Diabetic Kidney Disease Rats Research Articles

Astragaloside IV attenuates fatty acid-induced renal tubular injury in diabetic kidney disease by inhibiting fatty acid transport protein-2

BackgroundRenal tubular injury induced by free fatty acid bound to albumin is the key pathological basis for the progression of diabetic kidney disease. However, effective interventions are limited. Astragaloside IV, as a major bioactive component purified from Astragalus membranaceus (Fisch.) Bunge, possesses pharmacological properties of lowering blood glucose and proteinuria, and renal tubular protection in diabetic kidney disease. Further work is needed to understand the underlying molecular mechanisms. PurposeThis study was designed to investigate the mechanism of renal tubular protection by astragaloside IV in diabetic kidney disease. MethodsRats receiving high-fat diet combined with streptozotocin (30 mg/kg, i.p.) were gavaged with astragaloside IV (10 mg/kg/d or 20 mg/kg/d) or empagliflozin (1.72 mg/kg/d) for 8 weeks. In vitro, the NRK-52E cells were treated with free fatty acid-deleted BSA or palmitic acid-bound BSA in the presence or absence of astragaloside IV (5 μM, 10 μM, 20 μM) or 5 μM of mcc950. The effects of astragaloside IV on mitochondrial function, NLRP3/ASC/IL-18/IL-1β inflammatory cascade, and renal tubular injury were detected by pathological staining, immunoblotting, MitoSOX Red staining. Next, to investigate the mechanism of renal tubular protection by astragaloside IV, we transfected Fatp2 siRNA into BSA-PA-treated NRK-52E cells and injected lipofermata (a FATP2 inhibitor) intraperitoneally into free fatty acid-bound BSA overloaded rats with concomitant astragaloside IV treatment. ResultsTreatment with astragaloside IV for 8 weeks dose-dependently attenuated the blood glucose, ratio of urinary albumin to creatinine, disorder of lipid metabolism, and pathological injury in diabetic kidney disease rats. In addition, astragaloside IV dose-dependently attenuated mitochondrial-derived reactive oxygen species and subsequent inhibiting NLRP3-mediated inflammatory cascade in diabetic kidney disease rats and palmitic acid-bound BSA-treated NRK-52E cells, thereby exerting renal tubular protection. More importantly, the effects of astragaloside IV on restoration of mitochondrial function, inhibition of inflammatory response and amelioration of renal tubular injury in vivo and in vitro were further enhanced when used in combination with Fatp2 siRNA or lipofermata. ConclusionAstragaloside IV exerts antioxidant and anti-inflammatory effects in diabetic kidney disease by inhibiting FATP2-mediated fatty acid transport, thereby attenuating renal tubular injury.

Liraglutide ameliorates diabetic kidney disease by modulating gut microbiota and L-5-Oxoproline

The gut microbiome-metabolites-kidney axis is a potential target for treating diabetic kidney disease (DKD). Our previous study found that Liraglutide attenuated DKD in rats by decreasing renal tubular ectopic lipid deposition (ELD) and serum metabolites levels, including L-5-Oxoproline (5-OP). However, the response of gut microbiome-metabolites-kidney axis to Liraglutide in DKD rats and the effect of 5-OP on ELD remain unknown. In this study, Sprague-Dawley rats were used as an animal model of DKD. They were subjected to a high fat diet, streptozotocin and uninephrectomy, followed by Liraglutide treatment (0.4 mg/kg d). Additionally, HK-2 cells were incubated with 30 mM glucose and 200 μM palmitate for 24h, and exposed to different concentrations of 5-OP. In DKD rats, Liraglutide dramatically improved the renal tubule structure. It increased the Simpson index (F = 4.487, p = 0.035) and reduced the Actinobacteria-to-Bacteroidetes ratio (F = 6.189, p = 0.014). At the genus level, Liraglutide increased the relative abundance of Clostridium, Oscillospira, Sarcina, SMB53, and 02d06 while decreasing that of Allobaculum. Meanwhile, 13 metabolites were significantly altered after Liraglutide treatment. Multi-omics analysis found that 5-OP levels were positively correlated with Clostridium abundance but negatively correlated with renal injury related indicators. In HK-2 cells, 5-OP significantly reduced the ELD in a dose-dependent manner through inhibiting the expression of SREBP1 and FAS. Overall, the renoprotective effect of Liraglutide in DKD rats is linked to the improvement of the gut microbiota composition and increased serum 5-OP levels, which may reduce ELD in renal tubular cells by lowering lipid synthesis.

Supplemented Gegen Qinlian Decoction Formula attenuates podocyte mitochondrial fission and renal fibrosis in diabetic kidney disease by inhibiting TNF-α-mediated necroptosis, compared with empagliflozin

Ethnopharmacological relevanceRecently, podocyte mitochondrial dysfunction and necroptosis have been shown to play critical roles in renal fibrosis (RF) in diabetic kidney disease (DKD); however, these conditions lack effective treatment. In China, the supplemented Gegen Qinlian Decoction Formula (SGQDF), which originates from the classical prescription Gegen Qinlian Decoction, has been widely used to treat patients with DKD. However, it remains unclear whether SGQDF alleviates podocyte injury-associated RF in patients with DKD. Aim of studyThis study aimed to clarify the therapeutic effects of SGQDF compared with those of empagliflozin (EMPA) on podocyte mitochondrial fission and RF in DKD and its necroptosis-related mechanisms. Materials and methodsModified DKD rat models were developed through a combination of uninephrectomy, streptozotocin administration through intraperitoneal injection, and exposure to a high-fat diet. Following RF formation, the DKD rat models received either a high dose of SGQDF (H-SGQDF), a low dose of SGQDF (L-SGQDF), EMPA, or vehicle for 4 weeks. In our in vitro study, we subjected cultured murine podocytes to a high-glucose environment and various treatments including Mdivi-1, adalimumab, and necrostatin-1, with or without H-SGQDF or EMPA. SGQDF target prediction and molecular docking verification were performed. For the in vivo study, we focused on examining changes in the parameters associated with renal injury, RF, and oxidative stress (OS)-induced injuries in podocytes. Both in vivo and in vitro studies included an analysis of changes in podocyte mitochondrial fission, TNF-α-induced podocyte necroptosis, and the RIPK1/RIPK3/MLKL signaling pathway activation. ResultsSGQDF improved renal injury markers, including body weight, blood glucose, serum creatinine, blood urea nitrogen, and urinary albumin, in a dose-dependent manner. The beneficial effects of H-SGQDF in vivo were greater than those of L-SGQDF alone in vivo. Interestingly, similar to EMPA, H-SGQDF ameliorated RF and reduced OS-induced podocyte injury in diabetic kidneys. Furthermore, TNF-α signaling was shown to be important in the network construction of “the SGQDF-component-target.” Based on this, we also showed that the beneficial effects in vivo and in vitro of H-SGQDF were closely related to the improvement in mitochondrial dysfunction and the inhibition of TNF-α-induced necroptosis in podocytes. ConclusionIn the present study, we showed that H-SGQDF, similar to EMPA, attenuates podocyte mitochondrial fission and RF, and that the underlying therapeutic mechanisms are closely related to inhibiting the activation of the RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. Our findings provide new pharmacological evidence for the application of H-SGQDF in the RF treatment of DKD.

Acupuncture improve proteinuria in diabetic kidney disease rats by inhibiting ferroptosis and epithelial-mesenchymal transition

ObjectiveTo explore the mechanism of acupuncture to relieve diabetic proteinuria in Diabetic Kidney Disease (DKD). MethodsA total of 10 male Sprague-Dawley rats were randomly selected as the negative control group (NC), and a further 30 rats were fed a high-fat diet (HFD) and intraperitoneal streptozotocin (STZ). The DKD model rats in the model group (DKD) and the acupuncture group (DKD + Acu) were randomly assigned. After 4 weeks of intervention, collected urine, peripheral blood, and renal tissues from all rats, and assessed blood urea nitrogen, serum creatinine, triglyceride, 24-h urine protein quantification, and blood glucose, left kidney weight, kidney body ratio index, then observe changes in renal histology in the rats. The renal cortex tissues of three rats from each group were sent for transcriptomic analysis. According to the results of transcriptomic analysis, various kits were used to detect SOD, MDA, GSH, GSH-px, and iron concentration. The expression levels of GPX4 and System Xc-, members of the antioxidative stress pathway, and TfR 1, SLC39A14, FTH 1, and SLC40A1, involved in iron metabolism, in the kidney tissues were measured by western blotting and reverse transcription-quantitative PCR. The expression of mesenchymal phenotype markers and podocyte-specific markers were evaluated by immunofluorescence. ResultsAcupuncture promoted the levels of GSH, GSH-px, and SOD, decreased the level of MDA (P < 0.05), promoted the expression of GPX4 and System Xc- (P < 0.05), decreased the expression of TfR1 and SLC39A14 (P < 0.01), and increased the expression of FTH 1 and SLC40A1 (P < 0.05), inhibited the expression of TGF-β1, desmin, FSP-1, and α-SMA (P < 0.05), promoted the expression of Nephrin, Podocin, and CD2AP (P < 0.05). ConclusionImproving the ability of podocytes to prevent oxidative stress and restoring iron ion homeostasis, can improve Ferroptosis and block epithelial-mesenchymal transition, improve podocyte injury, restore filtration function, and reduce proteinuria in DKD rats.

Sleeve gastrectomy links the attenuation of diabetic kidney disease to the inhibition of renal tubular ferroptosis through down-regulating TGF-β1/Smad3 signaling pathway

PurposeTo investigate how sleeve gastrectomy (SG), a typical operation of bariatric surgery, attenuated symptom, and progression of diabetic kidney disease (DKD).MethodsDKD model was induced by high-fat diet (HFD) combined with streptozocin in Wistar rats. SG was performed, and the group subjected to sham surgery served as control. The animals were euthanized 12 weeks after surgery, followed by sample collection for the subsequent experiment. The HK-2, a renal proximal tubular epithelial cell line derived from human, was utilized to investigate the potential mechanisms.ResultsSG improved metabolic parameters and glucose homeostasis, and could alleviate DKD in terms of renal function indices as well as histological and morphological structures in DM rats, accompanied with a significant reduction in renal tubular injury. Compared with sham group, SG reduced the renal tubular ferroptosis. To further clarify the mechanism involved, in vitro experiments were performed. In the presence of high glucose, renal tubular TGF-β1 secretion was significantly increased in HK-2 cell line, which led to activation of ferroptosis through TGF-β1/Smad3 signaling pathway. Inhibition of TGF-β1 receptor and phosphorylation of Smad3 significantly ameliorated TGF-β1-mediated ferroptosis. In vivo experiments also found that SG improved the hyperglycemic environment, reduced renal TGF-β1 concentrations, and down-regulated the TGF-β1/Smad3 signaling pathway.ConclusionsWith the capacity to lower the glucose, SG could attenuate the ferroptosis by inhibiting TGF-β1/Smad3 signaling pathway in DKD rats, and eventually attenuated DKD.

Astragaloside IV attenuates renal tubule injury in DKD rats via suppression of CD36-mediated NLRP3 inflammasome activation.

In recent years, diabetic kidney disease (DKD) has emerged as a prominent factor contributing to end-stage renal disease. Tubulointerstitial inflammation and lipid accumulation have been identified as key factors in the development of DKD. Earlier research indicated that Astragaloside IV (AS-IV) reduces inflammation and oxidative stress, controls lipid accumulation, and provides protection to the kidneys. Nevertheless, the mechanisms responsible for its protective effects against DKD have not yet been completely elucidated. The primary objective of this research was to examine the protective properties of AS-IV against DKD and investigate the underlying mechanism, which involves CD36, reactive oxygen species (ROS), NLR family pyrin domain containing 3 (NLRP3), and interleukin-1β (IL-1β). The DKD rat model was created by administering streptozotocin along with a high-fat diet. Subsequently, the DKD rats and palmitic acid (PA)-induced HK-2 cells were treated with AS-IV. Atorvastatin was used as the positive control. To assess the therapeutic effects of AS-IV on DKD, various tests including blood sugar levels, the lipid profile, renal function, and histopathological examinations were conducted. The levels of CD36, ROS, NLRP3, Caspase-1, and IL-1β were detected using western blot analysis, PCR, and flow cytometry. Furthermore, adenovirus-mediated CD36 overexpression was applied to explore the underlying mechanisms through in vitro experiments. In vivo experiments demonstrated that AS-IV significantly reduced hyperglycemia, dyslipidemia, urinary albumin excretion, and serum creatinine levels in DKD rats. Additionally, it improved renal structural abnormalities and suppressed the expression of CD36, NLRP3, IL-1β, TNF-α, and MCP-1. In vitro experiments showed that AS-IV decreased CD36 expression, lipid accumulation, and lipid ROS production while inhibiting NLRP3 activation and IL-1β secretion in PA-induced HK-2 cells. AS-IV alleviated renal tubule interstitial inflammation and tubule epithelial cell apoptosis in DKD rats by inhibiting CD36-mediated lipid accumulation and NLRP3 inflammasome activation.

MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4.

Diabetic kidney disease (DKD) is a major complication of diabetes mellitus. Renal tubular epithelial cell (TEC) damage, which is strongly associated with the inflammatory response and mesenchymal trans-differentiation, plays a significant role in DKD; However, the precise molecular mechanism is unknown. The recently identified microRNA-630 (miR-630) has been hypothesized to be closely associated with cell migration, apoptosis, and autophagy. However, the association between miR-630 and DKD and the underlying mechanism remain unknown. To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats. Streptozotocin was administered to six-week-old male rats to create a hyperglycemic diabetic model. In the second week of modeling, the rats were divided into control, DKD, negative control of lentivirus, and miR-630 overexpression groups. After 8 wk, urine and blood samples were collected for the kidney injury assays, and renal tissues were removed for further molecular assays. The target gene for miR-630 was predicted using bioinformatics, and the association between miR-630 and toll-like receptor 4 (TLR4) was confirmed using in vitro investigations and double luciferase reporter gene assays. Overexpression of miR-630 in DKD rats led to changes in body weight, renal weight index, basic blood parameters and histopathological changes. The expression level of miR-630 was reduced in the kidney tissue of rats with DKD (P < 0.05). The miR-630 and TLR4 expressions in rat renal TECs (NRK-52E) were measured using quantitative reverse transcription polymerase chain reaction. The mRNA expression level of miR-630 was significantly lower in the high-glucose (HG) and HG + mimic negative control (NC) groups than in the normal glucose (NG) group (P < 0.05). In contrast, the mRNA expression level of TLR4 was significantly higher in these groups (P < 0.05). However, miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Furthermore, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 were significantly higher in the HG and HG + mimic NC groups than in NG group (P < 0.05). However, the levels of these cytokines were significantly lower in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Notably, changes in protein expression were observed. The HG and HG + mimic NC groups showed a significant decrease in E-cadherin protein expression, whereas TLR4, α-smooth muscle actin (SMA), and collagen IV protein expression increased (P < 0.05). Conversely, the HG + miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4, α-SMA, and collagen IV protein expression than in the HG + mimic NC group (P < 0.05). The miR-630 targets TLR4 gene expression. In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC. Additionally, rats treated with miR-630 agomir showed significant reductions in urinary albumin, blood glucose, TLR4, and proinflammatory markers (TNF-α, IL-1β, and IL-6) expression levels (P < 0.05). Moreover, these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells. MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4, and has a protective effect on DKD.

Quercetin improves diabetic kidney disease by inhibiting ferroptosis and regulating the Nrf2 in streptozotocin-induced diabetic rats

Diabetic kidney disease (DKD) is a leading factor in end-stage renal disease. The complexity of its pathogenesis, combined with the limited treatment efficacy, necessitates deeper insights into potential causes. Studies suggest that ferroptosis-driven renal tubular damage contributes to DKD's progression, making its counteraction a potential therapeutic strategy. Quercetin, a flavonoid found in numerous fruits and vegetables, has demonstrated DKD mitigation in mouse models, though its protective mechanism remains ambiguous. In this study, we delved into quercetin’s potential anti-ferroptotic properties, employing a DKD rat model and high glucose (HG)-treated renal tubular epithelial cell models. Our findings revealed that HG prompted unusual ferroptosis activation in renal tubular epithelial cells. However, quercetin counteracted this by inhibiting ferroptosis and activating NFE2-related factor 2 (Nrf2) expression in both DKD rats and HG-treated HK-2 cells, indicating its renal protective role. Further experiments, both in vivo and in vitro, validated that quercetin stimulates Nrf2. Thus, our research underscores quercetin’s potential in DKD treatment by modulating the ferroptosis process via activating Nrf2 in a distinct DKD rat model, offering a fresh perspective on quercetin’s protective mechanisms.

Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P&lt;0.05 or P&lt;0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P&lt;0.05 or P&lt;0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P&lt;0.05 or P&lt;0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P&lt;0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P&lt;0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.

High CD133 expression in proximal tubular cells in diabetic kidney disease: good or bad?

BackgroundProximal tubular cells (PTCs) play a critical role in the progression of diabetic kidney disease (DKD). As one of important progenitor markers, CD133 was reported to indicate the regeneration of dedifferentiated PTCs in acute kidney disease. However, its role in chronic DKD is unclear. Therefore, we aimed to investigate the expression patterns and elucidate its functional significance of CD133 in DKD.MethodsData mining was employed to illustrate the expression and molecular function of CD133 in PTCs in human DKD. Subsequently, rat models representing various stages of DKD progression were established. The expression of CD133 was confirmed in DKD rats, as well as in human PTCs (HK-2 cells) and rat PTCs (NRK-52E cells) exposed to high glucose. The immunofluorescence and flow cytometry techniques were utilized to determine the expression patterns of CD133, utilizing proliferative and injury indicators. After overexpression or knockdown of CD133 in HK-2 cells, the cell proliferation and apoptosis were detected by EdU assay, real-time cell analysis and flow analysis. Additionally, the evaluation of epithelial, progenitor cell, and apoptotic indices was performed through western blot and quantitative RT-PCR analyses.ResultsThe expression of CD133 was notably elevated in both human and rat PTCs in DKD, and this expression increased as DKD progressed. CD133 was found to be co-expressed with CD24, KIM-1, SOX9, and PCNA, suggesting that CD133+ cells were damaged and associated with proliferation. In terms of functionality, the knockdown of CD133 resulted in a significant reduction in proliferation and an increase in apoptosis in HK-2 cells compared to the high glucose stimulus group. Conversely, the overexpression of CD133 significantly mitigated high glucose-induced cell apoptosis, but had no impact on cellular proliferation. Furthermore, the Nephroseq database provided additional evidence to support the correlation between CD133 expression and the progression of DKD. Analysis of single-cell RNA-sequencing data revealed that CD133+ PTCs potentially play a role in the advancement of DKD through multiple mechanisms, including heat damage, cell microtubule stabilization, cell growth inhibition and tumor necrosis factor-mediated signaling pathway.ConclusionOur study demonstrates that the upregulation of CD133 is linked to cellular proliferation and protects PTC from apoptosis in DKD and high glucose induced PTC injury. We propose that heightened CD133 expression may facilitate cellular self-protective responses during the initial stages of high glucose exposure. However, its sustained increase is associated with the pathological progression of DKD. In conclusion, CD133 exhibits dual roles in the advancement of DKD, necessitating further investigation.

Placenta-derived mesenchymal stem cells protect against diabetic kidney disease by upregulating autophagy-mediated SIRT1/FOXO1 pathway

Diabetic kidney disease (DKD) is a common chronic microvascular complication of diabetes mellitus. Although studies have indicated the therapeutic potential of mesenchymal stem cells (MSCs) for DKD, the underlying molecular mechanisms remain unclear. Herein, we explored the renoprotective effect of placenta-derived MSCs (P-MSCs) and the potential mechanism of SIRT1/FOXO1 pathway-mediated autophagy in DKD. The urine microalbumin/creatinine ratio was determined using ELISA, and renal pathological changes were detected by special staining techniques. Immunofluorescence was used for detecting the renal tissue expression of podocin and nephrin; immunohistochemistry for the renal expression of autophagy-related proteins (LC3, Beclin-1, SIRT1, and FOXO1); and western blotting and PCR for the expression of podocyte autophagy- and pathway-related indicators. We found that P-MSCs ameliorated renal tubular injury and glomerular mesangial matrix deposition and alleviated podocyte damage in DKD rats. PMSCs enhanced autophagy levels and increased SIRT1 and FOXO1 expression in DKD rat renal tissue, whereas the autophagy inhibitor 3-methyladenine significantly attenuated the renoprotective effect of P-MSCs. P-MSCs improved HG-induced Mouse podocyte clone5(MPC5)injury, increased podocyte autophagy, and upregulated SIRT1 and FOXO1 expression. Moreover, downregulation of SIRT1 expression blocked the P-MSC-mediated enhancement of podocyte autophagy and improvement of podocyte injury. Thus, P-MSCs can significantly improve renal damage and reduce podocyte injury in DKD rats by modulating the SIRT1/FOXO1 pathway and enhancing podocyte autophagy.

Claudin-2 Mediates the Proximal Tubular Epithelial Cell-Fibroblast Crosstalk via Paracrine CTGF.

Proximal tubular epithelial cell (PTEC) is vulnerable to injury in diabetic kidney disease (DKD) due to high energy expenditure. The injured PTECs-derived profibrotic factors are thought to be driving forces in tubulointerstitial fibrosis (TIF) as they activate surrounding fibroblasts. However, the mechanisms remain unclear. The diabetes with uninephrectomy (DKD) rats were used to evaluated renal histological changes and the expression of Claudin-2 by immunofluorescence staining. Then, Claudin-2 expression in PTECs were modulated and subsequently determined the proliferation and activation of fibroblasts by building a transwell co-culture system in normal glucose (NG)or high glucose (HG) condition. Decreased expression of Claudin-2 in PTECs accompanied by tight junction disruption and increased interstitial fibrosis, were detected in DKD rats. In vitro, downregulated Claudin-2 in PTECs promoted proliferation and activation of fibroblasts, which coincided with elevated expression of profibrotic connective tissue growth factor (CTGF) in PTECs. Silenced CTGF inhibited the profibrotic of PTECs via Claudin-2 inhibition. Fibroblasts co-cultured with PTECs transitioned more to myofibroblasts and generated extracellular matrix (ECM) significantly in response to high glucose (HG) stimulation whereas overexpression of Claudin-2 in PTECs reversed the above results. Upregulating CTGF disrupted the beneficial anti-fibrosis effects obtained by overexpression of Claudin-2 in HG condition. Our study suggested that Claudin-2 in PTECs, a key mediator of paracellular cation and water transport, promotes the activation and proliferation of surrounding fibroblasts significantly via CTGF in a paracrine manner.

Central administration of Dapagliflozin alleviates a hypothalamic neuroinflammatory signature and changing tubular lipid metabolism in type 2 diabetic nephropathy by upregulating MCPIP1

BackgroundHypothalamic neuroinflammation is associated with disorders of lipid metabolism. Considering the anti-neuroinflammation effects of sodium-glucose cotransporter 2(SGLT2) inhibitors, a central administration of Dapagliflozin is postulated to provide hypothalamic protection and change lipid metabolism in kidney against diabetic kidney disease (DKD). MethodsBlood samples of DKD patients were collected. Male Sprague-Dawley (SD) rats with 30 mg/kg streptozotocin and a high-fat diet, db/db mice and palmitic acid (PA)-stimulated BV2 microglia were used for study models. 0.28 mg/3ul dapagliflozin was injected into the lateral ventricle in db/db mice. Genes and protein expression levels were determined by qPCR, western blotting, immunofluorescence, and immunohistochemistry staining. Secreted IL-1β and IL-6 were quantified by ELISA. Oil red O staining, lipidomic, and non-targeted metabolomics were performed to evaluate abnormal lipid metabolism in kidney. ResultsThe decrease of serum MCPIP1 was an independent risk factor for renal progression in DKD patients (OR=1.22, 95 %CI: 1.02–1.45, P = 0.033). Higher microglia marker IBA1 and lower MCPIP1 in the hypothalamus, as well as lipid droplet deposition increasing in the kidney were observed in DKD rats. Central dapagliflozin could reduce the blood sugar, hypothalamic inflammatory cytokines, lipid droplet deposition in renal tubular. Lipidomics and metabolomics results showed that dapagliflozin changed 37 lipids and 19 metabolites considered on promoting lipolysis. These lipid metabolism changes were attributed to dapagliflozin by upregulating MCPIP1, and inhibiting cytokines in the microglia induced by PA. ConclusionsCentral administrated Dapagliflozin elicits an anti-inflammatory effect by upregulating MCPIP1 levels in microglia and changes lipid metabolism in kidney of DKD.

Human placenta-derived mesenchymal stem cells ameliorate diabetic kidney disease by modulating the T helper 17 cell/ regulatory T-cell balance through the programmed death 1 / programmed death-ligand 1 pathway.

To investigate the therapeutic effects and immunomodulatory mechanisms of human placenta-derived mesenchymal stem cells (PMSCs) in diabetic kidney disease (DKD). Streptozotocin-induced DKD rats were administered an equivalent volume of saline or PMSCs (1 × 106 in 2 mL phosphate-buffered saline per rat) for 3 weeks. Eight weeks after treatment, we examined the biochemical parameters in the blood and urine, the ratio of T helper 17 cells (Th17) and regulatory T cells (Treg) in the blood, cytokine levels in the kidney and blood, and renal histopathological changes. In addition, we performed PMSC tracing and renal transcriptomic analyses using RNA-sequencing. Finally, we determined whether PMSCs modulated the Th17/Treg balance by upregulating programmed death 1 (PD-1) in vitro. The PMSCs significantly improved renal function, which was assessed by serum creatinine levels, urea nitrogen, cystatin C levels, urinary albumin-creatinine ratio, and the kidney index. Further, PMSCs alleviated pathological changes, including tubular vacuolar degeneration, mesangial matrix expansion, and glomerular filtration barrier injury. In the DKD rats in our study, PMSCs were mainly recruited to immune organs, rather than to the kidney or pancreas. PMSCs markedly promoted the Th17/Treg balance and reduced the levels of pro-inflammatory cytokines (interleukin [IL]-17A and IL-1β) in the kidney and blood of DKD rats. In vitro experiments showed that PMSCs significantly reduced the proportion of Th17 cells and increased the proportion of Treg cells by upregulating PD-1 in a cell-cell contact manner and downregulating programmed death-ligand 1 (PD-L1) expression in PMSCs, which reversed the Th17/Treg balance. We found that PMSCs improved renal function and pathological damage in DKD rats and modulated Th17/Treg balance through the PD-1/PD-L1 pathway. These findings provide a novel mechanism and basis for the clinical use of PMSCs in the treatment of DKD.

Effects and mechanisms of total flavones of Abelmoschus manihot in inhibiting podocyte necroptosis and renal fibrosis in diabetic kidney disease

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.

Repeated administration of adipose-derived mesenchymal stem cells added on beneficial effects of empagliflozin on protecting renal function in diabetic kidney disease rat

BackgroundDiabetic kidney disease (DKD) is one of the most significant public health burdens worldwide. This study explored the renal protections of combined adipose-derived mesenchymal stem cells (ADMSCs) and empagliflozin (EMPA) in DKD rats. MethodsAdult-male-SD rats were equally allocated into group 1 (sham-operated-control), group 2 (DKD), group 3 (DKD + EMPA/20 mg/kg/day since day-14 after CKD-induction), group 4 [DKD + ADMSCs (6.0 × 105/intrarenal-arterial-injection/post-day-28, followed by 1.2 × 106/intravenous injection post-days 35 and 42 after CKD-induction, i.e., defined as repeated administration)] and group 5 (DKD + ADMSCs + EMPA) and kidney was harvested post-day-60 CKD-induction. ResultsThe result showed that the blood sugar and circulatory levels of BUN/creatinine and the ratio of urine protein/creatinine at day 60 were greatly increased in group 2 as compared the SC (i.e., group 1), significantly increased in groups 3 and 4 than in groups 5, but these parameters showed the similar manner in groups 3 and 4, except for blood sugar that was significantly lower in group 3 than in group 4 (all p < 0.0001). The protein levels of inflammation (NF-κB/FNF-α/MMP-9)/oxidative-stress (NOX-1/NOX-2/oxidized protein/p22-phox)/apoptosis (cleaved-caspase-3/cleaved-PARP/mitochondrial-Bax)/fibrosis (TGF-β/Smad 3)/mitochondrial/DNA-damaged (p-DRP1/γ-H2AX) biomarkers revealed a similar manner of creatinine level among the groups (all p < 0.0001). Kidney injury score/fibrotic area/oxidative-stress score (8-OHdG) and cellular levels of kidney-damaged biomarkers (KIM-1/γ-H2AX) showed a unanimous manner. In contrast, the cellular expressions of podocyte components (ZO-1/synaptopodin) revealed an antithetical manner of creatinine among the groups (all p < 0.0001). ConclusionCombined ADMSCs-EMPA was superior to just one therapy for protecting kidney function and ultra-structural integrity in DKD rodents.

#4825 AUTOPHAGY REGULATES TUBULAR CELL SENESCENCE IN DIABETIC KIDNEY DISEASE

Abstract Background and Aims Cellular senescence commonly occurs in innate renal cells of diabetic kidney disease (DKD) and autophagy may be implicated. However, the characteristics of senescent cells in different pathological stages of DKD, and the driving force behind the cell senescence remain primarily unknown. This study aimed to examine kidney cellular senescence and its relationship to renal function in DKD and the effect of autophagy on high-glucose-induced cellular senescence. Method According to the DKD pathologic type, 46 patients diagnosed with renal biopsy were categorized into I IIa IIb III IV classes. The other four normal kidney specimens taken from patients with renal trauma were as the control group. Pathological changes in kidney tissues were detected by PAS staining, and the expressions of autophagy-related protein LC3 and senescence marker p21 were demonstrated by immunohistochemical staining. DKD rats were established by intraperitoneal injection with streptozotocin (STZ). HK-2 cells were cultured with 35mM glucose with or without autophagy inhibitor (3-methyladenine, 3-MA) and lysosomal inhibitor (chloroquine CQ). Immunohistochemistry and Western Blot were used to detect the expression of the senescence markers p53 and p21, as well as the autophagy-related proteins LC3 and p62. The electron microscopy technique was used to observe the number of autophagosomes in the DKD rats and each group of HK-2 cells. Results (1) In renal tissues of DKD patients, p21 infiltrated the interstitium and tubules, while LC3 was primarily expressed in the tubules. Both p21 and LC3 expression increased over time as the disease progressed (P&amp;lt;0.05) and were directly proportional to blood creatinine and proteinuria, respectively. Additionally, p21 and LC3 expression were positively correlated. (2) Compared with the control group, the renal tissue of DKD rats displayed higher levels of p21, p53, LC3, and p62 expression (P &amp;lt; 0.05), higher levels of renal tubular autophagosomes, and increased co-expression of p21 and LC3 (P&amp;lt; 0.05). (3) HK-2 cells treated with high glucose exhibited increased expression of p21, p53, p62 and LC3, as well as the number of autophagosomes (P&amp;lt;0.05). (4) 3-MA reduced p21 and p53 expression compared to the high glucose group (P&amp;lt; 0.05), whereas the opposite trend was observed in the CQ treatment (P&amp;lt;0.05). Conclusion Renal tubular cell senescence is closely associated with DKD progression. Autophagic flow may be involved in high glucose-induced renal tubular cell senescence.

MTOR/ULK1 signaling axis mediated ultrashort wave regulation of autophagy to alleviate diabetic kidney disease.

Diabetic kidney disease (DKD) is closely related to autophagy and inflammation. The mTOR/unc-51 like autophagy activating kinase 1 (ULK1) signaling axis is involved in the regulation of autophagy. Ultrashort wave (USW) therapy has been extensively studied in inflammatory diseases. However, the therapeutic effect of USW on DKD and the role of the mTOR/ULK1 signaling axis in USW interventions remain uncertain. This study aimed to explore the therapeutic effects of USW on DKD rats and the role of the mTOR/ULK1 signaling axis in USW interventions. A DKD rat model was established using a high-fat diet (HFD)/sugar diet and streptozocin (STZ) induction. The optimal duration of USW intervention was determined using different USW treatments. The levels of metabolism, inflammation and fibrosis associated with kidney injury in rats were measured. Western blot analysis was performed on the related indexes of autophagy and the mTOR/ULK1 signaling axis. In DKD rats, microalbuminuria (MAU), glucose (GLU), creatinine (CRE), and blood urea nitrogen (BUN) levels decreased after the USW intervention. Levels of interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), immunoglobulin M (IgM), immunoglobulin G (IgG), IL-18, tumor necrosis factor alpha (TNF-α), and IL-6 decreased in the USW group compared to the model group. The IL-10 and arginase (Arg-1) levels were increased in the USW group. The content of fibrosis-related indexes (vascular endothelial growth factor (VEGF), fibronectin (FN), type IV collagen, and type I collagen) decreased in the urine of the DKD rats. After USW treatment, LC3B and Beclin1 levels increased, while the level of p62 decreased. The levels of nephrin, podocin and synaptopodin increased. Ultrashort wave could reduce p-mTOR/mTOR ratios and increase ULK1 expression. After the overexpression of ULK1, the levels of LC3B and Beclin1 were higher in the overexpression (oe)-ULK1 group than in the oe-negative control (NC) group, while the level of p62 decreased. After mTOR activation, LC3B and ULK1 expression decreased, while CRE, BUN, MAU, and GLU levels increased. Ultrashort wave alleviated kidney injury induced by the HFD/sugar diet and STZ. The USW intervention reversed the decreased autophagy levels in the DKD rats. The mTOR/ULK1 signaling axis mediated USW to promote autophagy.

Early growth response factor 1 upregulates pro-fibrotic genes through activation of TGF-β1/Smad pathway via transcriptional regulation of PAR1 in high-glucose treated HK-2 cells

Tubulointerstitial fibrosis (TIF) makes a key role in diabetic kidney disease (DKD). In this study, we revealed that the expressions of Egr1 and protease-activated receptor 1 (PAR1) were increased in renal tissues of DKD rats. In vitro experiments demonstrated that both Egr1 overexpression and high glucose (HG) condition could promote the expressions of PAR1, fibronectin (FN) and collagen I (COL I). Furthermore, HG stimulation enhanced the binding capacity of Egr1 to PAR1 promoter. Both HG condition and Egr1 upregulation could increase, and thrombin inhibitor did not affect activity of TGF-β1/Smad pathway via PAR1. Collectively, Egr1 is involved in TIF of DKD partly through activating TGF-β1/Smad pathway via transcriptional regulation of PAR1 in HG treated HK-2 cells.

Evaluation of Early Diabetic Kidney Disease Using Ultrasound Localization Microscopy: A Feasibility Study.

The purpose of this study is to detect the hemodynamic changes of microvessels in the early stage of diabetic kidney disease (DKD) and to test the feasibility of ultrasound localization microscopy (ULM) in early diagnosis of DKD. In this study, streptozotocin (STZ) induced DKD rat model was used. Normal rats served as the control group. Conventional ultrasound, contrast-enhanced ultrasound (CEUS), and ULM data were collected and analyzed. The kidney cortex was divided into four segments, which are 0.25-0.5 mm (Segment 1), 0.5-0.75 mm (Segment 2), 0.75-1 mm (Segment 3), and 1-1.25 mm (Segment 4) away from the renal capsule, respectively. The mean blood flow velocities of arteries and veins in each segment were separately calculated, and also the velocity gradients and overall mean velocities of arteries and veins. Mann-Whitney U test was used for comparison of the data. Quantitative results of microvessel velocity obtained by ULM show that the arterial velocity of Segments 2, 3, and 4, and the overall mean arterial velocity of the four segments in the DKD group are significantly lower than those in the normal group. The venous velocity of Segment 3 and the overall mean venous velocity of the four segments in the DKD group are higher than those in the normal group. The arterial velocity gradient in the DKD group is lower than that in the normal group. ULM can visualize and quantify the blood flow and may be used for early diagnosis of DKD.