TangShenWeiNing Formula Prevents Diabetic Nephropathy by Protecting Podocytes Through the SIRT1/HIF-1α Pathway.

Background: Diabetic nephropathy (DN) represents a major complication of diabetes, and podocyte injury has a critical function in DN development. TangShenWeiNing formula (TSWN) has been demonstrated to efficiently decrease proteinuria and protect podocytes in DN. This work aimed to explore the mechanism by which TSWN alleviates DN and protects podocytes. Methods: The major bioactive components of TSWN were detected by mass spectrometry (MS) and pharmacological databases. Eight-week-old male C57BLKS/J db/m and db/db mice were provided pure water, valsartan, low dose TSWN, middle dose TSWN and high dose TSWN by gavage for 12 weeks, respectively. Results: MS and network pharmacology analyses suggested that TSWN might prevent DN through the sirtuin (SIRT)1/hypoxia-inducible factor (HIF)-1α pathway. Diabetic mice showed elevated urinary albumin in comparison with non-diabetic mice, and TSWN decreased urinary albumin in diabetic mice. Histological injury increased in the kidney in diabetic mice, which could be improved by TSWN. Fibrosis and collagen I expression were induced in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney; TSWN alleviated these effects. Apoptosis and cleaved caspase-3 were induced in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney, and TSWN blunted these effects. Podocytes were damaged in the diabetic mouse kidney, which was improved by TSWN. Podocin and nephrin amounts were decreased in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney, and podocalyxin was increased in urine of diabetic animals in comparison with non-diabetic counterparts. After TSWN treatment, podocin and nephrin were raised in the diabetic mouse kidney, and urinary podocalyxin was depressed in diabetic animals. Diabetic mice had lower SIRT1 and higher HIF-1α amounts in kidney specimens in comparison with non-diabetic mice, and TSWN promoted SIRT1 and inhibited HIF-1α in the diabetic mouse kidney. Moreover, co-staining of SIRT1 and podocin revealed that SIRT1 decreased in podocytes from diabetic mice in comparison with those from non-diabetic mice, and TSWN elevated SIRT1 in podocytes. Conclusions: This study indicated that TSWN alleviates DN by improving podocyte injury through the SIRT1/HIF-1α pathway in diabetic mouse kidneys.

PPAR-α Agonist Fenofibrate Prevented Diabetic Nephropathy by Inhibiting M1 Macrophages via Improving Endothelial Cell Function in db/db Mice.

Background: Diabetic nephropathy (DN) is one of the major diabetic microvascular complications, and macrophage polarization plays a key role in the development of DN. Endothelial cells regulate macrophage polarization. Peroxisome proliferator-activated receptor (PPAR)-α agonists were demonstrated to prevent DN and improve endothelial function. In this study, we aimed to investigate whether PPAR-α agonists prevented DN through regulating macrophage phenotype via improving endothelial cell function. Methods: Eight-week-old male C57BLKS/J db/m and db/db mice were given fenofibrate or 1% sodium carboxyl methylcellulose by gavage for 12 weeks. Results: Db/db mice presented higher urinary albumin-to-creatinine ratio (UACR) than db/m mice, and fenofibrate decreased UACR in db/db mice. Fibrosis and collagen I were elevated in db/db mouse kidneys compared with db/m mouse kidneys; however, they were decreased after fenofibrate treatment in db/db mouse kidneys. Apoptosis and cleaved caspase-3 were enhanced in db/db mouse kidneys compared to db/m mouse kidneys, while fenofibrate decreased them in db/db mouse kidneys. Db/db mice had a suppression of p-endothelial nitric oxide synthase (eNOS)/t-eNOS and nitric oxide (NO), and an increase of angiopoietin-2 and reactive oxygen species (ROS) in kidneys compared with db/m mice, and fenofibrate increased p-eNOS/t-eNOS and NO, and decreased angiopoietin-2 and ROS in db/db mouse kidneys. Hypoxia-inducible factor (HIF)-1α and Notch1 were promoted in db/db mouse kidneys compared with db/m mouse kidneys, and were reduced after fenofibrate treatment in db/db mouse kidneys. Furthermore, the immunofluorescence staining indicated that M1 macrophage recruitment was enhanced in db/db mouse kidneys compared to db/m mouse kidneys, and this was accompanied by a significant increase of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in kidneys and in serum of db/db mice compared with db/m mice. However, fenofibrate inhibited the renal M1 macrophage recruitment and cytokines associated with M1 macrophages in db/db mice. Conclusions: Our study indicated that M1 macrophage recruitment due to the upregulated HIF-1α/Notch1 pathway induced by endothelial cell dysfunction involved in type 2 diabetic mouse renal injury, and PPAR-α agonist fenofibrate prevented DN by reducing M1 macrophage recruitment via inhibiting HIF-1α/Notch1 pathway regulated by endothelial cell function in type 2 diabetic mouse kidneys.

Ferroptosis Enhanced Diabetic Renal Tubular Injury via HIF-1α/HO-1 Pathway in db/db Mice.

Background: Ferroptosis is a recently identified iron-dependent form of cell death as a result of increased reactive oxygen species (ROS) and lipid peroxidation. In this study, we investigated whether ferroptosis aggravated diabetic nephropathy (DN) and damaged renal tubules through hypoxia-inducible factor (HIF)-1α/heme oxygenase (HO)-1 pathway in db/db mice. Methods: Db/db mice were administered with or without ferroptosis inhibitor Ferrostatin-1 treatment, and were compared with db/m mice. Results: Db/db mice showed higher urinary albumin-to-creatinine ratio (UACR) than db/m mice, and Ferrostatin-1 reduced UACR in db/db mice. Db/db mice presented higher kidney injury molecular-1 and neutrophil gelatinase-associated lipocalin in kidneys and urine compared to db/m mice, with renal tubular basement membranes folding and faulting. However, these changes were ameliorated in db/db mice after Ferrostatin-1 treatment. Fibrosis area and collagen I were promoted in db/db mouse kidneys as compared to db/m mouse kidneys, which was alleviated by Ferrostatin-1 in db/db mouse kidneys. HIF-1α and HO-1 were increased in db/db mouse kidneys compared with db/m mouse kidneys, and Ferrostatin-1 decreased HIF-1α and HO-1 in db/db mouse kidneys. Iron content was elevated in db/db mouse renal tubules compared with db/m mouse renal tubules, and was relieved in renal tubules of db/db mice after Ferrostatin-1 treatment. Ferritin was increased in db/db mouse kidneys compared with db/m mouse kidneys, but Ferrostatin-1 reduced ferritin in kidneys of db/db mice. Diabetes accelerated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived ROS formation in mouse kidneys, but Ferrostatin-1 prevented ROS formation derived by NADPH oxidases in db/db mouse kidneys. The increased malondialdehyde (MDA) and the decreased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GSH-Px) were detected in db/db mouse kidneys compared to db/m mouse kidneys, whereas Ferrostatin-1 suppressed MDA and elevated SOD, CAT, and GSH-Px in db/db mouse kidneys. Glutathione peroxidase 4 was lower in db/db mouse kidneys than db/m mouse kidneys, and was exacerbated by Ferrostatin-1 in kidneys of db/db mice. Conclusions: Our study indicated that ferroptosis might enhance DN and damage renal tubules in diabetic models through HIF-1α/HO-1 pathway.