Tubular Injury In Diabetic Nephropathy Research Articles

Lipid nephrotoxicity mediated by HIF-1α activation accelerates tubular injury in diabetic nephropathy

This study is intended to explore the effect of hypoxia-inducible factor-1α (HIF-1α) activation on lipid accumulation in the diabetic kidney. A type 1 diabetic rat model was established by STZ intraperitoneal injection. Cobalt chloride (CoCl2) and YC-1 were used as the HIF-1α activator and antagonist, respectively. CoCl2 treatment significantly increased HIF-1α expression, accelerated lipid deposition, and accelerated tubular injury in diabetic kidneys. In vitro, CoCl2 effectively stabilized HIF-1α and increased its transportation from the cytoplasm to the nucleus, which was accompanied by significantly increased lipid accumulation in HK-2 cells. Furthermore, results obtained in vivo showed that HIF-1α protein expression in the renal tubules of diabetic rats was significantly downregulated by YC-1 treatment. Meanwhile, lipid accumulation in the tubules of the DM + YC-1 group was markedly decreased in comparison to the DM + DMSO group. Accordingly, PAS staining revealed that the pathological injury caused to the tubular epithelial cells was alleviated by YC-1 treatment. Furthermore, the blood glucose level, urine albumin creatinine ratio, and NAG creatinine ratio in the DM + YC-1 group were significantly decreased compared to the DM + DMSO group. Moreover, the protein expression levels of transforming growth factor β1 (TGF-β1) and connective tissue growth factor (CTGF) in diabetic kidneys were decreased by YC-1 treatment. Our findings demonstrate that the activation of HIF-1α contributed to interstitial injury in a rat model of diabetic nephropathy and that the underlying mechanism involved the induction of lipid accumulation.

Depression of LncRNA DANCR alleviates tubular injury in diabetic nephropathy by regulating KLF5 through sponge miR-214-5p

ObjectiveDiabetic nephropathy (DN) manifests a critical aspect in the form of renal tubular injury. The current research aimed to determine the function and mechanism of long non-coding ribonucleic acid (LncRNA) differentiation antagonising non-protein coding RNA (DANCR), with a focus on its impact on renal tubular injury.MethodsQuantitative reverse transcription polymerase chain reaction was employed to analyze the RNA levels of DANCR in the serum of patients with DN or human proximal tubular epithelial cells (human kidney 2 [HK2]). The diagnostic significance of DANCR was assessed using a receiver operating characteristic curve. A DN model was established by inducing HK-2 cells with high glucose (HG). Cell proliferation, apoptosis, and the levels of inflammatory factors, reactive oxygen species (ROS), and malondialdehyde (MDA) were detected using the Cell Counting Kit − 8, flow cytometry, and enzyme-linked immunosorbent assay. The interaction between microRNA (miR)-214-5p and DANCR or Krüppel-like factor 5 (KLF5) was investigated using RNA immunoprecipitation and dual-luciferase reporter assays.ResultsElevated levels of DANCR were observed in the serum of patients with DN and HG-inducted HK-2 cells (P < 0.05). DANCR levels effectively identified patients with DN from patients with type 2 diabetes mellitus. Silencing of DANCR protected against HG-induced tubular injury by restoring cell proliferation, inhibiting apoptosis, and reducing the secretion of inflammatory factors and oxidative stress production (P < 0.05). DANCR functions as a sponge for miR-214-5p, and the mitigation of DANCR silencing on HG-induced renal tubular injury was partially attenuated with reduced miR-214-5p (P < 0.05). Additionally, KLF5 was identified as the target of miR-214-5p.ConclusionDANCR was identified as diagnostic potential for DN and the alleviation of renal tubular injury via the miR-214-5p/KLF5 axis, following DANCR silencing, introduces a novel perspective and approach to mitigating DN.

An aldose reductase inhibitor, WJ-39, ameliorates renal tubular injury in diabetic nephropathy by activating PINK1/Parkin signaling

Renal tubular injury is a critical factor during the early stages of diabetic nephropathy (DN). Proximal tubular epithelial cells, which contain abundant mitochondria essential for intracellular homeostasis, are susceptible to disruptions in the intracellular environment, making them especially vulnerable to diabetic state disorders, which may be attributed to their elevated energy requirements and reliance on aerobic metabolism. It is widely thought that overactivation of the polyol pathway is implicated in DN pathogenesis, and inhibition of aldose reductase (AR), the rate-limiting enzyme in this pathway, represents a promising therapeutic avenue. WJ-39, a novel aldose reductase inhibitor, was investigated in this study for its protective effects on renal tubules in DN and the underlying mechanisms. Our findings revealed that WJ-39 significantly ameliorated the renal tubular morphology in high-fat diet (HFD)/streptozotocin (STZ)-induced DN rats, concurrently inhibiting fibrosis. Notably, WJ-39 safeguarded the structure and function of renal tubular mitochondria by enhancing mitochondrial dynamics. This involved the regulation of mitochondrial fission and fusion proteins and the promotion of PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy. Furthermore, WJ-39 demonstrated the inhibition of endogenous apoptosis by mitigating the production of mitochondrial reactive oxygen species (ROS). The protective effects of WJ-39 on mitochondria and apoptosis were countered in high glucose-treated HK-2 cells upon transfection with PINK1 siRNA. Overall, our findings suggest that WJ-39 protects the structural and functional integrity of renal tubules in DN, which may be attributed to its capacity to inhibit aldose reductase activity, activate the PINK1/Parkin signaling pathway, promote mitophagy, and alleviate apoptosis.

Crocin protects against endoplasmic reticulum stress-related tubular injury in diabetic nephropathy via the activation of the PI3K/AKT/Nrf2 pathway.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease, but the current treatment is not satisfactory. Crocin is a major bioactive compound of saffron with antioxidant and anti-endoplasmic reticulum stress (ERS) abilities used to treat diabetes. This study specifically investigated whether crocin has a regulatory role in renal injury in DN. The experiment was divided into control, (db/m mice), model (db/db mice), and experimental groups (db/db mice were intraperitoneally injected with 40 mg/kg crocin). Renal function-related indicators (Scr, BUN, FBG, UP, TG, TC, ALT, and AST) and oxidative stress-related indicators (ROS, MDA, GSH, SOD, and CAT) were assessed. The pathological changes of renal tissues were confirmed by HE, Masson, PAS, and TUNEL staining. The levels of ERS-related proteins (GRP78 and CHOP), apoptosis-related proteins, and PI3K/AKT and Nrf2 pathways-related proteins in renal tissue were detected. In db/db mice, renal function-related indicators, apoptotic cells of renal tissues, the contents of ROS and MDA as well as the expressions of CHOP, GRP78, and Bax were increased, the degree of renal tissue damage was aggravated, while the contents of GSH, SOD, and CAT, as well as the protein levels of Nrf2, PARP, anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xl) were decreased compared to the db/m mice. However, crocin treatment reversed the above-mentioned situation. The expressions of the PI3K/AKT and Nrf2 pathways-related proteins were also activated by crocin. Crocin inhibited oxidative stress and ERS-induced kidney injury in db/db mice by activating the PI3K/AKT and Nrf2 pathways.

Renalase regulates renal tubular injury in diabetic nephropathy via the p38MAPK signaling pathway.

Diabetic nephropathy (DN) is an important complication of diabetes and the leading cause of end-stage renal disease globally. Renal tubular damage occurs to varying degrees in the early stages of DN prior to glomerular damage. Renalase (RNLS) is an amine oxidase, which is produced and secreted by the renal tubular epithelial cells. RNLS is reportedly closely related to renal tubular injury in acute and chronic kidney diseases. Herein, we aimed to evaluate the changes in tubular RNLS expression in DN and its correlation with DN-associated renal tubular injury. Conditional permanent renal tubular epithelial rat-cell line NRK-52E was transfected with pcDNA3-RNLS plasmid or administered recombinant rat RNLS protein and high glucose (HG) dose. A total of 22 adult Sprague-Dawley rats were randomly divided into the control (CON, n = 10) or diabetic nephrology (DN, n = 12) group. Random blood glucose levels of the rats were measured by sampling of the caudal vein weekly. After 8 weeks, the rat's body weight, 24-h urinary albumin concentration, and right kidney were evaluated. Our study suggested the decreased expression levels of RNLS in renal tissue and renal tubular epithelial cells in DN rats, accompanied by renal tubulointerstitial fibrosis, apoptosis of renal tubular epithelial cells, and activation of the p38MAPK signal pathway. Reversing the low RNLS expression can reduce the level of p38MAPK phosphorylation and delay renal tubular injury. Thus, the reduction of renal tubular RNLS expression in DN mediates tubulointerstitial fibrosis and cell apoptosis via the activation of the p38MAPK signal pathway. RNLS plays a key mediating role in DN-associated tubular injury via p38MAPK, which provides new therapeutic targets and a theoretical basis for early prevention and treatment of DN.

DsbA-L alleviates tubular injury in diabetic nephropathy by activating mitophagy through maintenance of MAM integrity.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) regulate ATG14- and Beclin1-mediated mitophagy and play key roles in the development of diabetic nephropathy (DN). DsbA-L is mainly located in MAMs and plays a role in renoprotection, but whether it activates mitophagy by maintaining MAM integrity remains unclear. In the present study, we found that renal tubular damage was further aggravated in diabetic DsbA-L-/- mice compared with diabetic mice and that this damage was accompanied by disrupted MAM integrity and decreased mitophagy. Furthermore, notably decreased expression of ATG14 and Beclin1 in MAMs extracted from the kidneys of diabetic DsbA-L-/- mice was observed. In vitro, overexpression of DsbA-L reversed the disruption of MAM integrity and enhanced mitophagy in HK-2 cells, a human proximal tubular cell line, after exposure to high-glucose (HG) conditions. Additionally, compared with control mice, DsbA-L-/- mice were exhibited down-regulated expression of helicase with zinc finger 2 (HELZ2) in their kidneys according to transcriptome analysis; HELZ2 serves as a cotranscription factor that synergistically functions with PPARα to promote the expression of mitofusin 2 (MFN-2). Treatment of HK-2 cells with MFN-2 siRNA resulted in MAM uncoupling and decreased mitophagy. Moreover, HG notably reduced the expression of HELZ2 and MFN-2 and inhibited mitophagy, and these effects were partially blocked by overexpression of DsbA-L and altered upon cotreatment with HELZ2 siRNA, HELZ2 overexpression or MK886 (PPARα inhibitor) treatment. These data indicate that DsbA-L alleviates diabetic tubular damage by activating mitophagy through maintenance of MAM integrity via the HELZ2/MFN-2 pathway.

The Role of the NLRP3 Inflammasome in Mediating Glomerular and Tubular Injury in Diabetic Nephropathy.

The NOD-like receptor protein 3 (NLRP3) inflammasome is a multi-protein signalling complex integral to the chronic inflammatory response, activated in response to sterile and non-sterile cellular damage. The assembly and activation of the NLRP3 inflammasome comprise a two-step process involving nuclear factor kappa B (NFkB)-mediated priming, followed by canonical, non-canonical or alternative signalling pathways. These result in the maturation and release of inflammatory cytokines interleukin 1 beta (IL1ß) and interleukin-18 (IL18), which are associated with chronic inflammatory conditions including diabetic kidney disease. Diabetic nephropathy is a condition affecting ∼40% of people with diabetes, the key underlying pathology of which is tubulointerstitial inflammation and fibrosis. There is growing evidence to suggest the involvement of the NLRP3 inflammasome in this chronic inflammation. Early deterioration of kidney function begins in the glomerulus, with tubular inflammation dictating the progression of late-stage disease. Priming and activation of the NLRP3 inflammasome have been linked to several clinical markers of nephropathy including proteinuria and albuminuria, in addition to morphological changes including mesangial expansion. Treatment options for diabetic nephropathy are limited, and research that examines the impact of directly targeting the NLRP3 inflammasome, or associated downstream components are beginning to gain favour, with several agents currently in clinical trials. This review will explore a role for NLRP3 inflammasome activation and signalling in mediating inflammation in diabetic nephropathy, specifically in the glomerulus and proximal tubule, before briefly describing the current position of therapeutic research in this field.

Formononetin Attenuates Renal Tubular Injury and Mitochondrial Damage in Diabetic Nephropathy Partly via Regulating Sirt1/PGC-1α Pathway.

Mitochondrial abnormality is one of the main factors of tubular injury in diabetic nephropathy (DN). Formononetin (FMN), a novel isoflavonoid isolated from Astragalus membranaceus, has diverse pharmacological activities. However, the beneficial effects of FMN on renal tubular impairment and mitochondrial dysfunction in DN have yet to be studied. In this study, we performed in vivo tests in Streptozotocin (STZ) -induced diabetic rats to explore the therapeutic effects of FMN on DN. We demonstrated that FMN could ameliorate albuminuria and renal histopathology. FMN attenuated renal tubular cells apoptosis, mitochondrial fragmentation and restored expression of mitochondrial dynamics-associated proteins, such as Drp1, Fis1 and Mfn2, as well as apoptosis-related proteins, such as Bax, Bcl-2 and cleaved-caspase-3. Moreover, FMN upregulated the protein expression of Sirt1 and PGC-1α in diabetic kidneys. In vitro studies further demonstrated that FMN could inhibit high glucose-induced apoptosis of HK-2 cells. FMN also reduced the production of mitochondrial superoxide and alleviated mitochondrial membrane potential (MMP) loss. Furthermore, FMN partially restored the protein expression of Drp1, Fis1 and Mfn2, Bax, Bcl-2, cleaved-caspase-3, Sirt1 and PGC-1α in HK-2 cells exposure to high glucose. In conclusion, FMN could attenuate renal tubular injury and mitochondrial damage in DN partly by regulating Sirt1/PGC-1α pathway.

PACS-2 Ameliorates Tubular Injury by Facilitating Endoplasmic Reticulum-Mitochondria Contact and Mitophagy in Diabetic Nephropathy.

Mitochondria-associated endoplasmic reticulum membrane (MAM) may have a role in tubular injury in diabetic nephropathy (DN), but the precise mechanism remains unclear. Here, we demonstrate that the expression of phosphofurin acidic cluster sorting protein 2 (PACS-2), a critical regulator of MAM formation, is significantly decreased in renal tubules of patients with DN, and PACS-2 expression is positively correlated with renal function and negatively correlated with degrees of tubulointerstitial lesions. Conditional deletion of Pacs-2 in proximal tubules (PTs) aggravates albuminuria and tubular injury in a streptozotocin-induced mouse model of diabetes. Mitochondrial fragmentation, MAM disruption, and defective mitophagy accompanied by altered expression of mitochondrial dynamics and mitophagic proteins, including Drp1 and Becn1, are observed in tubules of diabetic mice; these changes are more pronounced in PT-specific Pacs-2 knockout mice. In vitro, overexpression of PACS-2 in HK-2 cells alleviates excessive mitochondrial fission induced by high glucose concentrations through blocking mitochondrial recruitment of DRP1 and subsequently restores MAM integrity and enhances mitophagy. Mechanistically, PACS-2 binds to BECN1 and mediates the relocalization of BECN1 to MAM, where it promotes the formation of mitophagosome. Together, these data highlight an important but previously unrecognized role of PACS-2 in ameliorating tubular injury in DN by facilitating MAM formation and mitophagy.

The CXCL1-CXCR2 Axis Mediates Tubular Injury in Diabetic Nephropathy Through the Regulation of the Inflammatory Response.

Diabetic nephropathy (DN) is one of the most severe complications of diabetes. Inflammation mediated by inflammatory factors is thought to accelerate the progression of renal damage in DN. However, which inflammatory factors mediate the inflammatory response in DN remains unclear. In this study, we determined that the CXCL1-mediated inflammatory response may play an essential role in DN progression through bioassays. Subsequently, we observed that the expression of CXCL1 and its receptor (CXCR2) was significantly increased in the kidneys of mice with HFD + STZ induced diabetes and DN patients. In addition, inhibition of the CXCL1/CXCR2 axis by repertaxin alleviates renal inflammation and pathological damage in the kidneys of db/db mice. Finally, we noted that the CXCL1/CXCR2 axis might lead to inflammatory damage through phosphorylated NF-κB and further activate the NLRP3 inflammasome. Our results revealed the role of the CXCL1/CXCR2 axis in DN progression for the first time, which may be a novel therapeutic target for DN.

Plasma levels of tissue inhibitor of metalloproteinase-1 in patients with type 1 diabetes mellitus associate with early diabetic neuropathy and nephropathy.

Background:Tissue inhibitor of metalloproteinase-1 (TIMP-1) has been suggested as a marker for abnormal regulation of tissue remodelling in type 1 diabetes. Metalloproteinase-9 (MMP-9) has been associated with matrix turnover, and Neutrophil gelatinase associated lipocalin (NGAL) is a marker of tubular injury in diabetic nephropathy. The aim was to analyse these biomarkers to unmask early diabetic complications.Methods:Thirty-three type 1 diabetes patients, aged 20–35 years, and disease duration 20 ± 5.3 years were included. Along with clinical examination, neurophysiological measurements, routine biochemistry, plasma concentrations of TIMP-1, MMP-9 and NGAL were determined with immunoenzymatic techniques.Results:TIMP-1 correlated with abnormal unilateral and bilateral vibratory sense foot perception (r = −0.49 and r = −0.51, respectively), foot neuropathy impairment assessment score (NIA; r = −0.55), neuropathy symptom assessment score (r = 0.42), microalbuminuria (r = 0.50) and eGFR (r = −0.45). MMP-9 correlated with impaired foot NIA (r = 0.51). Multiple regression analysis showed an association for TIMP-1 (p = 0.004) with impaired neurophysiological examinations and renal dysfunction along with NGAL (p = 0.016 and p = 0.015 respectively).Conclusions:This study suggests that plasma levels of TIMP-1, MMP-9 and NGAL may serve as useful biomarkers in unravelling subclinical neuropathy and nephropathy in type 1 diabetes.

HIF-1α ameliorates tubular injury in diabetic nephropathy via HO-1-mediated control of mitochondrial dynamics.

ObjectivesIn diabetic nephropathy (DN), hypoxia‐inducible factor‐1α (HIF‐1α) activation in tubular cells plays an important protective role against kidney injury. The effects may occur via the target genes of HIF‐1α, such as haem oxygenase‐1 (HO‐1), but the exact mechanisms are incompletely understood.Materials and methodsMice with proximal tubule‐specific knockout of HIF‐1α (PT‐HIF‐1α−/− mice) were generated, and diabetes was induced in these mice by streptozotocin (STZ) injection. In addition, to mimic a hypoxic state, cobaltous chloride (CoCl2) was applied to HK‐2 cells.ResultsOur study first verified that conditional knockout of HIF‐1α worsened tubular injury in DN; additionally, aggravated kidney dysfunction, renal histopathological alterations, mitochondrial fragmentation, ROS accumulation and apoptosis were observed in diabetic PT‐HIF‐1α−/− mice. In vitro study showed that compared to control group, HK‐2 cells cultured under hypoxic ambiance displayed increased mitochondrial fragmentation, ROS production, mitochondrial membrane potential loss and apoptosis. These increases were reversed by overexpression of HIF‐1α or treatment with a HO‐1 agonist. Importantly, cotreatment with a HIF‐1α inhibitor and a HO‐1 agonist rescued the HK‐2 cells from the negative impacts of the HIF‐1α inhibitor.ConclusionsThese data revealed that HIF‐1α exerted a protective effect against tubular injury in DN, which could be mediated via modulation of mitochondrial dynamics through HO‐1 upregulation.

AdipoRon Protects against Tubular Injury in Diabetic Nephropathy by Inhibiting Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress has been reported to play a pivotal role in diabetic nephropathy (DN). AdipoRon is a newly developed adiponectin receptor agonist that provides beneficial effects for diabetic mice; however, its underlying mechanism remains to be delineated. Here, we demonstrated increased expression levels of ER stress markers, accompanied by upregulated levels of proinflammatory cytokines and increased expression of collagen I, fibronectin, Bax, and cleaved caspase 3 in the kidneys of db/db mice compared with control mice. Decreased expression of adiponectin receptor 1 (AdipoR1) and phosphorylated 5′AMP-activated kinase (p-AMPK) was also observed in the kidneys of db/db mice. However, these alterations were partially reversed by intragastric gavage with AdipoRon. In vitro, AdipoRon alleviated high-glucose-induced ER stress, oxidative stress, and apoptosis in HK-2 cells, a human tubular cell line. Moreover, AdipoRon restored the expression of AdipoR1 and p-AMPK in HK-2 cells exposed to high-glucose conditions. Additionally, these effects were partially abrogated by pretreatment with AdipoR1 siRNA, but this abrogation was ameliorated by cotreatment with AICAR, an AMPK activator. Furthermore, the effects of AdipoRon were also partially abolished by cotreatment with compound C. Together, these results suggest that AdipoRon exerts favorable effects on diabetes-induced tubular injury in DN by inhibiting ER stress mediated by the AdipoR1/p-AMPK pathway.

Pathological significance of urinary complement activation in diabetic nephropathy: A full view from the development of the disease.

Aims/IntroductionThe aim of the present study was to obtain a full view of the changes of urinary complement activation products in the development of diabetic nephropathy and explore their possible significance in the disease process.Materials and methodsA total of 62 patients at different stages of diabetic nephropathy, 20 diabetes patients without nephropathy and 20 healthy persons were enrolled. Urinary complement activation products, including C3a, C5a and C5b‐9, were measured, and their associations with the progression of the disease were analyzed.ResultsThe urinary complement activation products increased markedly since the proteinuria stage, and were parallel with the progression of diabetic nephropathy. More severe renal tubular damage was observed in patients with higher levels of urinary complement activation products. The urinary complement activation products levels correlated closely with renal tubulointerstitial injury score and relative tubular interstitial volume. Multivariate regression analysis showed that elevated urinary complement activation products were independent risk factors for tubular injury in diabetic nephropathy patients.ConclusionsUrinary complement activation might have a role in renal tubular interstitial injury in patients with diabetic nephropathy, especially in patients at a later stage of the disease.

The Association between Urinary N-Acetyl-ß-D-Glucosaminidase and Cardiovascular Autonomic Neuropathy in Patients with Diabetes

Background: Cardiovascular autonomic neuropathy (CAN) is a microvascular complication of diabetes, which involves autonomic nerve fibers innervating the heart and vessels, resulting in increased cardiovascular morbidity and mortality. Previous studies reported that diabetic CAN is related to microalbuminuria. However, it is unclear whether CAN is related with urinary N-acetyl-β-D-glucosaminidase (uNAG), which was suggested as an early marker of renal tubular injury in diabetic nephropathy recently. Methods: Cross-sectional data of 1052 patients with diabetes were analyzed who had tests for uNAG and autonomic function test at outpatient clinic. The presence and severity of CAN were assessed by the five autonomic function tests. Results: The association between uNAG and the presence of CAN in patients with type 1 diabetes (T1D) (n=161) was significant in multivariate analysis (OR 1.20, 95% CI 1.03-39; p = 0.018) after correction of variables including age, duration of diabetes, glycemic index and albuminuria, but it was insignificant in patients with type 2 diabetes (T2D) (n=891). Among quartile groups divided by uNAG levels, prevalence of CAN was significantly increased across with higher quartiles of uNAG level only in patients with T1D (p = 0.013). The association between uNAG and the total CAN score, assessed as the severity of CAN, was significant in both patients with T1D (p = 0.033) and T2D (p = 0.006) in multivariate analyses. Conclusion: Elevated uNAG is associated with the prevalence of CAN only in patients with T1D, and it is also related to the severity of CAN in both patients with T1D and T2D. It suggests that elevated uNAG might be a potential marker for the development and progression of CAN in patients with diabetes. Disclosure M. Choi: None. J. Jun: None. G. Kim: None. S. Jin: None. K. Hur: None. M. Lee: None. J. Kim: None.

Reactive oxygen species promote tubular injury in diabetic nephropathy: The role of the mitochondrial ros-txnip-nlrp3 biological axis

NLRP3/IL-1β activation via thioredoxin (TRX)/thioredoxin-interacting protein (TXNIP) following mitochondria ROS (mtROS) overproduction plays a key role in inflammation. However, the involvement of this process in tubular damage in the kidneys of patients with diabetic nephropathy (DN) is unclear. Here, we demonstrated that mtROS overproduction is accompanied by decreases in TRX expression and TXNIP up-regulation. In addition, we discovered that mtROS overproduction is also associated with increases in NLRP3/IL-1β and TGF-β expression in the kidneys of patients with DN and db/db mice. We reversed these changes in db/db mice by administering a peritoneal injection of MitoQ, an antioxidant targeting mtROS. Similar results were observed in human tubular HK-2 cells subjected to high-glucose (HG) conditions and treated with MitoQ. Treating HK-2 cells with MitoQ suppressed the dissociation of TRX from TXNIP and subsequently blocked the interaction between TXNIP and NLRP3, leading to the inhibition of NLRP3 inflammasome activation and IL-1β maturation. The effects of MitoQ were enhanced by pretreatment with TXNIP siRNA and abolished by pretreatment with monosodium urate (MSU) and TRX siRNA in vitro. These results suggest that mitochondrial ROS-TXNIP/NLRP3/IL-1β axis activation is responsible for tubular oxidative injury, which can be ameliorated by MitoQ via the inhibition of mtROS overproduction.

SIRT1 activator ameliorates the renal tubular injury induced by hyperglycemia in vivo and in vitro via inhibiting apoptosis.

We aimed to explore the role of SIRT1 in apoptosis in human kidney proximal tubule epithelial (HK-2) cells, and to determine whether resveratrol (RSV, a SIRT1 activator) could ameliorate apoptosis in rats with streptozotocin-induced diabetes mellitus (DM) and/or in high glucose (HG, 30mM) - stimulated HK-2 cells. Rats were distributed randomly into three groups: 1) control group, 2) DM group, and 3) DM with RSV group (DM+RSV; rats treated with 30mg/kg/d of RSV for 16 weeks). The physical, biochemical, and morphological parameters were then examined. Additionally, the deacetylase activity of SIRT1, and the expression levels of SIRT1 and of representative apoptosis markers, such as p53, acetylated p53, cleaved caspase-3, caspase-9, and cleaved PARP, were measured. HK-2 cells were stimulated by HG for different lengths of time to study the effect of HG on apoptosis. HK-2 cells were treated with or without RSV (25μM) to investigate if RSV has a protective effect on HG-induced apoptosis. A gene-specific small interfering RNA against SIRT1 was used to study the role of SIRT1 in apoptosis. More apoptosis was found in the DM rats than in the control rats. Similarly, the expression levels of cleaved caspase-3, cleaved PARP, and acetylated p53 were significantly higher, and the level of SIRT1 was significantly lower, in the HK-2 cells that were cultured under HG conditions than those in the HK-2 cells that were cultured under low glucose (5.5mM) conditions. Notably, treatment with RSV lessened the HG-induced changes in the levels of apoptosis indicators, and this inhibition of HG-induced apoptosis in HK-2 cells by RSV treatment was abolished by SIRT1 silencing. Our study showed that hyperglycemia contributes to apoptosis in rat kidney and HK-2 cells. SIRT1 activation by RSV can reduce urinary albumin excretion and proximal tubule epithelial apoptosis both in vitro and in vivo. Based on our study, SIRT1/p53 axis played an important role in the hyperglycemia induced apoptosis. These findings indicated that the increased expression of SIRT1, mediated by RSV, is a possible mechanism by which RSV prevents renal tubular injury in diabetic nephropathy (DN). So RSV has great clinical significance and could provide the basis for the new way to effective treatment to contain the morbidity and mortality associated with DN.

Atrasentan increased the expression of klotho by mediating miR-199b-5p and prevented renal tubular injury in diabetic nephropathy

Atrasentan is a promising therapy for treating diabetic nephropathy (DN). Here we evaluated whether atrasentan down-regulated the miR-199b-5p expression, thereby increasing klotho and preventing renal tubular injury in DN. One-hundred patients with type 2 diabetes mellitus (T2DM) and 40 healthy subjects were included. A DN mice model was established by an injection of streptozotocin (STZ). Human renal proximal tubular epithelial HK-2 cells were exposed to high glucose (20 mmol/L). Treated the mice and HK-2 cells with atrasentan, and we then investigated whether and how miR-199b-5p and Klotho were involved in preventing renal tubular injury in DN. In patients, the serum miR-199b-5p level increased and the klotho concentration decreased in accordance with elevated albuminuria. Atrasentan down-regulated miR-199b-5p and up-regulated klotho of the DN mice and HK-2 cells exposed to high glucose. High glucose promoted the binding of histone H3 to the miR-199b-5p promoter, and atrasentan canceled this effect. MiR-199b-5p targeted the 3′ UTR of klotho. Overexpression of miR-199b-5p canceled the effects of atrasentan on klotho expression and apoptosis of renal tubular cells in both in vivo and in vitro. The increased serum klotho, mediated by miR-199b-5p, is a possible mechanism by which atrasentan prevents renal tubular injury in DN.

Increased sphingosine 1-phosphate mediates inflammation and fibrosis in tubular injury in diabetic nephropathy.

Hyperglycemia induces all isoforms of transforming growth factor β (TGFβ), which in turn play key roles in inflammation and fibrosis that characterize diabetic nephropathy. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid, derived from sphingosine by the action of sphingosine kinase (SK). S1P mediates many biological processes, which mimic TGFβ signaling. To determine the role of SK1 and S1P in inducing fibrosis and inflammation, and the interaction with TGFβ-1, 2 and 3 signalling in diabetic nephropathy, human proximal tubular cells (HK2 cells) were exposed to normal (5mmol/L) or high (30mmol/L) glucose or TGFβ-1, -2, -3 ± an SK inhibitor (SKI-II) or SK1 siRNA. Control and diabetic wild type (WT) and SK1(-/-) mice were studied. Fibrotic and inflammatory markers, and relevant downstream signalling pathways were assessed. SK1 mRNA and protein expression was increased in HK2 cells exposed to high glucose or TGFβ1,-2,-3. All TGFβ isoforms induced fibronectin, collagen IV and macrophage chemoattractant protein 1 (MCP1), which were reversed by both SKI-II and SK1 siRNA. Exposure to S1P increased phospho-p44/42 expression, AP-1 binding and NFkB phosphorylation. WT diabetic mice exhibited increased renal cortical S1P, fibronectin, collagen IV and MCP1 mRNA and protein expression compared to SK1(-/-) diabetic mice. In summary, this study demonstrates that inhibiting the formation of S1P reduces tubulointerstitial renal inflammation and fibrosis in diabetic nephropathy.

Urinary angiotensinogen level and its risk factors in patients with " subclinical" diabetic nephropathy

Objective To evaluate the level and related factors of urinary angiotensinogen in patients with subclinical diabetic nephropathy. Methods (1)A total of 80 type 2 diabetic patients without angiotensin-converting enzyme inhibitor (ACEI) or angiotensin Ⅱ receptor blocker(ARB) treatment and hypertension were divided into 3 groups according to 24-hour urinary albuminuria and estimated glomerular filtration rate(eGFR): normoalbuminuria with normal glomerular filtration rate group[urinary microalbuminuria(UMA) <30 mg/24 h, 90≤eGFR <120 ml·min-1·(1.73 m2)-1, NC group, n=28], normoalbuminuria with glomerular hyperfiltration group[UMA<30 mg/24 h, eGFR≥120 ml·min-1·(1.73 m2)-1, SDN group, n=36], microalbuminuria group[30 h≤UMA<300 mg/24 h, 90≤eGFR<120 ml·min-1·(1.73 m2)-1, EDN group, n=16]. The levels of urinary angiotensinogen among various groups were compared.(2)The levels of urine retinol binding protein(RBP), N-acetyl-β-D-glucosaminadase, α-galactosidase, and β2-microglobubin were used to evaluate the renal tubular function. Then all the patients were divided into 4 groups according to the degree of renal tubular injury; the urinary angiotensinogen was compared and the independent related factors were analyzed. (3)20 type 2 diabetic patients with microalbuminuria and ACEI or ARB treatment were also enrolled in this study. The level of urinary angiotensinogen was compared between patients with and without ACEI or ARB treatment. Results Compared with NC group, the level of urinary angiotensinogen in the SDN group was lowered[(1.47±0.11 vs 1.59±0.11)ng/ml, P<0.05], but was raised in the EDN group[(1.62±0.15)ng/ml, P<0.05]. With the aggravation of renal tubular injury, the level of urinary angiotensinogen was increased. Multiple regression analysis showed that urinary angiotensinogen was positively correlated with RBP(P<0.05)and negatively correlated with eGFR(P<0.05). Compared with the patients without ACEI or ARB treatment, the level of urinary angiotensinogen was decreased in patients with ACEI or ARB treatment [(1.43±0.16 vs 1.62±0.15)ng/ml, P<0.05]. Conclusion The level of urinary angiotensinogen in patients with subclinical diabetic nephropathy is lowered, but is then raised in patients with microalbuminuria, which may participate in the progression of renal glomerular and tubular injury in diabetic nephropathy. The renin-angiotensin system inhibitors may delay the progression of diabetic nephropathy via decreasing urinary angiotensinogen level. (Chin J Endocrinol Metab, 2015, 31: 395-399) Key words: Subclinical; Diabetic nephropathy; Urinary angiotensinogen; Renal tubular injury