Sestrin2 remedies podocyte injury via orchestrating TSP-1/TGF-β1/Smad3 axis in diabetic kidney disease

www.nature.com/cddis ARTICLE OPEN Sestrin2 remedies podocyte injury via orchestrating TSP-1/TGF-β1/Smad3 axis in diabetic kidney disease Shan Song 1,2,3, Chonglin Shi1, Yawei Bian1, Zhaohua Yang1, Lin Mu1,3,4, Haijiang Wu ✉ Yonghong Shi 1,2,3 1,2,3 , Huijun Duan1,2,3 and 1234567890();,: © The Author(s) 2022 Sestrin2 is identified as a stress-induced protein and could functionate in many aspects. In our study, we investigated the latent impact of Sestrin2 on podocyte injury and its molecular mechanism in vivo and in vitro in diabetic kidney disease (DKD). Sestrin2 was low-expressed in renal biopsies from individuals with DKD, the glomeruli from diabetic mice, and mouse podocytes exposed to high glucose (HG). Sestrin2 overexpression ameliorated HG-induced phenotypic alterations, apoptosis, and oxidative stress in conditionally immortalized mouse podocytes and modulated the activity of Thrombospondin-1 (TSP-1)/transforming growth factor (TGF-β1)/Smad3 pathway in podocytes. Moreover, TSP-1 inhibitor LSKL or TGF-β blocker Pirfenidone arrested podocyte injury induced by HG. Streptozotocin (STZ) was employed to render equivalent diabetes in B6-TgN (CMV-Sestrin2) (TgN) and wild-type (WT) control mice. Sestrin2 alleviated increased levels of 24‐h urinary protein, blood urea nitrogen, serum creatinine and triglyceride, and urine 8-OHdG in diabetic mice. Podocyte phenotypic alterations, increased expression of apoptosis-associated proteins and podocyte loss were observed in WT but not in diabetic TgN mice, as well as oxidative stress. Additionally, TSP-1/TGFβ1/Smad3 signaling pathway was also suppressed in glomeruli of diabetic TgN mice. Thus, Sestrin2 mitigates podocyte injury in DKD via orchestrating TSP-1/TGF-β1/Smad3 pathway, underlining Sestrin2 as a promising therapeutic target for DKD. Cell Death and Disease (2022)13:663 ; https://doi.org/10.1038/s41419-022-05120-0 INTRODUCTION Podocytes are critical constituents of the glomerular filtration apparatus; furthermore, the integrity of podocytes and their foot processes, along with the slit diaphragm, is vital for inhibiting proteinuria [1, 2]. Previously, clinical and basic research demonstrated that podocyte injury is a principal determinant of diabetic kidney disease (DKD) [3, 4]. Podocytes originate from cells undergoing mesenchymal-to-epithelial transition (MET); however, these cells can undergo epithelial-to-mesenchymal transition (EMT), a reverse form of embryogenesis characterized by loss of podocyte-specific marker expression and gain of transitional marker expression in the kidneys under pathological conditions [5]. Since renal tubular epithelial cells are likely to undergo EMT after chronic injury, we hypothesized that podocyte EMT had a strong effect on podocyte dysfunction, which might lead to glomerular filtration deficiency. In addition, previous studies have shown that high glucose (HG) can induce renal podocyte apoptosis [6–8]. Therefore, it is clinically important to identify potential therapeutic targets to efficiently ameliorate podocyte injury. Although hyperglycemia is considered as the driving force of DKD development, some studies indicate that hyperglycemia might not be the dominant underlying culprit in DKD [9, 10]. Evidence suggests that oxidative stress is closely linked to renal dysfunction in DKD [11, 12]. The Sestrin family is a group of highly conserved proteins that have a variety of biological functions and whose expression can be induced by stress [13]. Sestrin2, which is known as hypoxia-inducible gene 95, is an important member of Sestrin family that can be cloned from A172 human glioma cell line and is located on human chromosome 1p35.3 [14]. Sestrin2 can be activated by various metabolic stresses, including hypoxia, DNA damage, oxidative stress, and endoplasmic reticulum stress [14]. Additionally, Sestrin2 plays a critical role in mitigating the accumulation of reactive oxygen species (ROS), maintaining energy balance, enhancing autophagy, reducing protein synthesis, and slowing the progression of metabolic diseases [15]. Recently, novel functions of Sestrin2 and their relevant mechanisms have been reported; notably, the role of Sestrin2 in DKD has been emphasized. Sestrin2 overexpression may relieve albumininduced EMT in renal tubular epithelial cells [16]. In addition, HG can downregulate the expression of Sestrin2 and decrease the phosphorylation level of adenylate-activated protein kinase (AMPK), resulting in Nox4-dependent endothelial nitric oxide synthase dysfunction and glomerular mesangial cell fibrosis [17]. Thus, these studies indicate that Sestrin2 may be involved in the pathogenesis of DKD, but the mechanism is still unclear. Here, we identified a novel role for Sestrin2 in modulating HG-induced podocyte injury via thrombospondin-1 (TSP-1)/ 1 Department of Pathology, Hebei Medical University, Shijiazhuang, China. 2Center of Metabolic Diseases and Cancer Research, Institute of Medical and Health Science, Hebei Medical University, Shijiazhuang, China. 3Hebei Key Laboratory of Kidney Diseases, Shijiazhuang, China. 4The Second Hospital of Hebei Medical University, Shijiazhuang, China. ✉email: Edited by Professor Alessandro Finazzi-Agrò Received: 9 April 2022 Revised: 17 July 2022 Accepted: 20 July 2022 Official journal of CDDpress S. Song et al. 2 transforming growth factor-β1 (TGF-β1)/Smad3 axis. Similar results were observed in diabetic mice. These results suggest that Sestrin2 might serve as a new therapeutic target for treating podocyte injury in DKD. RESULTS The level of Sestrin2 is diminished in glomeruli from mice with DKD and renal biopsies from individuals with DKD The immunohistochemical staining results showed that the expression of Sestrin2 was decreased in glomeruli and renal tubules of DKD patients compared to those of normal subjects (Fig. 1A, C). Subsequently, double immunofluorescence staining was used to assess the expression of Sestrin2 in the glomeruli of wild-type (WT) mice and streptozotocin (STZ)-induced diabetes model mice. Synaptopodin is an actin-binding protein that is expressed in crucial cell compartments, such as neuronal dendritic spines in the brain and kidney podocyte foot processes [18]. Here, synaptopodin was used to label renal podocytes in mice. Notably, Sestrin2 expression was downregulated in the glomeruli of diabetic mice at 8, 12, and 16 weeks (Fig. 1B, D). Additionally, we observed decreased serum levels of Sestrin2 in diabetic mice compared with WT mice (Fig. 1E). In vitro, Sestrin2 expression was examined in cultured mouse podocytes exposed to HG for different times (Fig. 1F, G). The protein level of Sestrin2 gradually decreased with time and tended to be the lowest at 24 h (Fig. 1F, G). Fig. 1 The level of Sestrin2 is decreased in glomeruli from mice with DKD and renal biopsies from individuals with DKD. A Representative immunohistochemical staining images of Sestrin2 in renal biopsies from normal subjects and diabetic kidney (...truncated)