progression to chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) are highly susceptible to injury in AKI resulting in proliferation, which is suggested to be important for tubule recovery and prevention of CKD progression. Roxadustat, a hypoxia-inducible factor (HIF) stabilizer, is a novel drug for the treatment of CKD-related anemia. Some studies have reported the protective effect of Roxadustat on animal AKI models. However, it remains unclear whether Roxadustat induces proliferation and is associated with recovery of renal tubules. Here, we evaluated the effect of Roxadustat on proliferation of PTECs in vitro and in glycerol-induced AKI mice. Method Male C57BL/6J mice were randomly divided into four groups (n = 5–11): a control (i) and a glycerol (Gly) (ii) group received intramuscular injections of PBS or 50% glycerol (5 ml/kg) and a PBS+Rox (iii) and a Gly+Rox (iv) group received intraperitoneal injection of Roxadustat 10 mg/kg/day 2 days prior to glycerol or PBS injection. Urine, kidneys, and blood samples were collected 2 days after glycerol injection and renal dysfunction was evaluated (serum urea nitrogen and creatinine) as well as tubular damage (PAS staining, renal NGAL and Kim-1 expression, and urine NGAL excretion). Renal expression of PCNA, a proliferation marker, was determined by western blotting and immunohistochemistry. PTECs proliferation was analyzed in immortalized proximal tubular cells (RPTEC/TERT1, ATCC) treated with roxadustat by a CCK-8 assay, a wound-healing assay, and western blotting (Ki-67, cyclin A, and phosphorylated Rb). Results Roxadustat treatment significantly suppressed AKI markers (renal NGAL and Kim-1 expression and urine NGAL excretion) and tubular injury score (PAS staining) in glycerol-injected AKI mice. Additionally, Roxadustat treatment reduced serum urea nitrogen and creatinine levels in glycerol injected mice suggesting that it protects proximal tubules against injury, which leads to rescue of renal function. To investigate if the renoprotective effect of Roxadustat was mediated by stimulation of proliferation PCNA-positive PTECs were analyzed. Glycerol injected mice had an increase in PCNA positive cells compared to PBS treated mice as expected. The Gly+Rox group had the same level of PCNA positive PTEC cells as the Gly group despite less renal damage. These results indicate that there may be either a direct proliferative effect of Roxadustat or that the treatment by other mechanisms protect the kidney resulting in less proliferation. To investigate if there is a direct effect of Roxadustat on proliferation we analyzed Roxadustat treatment in RPTEC/TERT1 cells. Roxadustat treatment significantly induced the increase in HIF-1α protein in RPTEC/TERT1 cells. CCK-8 and wound-healing assays revealed that Roxadustat-treated cells showed significant increase in cell proliferation, which was supported by western blotting of Ki-67, cyclin A, and phosphorylated Rb. Furthermore, HIF-1α-knockdown by small interfering RNA tended to suppress the expression of proliferation markers. Conclusion The present study indicates that HIF-1α activation by Roxadustat treatment accelerates cell proliferation in PTECs as a part of the protective mechanism of Roxadustat on tubule damage and AKI.