key: cord-0327785-uc3slu8q authors: Wu, Ming; Xu, Lin; Tan, Bo; Huang, Di; Yuan, Meijie; Ye, Chaoyang title: Remdesivir inhibits renal fibrosis in obstructed kidneys date: 2020-04-03 journal: bioRxiv DOI: 10.1101/2020.04.01.019943 sha: a44d13c1d27735e53194f8f86d865c690bd923d9 doc_id: 327785 cord_uid: uc3slu8q Aim Kidney impairment is observed in patients with COVID-19. We aimed to demonstrate the effect of anti-COVID-19 agent remdesivir on renal fibrosis. Methods Remdesivir and its active nucleoside metabolite GS-441524 were used to treat TGF-β stimulated renal fibroblasts (NRK-49F) and human renal epithelial cells (HK2). Cell viability was determined by CCK8 assay, and fibrotic markers were measured by Western blotting. Vehicle or remdesivir were given by intraperitoneal injection or renal injection through the left ureter in unilateral ureteral obstruction (UUO) mice. Serum and kidneys were harvested. The concentrations of remdesivir and GS-441524 were measured using LC-MS/MS. Renal and liver function were assessed. Renal fibrosis was evaluated by Masson’s trichrome staining and Western blotting. Results Remdesivir and GS-441524 inhibited cell proliferation and the expression of fibrotic markers (fibronectin, pSmad3, and aSMA) in NRK-49F and HK2 cells. Intraperitoneal injection or renal injection of remdesivir attenuated renal fibrosis of UUO kidneys. Renal and liver function were not changed in remdesivir treated UUO mice. Remdesivir can not be detected, but two remdesivir metabolites were detected after injection. Conclusion Remdesivir inhibits renal fibrosis in obstructed kidneys. A novel coronavirus (2019-nCoV) reported in Wuhan in late December 2019 has rapidly spread to the rest of the world (1, 2) . Novel coronavirus pneumonia is becoming a worldwide public health event due to the rapid increase in new cases and the high severity and mortality (1) (2) (3) . A latest research shows that COVID-19 patients in the intensive care unit were older (median age 66 vs 51) and more likely to have comorbidities (72.2% vs 37.3%) than patients not in the intensive care unit, suggesting that elderly people or patients with underlying disease have higher disease severity (3). Chronic kidney disease (CKD) is a common disorder and the prevalence of CKD is around 10% in adults (4, 5). Thus, CKD patients combined with COVID-19 should be drawn enough attention from nephrologists. It is worth noting that the 2019-nCoV can not only cause pneumonia, but also damage other organs, such as the heart, liver and kidneys (6). Angiotensin-converting enzyme 2 (ACE2) mediates the entry of 2019-nCoV coronavirus into human cells (7). It has been found that ACE2 is highly expressed in renal tubular cells, implying that 2019-nCoV may directly bind to ACE2-positive cells in the kidney and thus induce kidney injuries (7). Indeed, a clinical study reported that 27.06% of patients with COVID-19 exhibited acute renal failure (ARF), while elderly patients (≥60 years) were more likely to develop ARF (65.22% vs 24.19%) (8) . A further immunohistochemistry analysis revealed that the antigen for 2019-nCoV accumulates in renal tubules (8) . Another clinical study with 59 COVID-19 patients showed that proteinuria occurred in 63% of patients (9) . 19% and 27% COVID-19 patients have elevated plasma creatinine and urea nitrogen levels, respectively (9) . A consecutive cohort study with 710 COVID-19 patients further shows that the prevalence of renal impairment is high, which is associated with in-hospital death (10) . Therefore, enough attentions should be paid to kidney protection of COVID-19 patients, and the effect of anti-COVID-19 agents on the kidney should also be concerned. Renal interstitial fibrosis is a common pathway and main pathological basis for the progression of various chronic kidney diseases to the end-stage renal disease (ESRD) (11, 12) . It is characterized by excessive deposition of extracellular matrix in the kidney leading to completely loss of renal function (11, 12) . Loss of renal tubule drives the development of renal interstitial fibrosis by producing a large number of profibrotic factors such as TGF-β (12, 13) . It has been shown by several animal models that the TGF-β / Smad3 signaling pathway play a key role in renal fibrosis (14, 15) . Remdesivir (GS-5734) is a nucleoside analogue designed for the treatment of severe acute respiratory syndrome coronavirus (SARS), the Middle East respiratory syndrome (MERS) and Ebola virus (16, 17) . It can be anabolized to the active triphosphate metabolite and then incorporated into the newly synthesized RNA strand of the virus as a substrate for viral RNA-dependent RNA synthetase (RdRp), thereby prematurely terminating viral RNA transcription (16, 17) . In vitro study shows that remdesivir can effectively inhibit the infection of 2019-nCoV (18) . A single case study shows that treatment with remdesivir improved the clinical condition of the first severely infected COVID-19 patient in the United States in 24 hours (19) . Currently, several clinical trials using remdesivir as a treatment for infected COVID-19 patients are undergoing around the world. The first phase III clinical trial which is conducted in China is expected to be completed in the end of April 2020 (ClinicalTrials.gov Identifier: NCT04257656). The aim of the current study is to determine the effect remdesivir on renal fibrosis. GS-441524, the active metabolite of remdesivir, was used to treat TGF-β stimulated rat renal interstitial fibroblasts (NRK-49F) and huamn renal epithelial cells (HK2) (20) . 24 hours stimulation with 2.5 ng/ml TGF-β increased the cell proliferation of NRK-49F cells as shown by CCK8 assay, and treatment with the Figure 2D ). Mouse renal fibrosis model was established by UUO operation. One day after sham or UUO operations, mice were treated with vehicle or remdesivir for 10 days. Treatment with remdesivir had no effect on body weight of sham and UUO mice, and all mice were survived during the treatment (data not shown). Mild interstitial fibrosis was observed in vehicle treated UUO mice, which was attenuated by remdesivir ( Figure 3A ). The protein expression of FN, collagen-I (Col-I), pSmad3, and α -SMA were up-regulated in UUO mouse kidneys as compared with that in sham operated mouse kidneys, and the treatment with remdesivir significantly reduced the expression of these pro-fibrotic proteins in UUO mouse kidneys ( Figure 3B ). Liver function (ALT and AST) and renal function (Scr and BUN) were determined. Remdesivir has no effect on either liver function or renal function ( Figure 3C Figure 3D ). Similarly, serum or kidney Ala-Met can only be detected in remdesivir treated sham or UUO mice ( Figure 3D ). Remdesivir (1 mg/mL, 50 μ l/mouse) or vehicle was infused retrogradely through ureter to the left kidney which was subjected to unilateral utero ligation (UUO) operation. Masson staining shows that interstitial fibrosis was attenuated in UUO kidneys at day7 by local remdesivir injection ( Figure 4A ). The expression of FN, Col-I, pSmad3, and α -SMA were reduced in remdesivir treated UUO kidneys as compared with that in vehicle treated control kidneys at day7 as shown by Western blotting ( Figure 4B ). Liver function (ALT and AST) and renal function (Scr and BUN) were not changed by remdesivir in UUO mice ( Figure 4C ). The remdesivir metabolite GS-441524 can only be detected in serum and kidney at 1 hour after injection only in remdesivir treated mice, which can not be detected at day7 ( Figure 4D ). The Ala-Met is abundant in remdesivir treated kidneys at 1 hour after injection and it was reduced to background level at day7 ( Figure 4D ). The effect of remdesivir on renal fibrosis is currently unknown. In the present study, we showed that remdesivir inhibits renal fibrosis. First, treatment with remdesivir or its active metabolite GS-441524 inhibited fibrosis in two cellular models for renal fibrosis. Second, systematic administration of remdesivir inhibited renal fibrosis as shown by Masson staining and Western blotting. Third, local infusion of remdesivir into UUO kidneys further revealed the anti-fibrotic effect of remdesivir. The limitation of this study is that UUO model is not suitable to study the pharmaceutical effect on renal function. UUO model is a classic model to study renal fibrosis, although we did observe a slightly increase in Scr and BUN levels in UUO mice as compared with that in sham mice (21) . The renal and liver function were assessed in this study to exclude the potential toxic effect of remdesivir, and we show no effect of remdesivir on renal and liver function in this study. The concentration of remdesivir was measured to assess the dosing efficacy. However, we are not able to detect remdesivir in serum or kidneys in two different dosing regimens or at different time points. This is probably due to the rapid turnover of remdesivir by esterases which are abundant in blood and tissues (17, 22) . Next, we measured the concentration of two remdesivir metabolites, alanine metabolite (Ala-Met) and nucleoside metabolite (GS-441524) (17) . GS-441524 and Ala-Met can be detected at 1 hour after IP injection in serum and kidneys of sham and UUO mice. GS-441524 was detected in the serum and kidney at 1 hour after renal injection but not at day 7 post-injection. To prove the specificity of renal infusion of remdesivir, we further measured Ala-Met which can only be detected in kidneys but not in serum at 1 hour after renal injection, and it was not detectable at day 7 after renal injection. In conclusion, we showed that remdesivir inhibits renal fibrosis in obstructed kidneys. Male C57 mice (SPF grade, 20-25g) were purchased and housed in Shanghai Model Organisms Center Inc (SMOC) according to local regulations and guidelines. After anesthesia with sodium pentobarbital (8mg/kg, i.p.), the left mouse kidney Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study Human Kidney is a Target for Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection Caution on Kidney Dysfunctions of 2019-nCoV Patients Kidney impairment is associated with in-hospital death of COVID-19 patients Progression of Tubulointerstitial Fibrosis and the Chronic Kidney Disease Phenotype -Role of Risk Factors and Epigenetics Progression of chronic kidney disease: too much cellular talk causes damage Renal tubule injury: a driving force toward chronic kidney disease Mechanism of chronic aristolochic acid nephropathy: role of Smad3 Role of Smad signaling in kidney disease Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Novel Coronavirus in the United States Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. mBio Unilateral Ureteral Obstruction as a Model to Investigate Fibrosis-Attenuating Treatments Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses Remdesivir inhibited renal fibrosis in vitro.