Response In Diabetic Nephropathy Research Articles

Ferroptosis: an important player in the inflammatory response in diabetic nephropathy.

Diabetic nephropathy (DN) is a chronic inflammatory disease that affects millions of diabetic patients worldwide. The key to treating of DN is early diagnosis and prevention. Once the patient enters the clinical proteinuria stage, renal damage is difficult to reverse. Therefore, developing early treatment methods is critical. DN pathogenesis results from various factors, among which the immune response and inflammation play major roles. Ferroptosis is a newly discovered type of programmed cell death characterized by iron-dependent lipid peroxidation and excessive ROS production. Recent studies have demonstrated that inflammation activation is closely related to the occurrence and development of ferroptosis. Moreover, hyperglycemia induces iron overload, lipid peroxidation, oxidative stress, inflammation, and renal fibrosis, all of which are related to DN pathogenesis, indicating that ferroptosis plays a key role in the development of DN. Therefore, this review focuses on the regulatory mechanisms of ferroptosis, and the mutual regulatory processes involved in the occurrence and development of DN and inflammation. By discussing and analyzing the relationship between ferroptosis and inflammation in the occurrence and development of DN, we can deepen our understanding of DN pathogenesis and develop new therapeutics targeting ferroptosis or inflammation-related regulatory mechanisms for patients with DN.

Total glucosides of peony regulate toll-like receptor to inhibit inflammatory response in diabetic nephropathy

Diabetic nephropathy (DN) is a common chronic disease affecting people’s life. Total glucosides of peony (TGP) regulates TLR4/NF-κB activation. This study aims at investigating the underlying mechanism of TGP in DN treatment. A retrospective analysis of clinical data was conducted to observe periodic acid-silver metheramine (PASM) staining and detect the expression of TLR4 and VEGF in DN. Additionally, rats were injected with Streptozocin (STZ) (30–60 mg) according to the ratio of 45–65 mg/kg to establish DN model. 8 weeks later, rats of experimental group were intraperitoneally injected with TGP. Kidney was collected for HE staining to observe morphological changes. Fibronectin expression was detected by histochemistry and the level of TLR4/NF-κB pathway-related proteins and apoptosis were detected by Western blot. PASM staining revealed focal tubular atrophy and interstitial fibrosis in DN patients. The renal medulla gradually disappeared and infiltration increased in control group, while inflammatory cells decreased in experimental group. The expression of fibronectin, TLR4 and p-p65 was decreased after TGP treatment. TGP treatment significantly decreased fibronectin expression and TGP treatment-induced inhibition of NF-κB signaling enhanced apoptotic activation of HK-2 cells, which was partially reversed by TLR4 and NF-κB inhibitors. TGP can inhibit inflammatory response of DN via regulating TLR4/NF-κB.

Immunological Approaches in the Treatment of Diabetic Nephropathy

Abstract: Diabetic nephropathy (DN), the leading cause of end-stage renal disease, has no definite treatment so far. In fact, a combination of metabolic, hemodynamic, and immunological factors are involved in the pathogenesis of DN; therefore, effective disease management requires a holistic approach to all predisposing contributors. Due to the recent findings about the role of inflammation in the initiation and progression of kidney injury in diabetic patients and considerable advances in immunotherapy methods, it might be useful to revise and reconsider the current knowledge of the potential of immunomodulation in preventing and attenuating DN. In this review, we have summarized the findings of add-on therapeutic methods that have concentrated on regulating inflammatory responses in diabetic nephropathy, including phosphodiesterase inhibitors, nuclear factor-kB inhibitors, Janus kinase inhibitors, chemokine inhibitors, anti-cytokine antibodies, cell therapy, and vaccination.

Network Meta-analysis of efficacy of Chinese patent medicine in treatment of inflammatory response in diabetic nephropathy

This study aimed to evaluate the efficacy and safety of various Chinese patent medicines in the treatment of inflammatory response in diabetic nephropathy(DN) based on network Meta-analysis. Randomized controlled trial(RCT) of oral Chinese patent medicines for improving inflammatory response in patients with DN was retrieved from CNKI, Wanfang, VIP, SinoMed, PubMed, Cochrane Library, EMbase, Web of Science, and other databases from database inception to October 2022. All investigators independently screened the literature, extracted data, and evaluated the quality. Stata 16.0 software and RevMan 5.4.1 were used to analyze the data of the literature that met the quality standards. Finally, 53 RCTs were included, involving 6 Chinese patent medicines. The total sample size was 4 891 cases, including 2 449 cases in the test group and 2 442 cases in the control group. The network Meta-analysis showed that(1) in terms of reducing TNF-α, the top 3 optimal interventions according to the surface under the cumulative ranking curve(SUCRA) were Shenshuaining Capsules/Granules/Tablets + conventional western medicine, Jinshuibao Capsules + conventional western medicine, and Niaoduqing Granules + conventional western medicine.(2) In terms of reducing hs-CRP, the top 3 optimal interventions according to SUCRA were Bailing Capsules + conventional western medicine, Tripterygium Glycosides Tablets + conventional western medicine, and Shenshuaining Capsules/Granules/Tablets + conventional western medicine.(3) In terms of reducing IL-6, the top 3 optimal interventions according to SUCRA were Bailing Capsules + conventional western medicine, Tripterygium Glycosides Tablets + conventional western medicine, and Jinshuibao Capsules + conventional western medicine.(4) In terms of reducing UAER, the top 3 optimal interventions according to SUCRA were Shenshuaining Capsules/Granules/Tablets + conventional western medicine, Huangkui Capsules + conventional western medicine, and Jinshuibao Capsules + conventional western medicine.(5) In terms of reducing Scr, the top 3 optimal interventions according to SUCRA were Jinshuibao Capsules + conventional western medicine, Niaoduqing Granules + conventional wes-tern medicine, and Tripterygium Glycosides Tablets + conventional western medicine.(6) In terms of reducing BUN, the first 3 optimal interventions according to SUCRA were Niaoduqing Granules + conventional western medicine, Tripterygium Glycosides Tablets + conventional western medicine, and Huangkui Capsules + conventional western medicine.(7) In terms of improving the clinical total effective rate, the first 3 optimal interventions according to SUCRA were Jinshuibao Capsules + conventional western medicine, Niaoduqing Granu-les + conventional western medicine, and Huangkui Capsules + conventional western medicine. The results showed that the combination of western medicine and Chinese patent medicine could reduce the expression of serum inflammatory factors TNF-α, hs-CRP, and IL-6 and inhibit the inflammatory response. The combination of western medicine and Chinese patent medicine was superior to western medicine alone in reducing Scr, BUN, and UAER, and improving the total effective rate of treatment. Due to the limitation of the quantity and quality of literature included, the above conclusions need to be validated by more high-quality studies.

Immune responses in diabetic nephropathy: Pathogenic mechanisms and therapeutic target

Diabetic nephropathy (DN) is a chronic, inflammatory disease affecting millions of diabetic patients worldwide. DN is associated with proteinuria and progressive slowing of glomerular filtration, which often leads to end-stage kidney diseases. Due to the complexity of this metabolic disorder and lack of clarity about its pathogenesis, it is often more difficult to diagnose and treat than other kidney diseases. Recent studies have highlighted that the immune system can inadvertently contribute to DN pathogenesis. Cells involved in innate and adaptive immune responses can target the kidney due to increased expression of immune-related localization factors. Immune cells then activate a pro-inflammatory response involving the release of autocrine and paracrine factors, which further amplify inflammation and damage the kidney. Consequently, strategies to treat DN by targeting the immune responses are currently under study. In light of the steady rise in DN incidence, this timely review summarizes the latest findings about the role of the immune system in the pathogenesis of DN and discusses promising preclinical and clinical therapies.

Effects of Metformin on Renal Function, Cardiac Function, and Inflammatory Response in Diabetic Nephropathy and Its Protective Mechanism.

Objective To investigate the effect of metformin on renal function, cardiac function, and inflammatory response in diabetic nephropathy and its protective mechanism. Methods A total of 88 patients with diabetic nephropathy who were admitted to our hospital from April 2019 to October 2020 were recruited and grouped according to different treatment methods, namely, the experimental group (n = 44) and the control group (n = 44). The patients in the experimental group were treated with metformin, and the patients in the control group were treated with liraglutide injection (nonmetformin). Left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), left ventricular end-systolic diameter (LVESD), and inflammatory response (hs-CRP, TNF-α, IL-6) were compared. Results Compared with corresponding values before treatment, BUN, Scr, hs-CRP, TNF-α, IL-6, LVEDD, and LVESD were decreased after treatment, whereas LVEF was increased (all P < 0.05), with significant change in the experimental group (all P < 0.001). Conclusion Metformin can effectively improve the level of renal function and cardiac function in patients with diabetic nephropathy and help patients control and reduce the body's inflammatory response, and its therapeutic efficacy is superior to that of liraglutide injection.

Regulation of the Homeostatic Unfolded Protein Response in Diabetic Nephropathy

A growing body of scientific evidence indicates that protein homeostasis, also designated as proteostasis, is causatively linked to chronic diabetic nephropathy (DN). Experimental studies have demonstrated that the insulin signaling in podocytes maintain the homeostatic unfolded protein response (UPR). Insulin signaling via the insulin receptor non-canonically activates the spliced X-box binding protein-1 (sXBP1), a highly conserved endoplasmic reticulum (ER) transcription factor, which regulates the expression of genes that control proteostasis. Defective insulin signaling in mouse models of diabetes or the genetic disruption of the insulin signaling pathway in podocytes propagates hyperglycemia induced maladaptive UPR and DN. Insulin resistance in podocytes specifically promotes activating transcription factor 6 (ATF6) dependent pathogenic UPR. Akin to insulin, recent studies have identified that the cytoprotective effect of anticoagulant serine protease-activated protein C (aPC) in DN is mediated by sXBP1. In mouse models of DN, treatment with chemical chaperones that improve protein folding provides an additional benefit on top of currently used ACE inhibitors. Understanding the molecular mechanisms that transmute renal cell specific adaptive responses and that deteriorate renal function in diabetes will enable researchers to develop new therapeutic regimens for DN. Within this review, we focus on the current understanding of homeostatic mechanisms by which UPR is regulated in DN.

Gene Regulatory Effect of Pyruvate Kinase M2 is Involved in Renal Inflammation in Type 2 Diabetic Nephropathy

The inflammation of glomerular endothelial cells induces and promotes the activation of macrophages and contributes to the development of diabetic nephropathy. Thus, this study aimed to investigate the gene regulatory effect and potential role of pyruvate kinase M2 (PKM2) in inflammatory response in diabetic nephropathy. The plasma PKM2 levels of patients with diabetes were evaluated. Eight-week-old mice were divided into three groups (WT, db/db mice, and db/db mice treated with TEPP-46) and raised for 12 weeks. Blood and kidney samples were collected at the end of the experiment. Endothelial cells were stimulated with high glucose with or without TEPP-46. The expression of intercellular adhesion molecule 1 (ICAM-1), interleukin 6 (IL-6), interleukin 1 beta (IL-1β), phospho-PKM2, PKM2, phospho-STAT3(signal transducer and activator of transcription), STAT3, nuclear factor kappa B (NF-kB), and phospho-NF-kB in vivo and in vitro were determined using Western blot. The activation of macrophages (CD68+CD86+) in the glomeruli was assessed via fluorescent double staining. Moreover, immune endothelial adhesion experiments were performed. The plasma PKM2 levels of patients with type 2 diabetes increased. P-PKM2 was up-regulated in vivo and in vitro. TEPP-46 decreased inflammatory cell infiltration and ICAM-1 expression in vivo and in vitro and inhibited the differentiation of macrophages to M1 cells in db/db mice with diabetic nephropathy. PKM2 regulated the phosphorylation of STAT3 and NF-kB. Furthermore, high glucose levels induced the transition from tetramer to dimer and the nuclear translocation of PKM2. The gene regulatory effect of PKM2 is involved in renal inflammation in type 2 diabetic nephropathy by promoting the phosphorylation of STAT3 and NF-kB and the expression of intercellular adhesion molecule 1. Thus, the down-regulation of phosphorylated PKM2 may have protective effects against diabetic nephropathy by inhibiting renal inflammation.

Long non-coding RNA MEG3 promotes fibrosis and inflammatory response in diabetic nephropathy via miR-181a/Egr-1/TLR4 axis.

Long non-coding RNAs (lncRNAs) play vital roles in diabetic nephropathy (DN). This research aimed to study the potential role and underlying molecular mechanisms of long non-coding RNA MEG3 in DN. We found that MEG3 was upregulated in DN in vivo and in vitro and could enhance cell fibrosis and inflammatory response in DN. MEG3 functioned as an endogenous sponge for miR-181a in mesangial cells (MCs) via direct targeting and in an Ago2-dependent manner. MiR-181a inhibition promoted MC fibrosis and inflammatory response. In addition, Egr-1 was confirmed as a target gene of miR-181a. Further investigations verified that MEG3 promotes fibrosis and inflammatory response via the miR-181a/Egr-1/TLR4 axis in vitro and in vivo. These results provide new insights into the regulation between MEG3 and the miR-181a/Egr-1/TLR4 signaling pathway during DN progression.

MiR-874 alleviates renal injury and inflammatory response in diabetic nephropathy through targeting toll-like receptor-4.

Diabetic nephropathy (DN) is a kind of diabetic complication with capillary damage, and its pathogenesis remains obscure. Recently, microRNAs have been identified as diagnostic biomarkers in various diseases including DN. Toll-like receptor 4 (TLR4) contributes to inflammation, and it has been implicated in diabetes pathophysiology. This study was designed to investigate the role of miR-874 and TLR4 in a streptozotocin (STZ)-induced DN rat model and glucose-induced mouse podocyte model. In the current study, we reported that miR-874 was markedly downregulated in DN rats and glucose-induced mouse podocytes compared with the corresponding control groups with the activation of TLR4. In addition, we observed that overexpression of miR-874 was able to alleviate renal injury in DN rats. The cell counting kit (CCK-8) assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay demonstrated that glucose simulation significantly inhibited podocyte proliferation and induced cell apoptosis, which can be reversed by miR-874 mimics significantly. Notably, miR-874 overexpression dramatically attenuated the inflammatory response, indicated by the decreased levels of interleukin-6, L-1β, and tumor necrosis factor α (TNF-α). Finally, the binding correlation between miR-874 and TLR4 was confirmed by carrying out dual-luciferase reporter assay in our study. It was found that overexpression of miR-874 depressed TLR4 levels in podocytes. These findings implied for the first time that the overexpression of miR-874 repressed glucose-triggered podocyte injury through targeting TLR4 and suggested that miR-874/TLR4 axis might represent a pathological mechanism of DN.

MiR-374a Regulates Inflammatory Response in Diabetic Nephropathy by Targeting MCP-1 Expression.

The microRNA (mir)-374a has been implicated in several types of human cancer; however, its role in diabetic nephropathy (DN) remains unclear. Monocyte chemoattractant protein (MCP)-1 is a chemokine that recruits macrophages to inflammatory sites and is important for the development and progression of DN. However, the relationship between miR-374a and MCP-1 in DN is unknown. We addressed this in the present study by examining the expression of these factors in kidney tissue samples from DN patients and through loss- and gain-of-function experiments using HK2 human renal tubular epithelial cells. We found that miR-374a was downregulated whereas MCP-1 was upregulated in DN tissue. A bioinformatics analysis revealed that MCP-1 is a putative target of miR-374a. To confirm this relationship, HK2 cells treated with normal glucose (5.6 mmol/l D-glucose), high glucose (HG) (30 mmol/l D-glucose), or high osmotic pressure solution (5.6 mmol/l D-glucose + 24.4 mmol/l D-mannitol) were transfected with miR-374a mimic or inhibitor. miR-374a mimic reduced MCP-1 mRNA expression and migration of co-cultured U937 cells, whereas miR-374a inhibition had the opposite effects. Additionally, interleukin-6 and -18 and tumor necrosis factor-α levels were downregulated by transfection of miR-374a mimic. On the other hand, MCP-1 overexpression reversed the inhibitory effects of miR-374a in HK2 cells. Thus, miR-374a suppresses the inflammatory response in DN through negative regulation of MCP-1 expression. These findings suggest that therapeutic strategies that target the miR-374a/MCP-1 axis can be an effective treatment for DN.

Oridonin protects against the inflammatory response in diabetic nephropathy by inhibiting the TLR4/p38-MAPK and TLR4/NF-κB signaling pathways

Inflammation plays a pivotal role in the development and progression of diabetic nephropathy (DN). Oridonin (Ori), a component isolated from Rabdosia rubescens, possesses remarkable anti-inflammatory, immunoregulatory and antitumor properties. However, the renoprotective effects of Ori and the underlying molecular mechanisms have not been explored in DN. In this study, we aimed to investigate the protective effects and potential mechanisms responsible for the anti-inflammatory effects of Ori in diabetes-induced renal injury in vivo and in vitro. Our results showed that Ori significantly attenuated diabetes-induced renal injury and markedly decreased urinary protein excretion levels, serum creatinine concentrations and blood urea nitrogen concentrations in rats. Ori also significantly alleviated infiltration of inflammatory cells (cluster of differentiation (CD)68) in kidney tissues and reduced the levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and monocyte chemotactic protein 1 (MCP-1), both in vivo and in vitro. TLR4 is a principal mediator of innate immune and inflammatory responses and participates in the development of DN. Our molecular studies indicated that Ori administration significantly down-regulated TLR4 overexpression in DN. Additional studies were conducted to investigate the effect of Ori on the p38-mitogen-activated protein kinase (p38-MAPK) and nuclear factor (NF)-κB pathways. The results showed that Ori inhibited IκBα, p65, and p38 phosphorylation, as well as NF-κB DNA-binding activity. In conclusion, these results demonstrated that Ori exerts protective effects in diabetes-induced renal injury in vivo and in vitro. These effects may be ascribed to its anti-inflammatory and modulatory effects on the TLR4/p38-MAPK and TLR4/NF-κB signaling pathways.

The cellular selection between apoptosis and autophagy: roles of vitamin D, glucose and immune response in diabetic nephropathy.

Apoptosis, autophagy and cell cycle arrest are cellular responses to injury which are supposed to play fundamental roles in initiation and progression of diabetic nephropathy (DN). The aims of the present study is to shed light on the potential effects of vitamin D analog 22-oxacalcitriol (OCT) on different cell responses during DN, and the possible interplay between both glucose, immune system and vitamin D in determining the cell fate. All rats were randomly allocated into one of three groups: control, vehicle-treated DN group and OCT-treated DN group. Eight weeks after induction of diabetes, the rats were killed. Fasting blood glucose levels, serum 25 (OH) D, renal functions, cytokines and gene expression of autophagy, apoptotic and cell cycle arrest markers were assessed. In addition, the histological assessment of renal architecture was done. OCT treatment remarkably improved the renal functions and albuminuria. The reductions in mesangial cell hypertrophy, extracellular matrix as well as cell loss were significantly associated with upregulation of pro-autophagy gene expressions and downregulation of both pro-apoptotic and G1-cell cycle arrest genes expression. The reno-protective effects of OCT treatment were associated with significant attenuation of the fasting blood glucose, serum IL-6, renal TLR-4 and IFN-g gene expression. Modulator effects of OCT on glucose and immune system play important roles in renal cell fate decision and chronic kidney disease progression.

Renal functional responses in diabetic nephropathy following chronic bilateral renal denervation.

Renal innervation operates in conjunction with the intrarenal renin-angiotensin system (RAS) to control tubular reabsorption of sodium and water. This relationship remains unexplored in diabetic nephropathy. This study investigates the effects of acute RAS inhibition and chronic renal denervation on renal function in diabetic rats. Diabetes was induced in mRen-2 rats prior to conducting chronic bilateral denervation in diabetic and normoglycaemic animals. At 12-weeks post-diabetic induction, renal haemodynamics and tubular handling of sodium and water were measured before and after acute captopril infusion. Neither GFR nor renal blood flow were affected by diabetes or chronic renal denervation alone. While captopril produced natriuretic and diuretic responses in chronically-denervated diabetic animals, shown by increases (P<0.05) of 38±14% in absolute (UNaV), and 71±20% in fractional sodium excretion (FENa), and 68±17% in urine volume (UV); in the innervated-diabetic group captopril produced anti-natriuretic effects (UNaV and FENa reduced by 41±10% and 29±13%, respectively; all P<0.05). This difference was not observed however in normoglycaemic groups where RAS inhibition produced anti-natriuretic (normoglycaemic denervated vs. innervated: 56±14% vs. 49±14% UNaV; 45±13% vs. 37±14% FENa) and anti-diuretic (normoglycaemic-denervated vs. innervated: 34±8% vs. 38±10% UV) effects in both denervated and innervated animals. These data indicate that renal neuronal control is altered in chronic hyperglycaemia. The role of the RAS in sodium conservation in the diabetic kidney, appears to be more significant in the absence of renal innervation, suggesting that the interaction between the RAS and renal sympathetic nervous system is responsible for changes in renal function in diabetic nephropathy.

Defective podocyte insulin signalling through p85-XBP1 promotes ATF6-dependent maladaptive ER-stress response in diabetic nephropathy.

Endoplasmic reticulum (ER) stress is associated with diabetic nephropathy (DN), but its pathophysiological relevance and the mechanisms that compromise adaptive ER signalling in podocytes remain unknown. Here we show that nuclear translocation of the transcription factor spliced X-box binding protein-1 (sXBP1) is selectively impaired in DN, inducing activating transcription factor-6 (ATF6) and C/EBP homology protein (CHOP). Podocyte-specific genetic ablation of XBP1 or inducible expression of ATF6 in mice aggravates DN. sXBP1 lies downstream of insulin signalling and attenuating podocyte insulin signalling by genetic ablation of the insulin receptor or the regulatory subunits phosphatidylinositol 3-kinase (PI3K) p85α or p85β impairs sXBP1 nuclear translocation and exacerbates DN. Corroborating our findings from murine DN, the interaction of sXBP1 with p85α and p85β is markedly impaired in the glomerular compartment of human DN. Thus, signalling via the insulin receptor, p85, and XBP1 maintains podocyte homeostasis, while disruption of this pathway impairs podocyte function in DN.

Renal Kallikrein Activation and Renoprotection after Dual Blockade of Renin-Angiotensin System in Diet-Induced Diabetic Nephropathy

Purpose. The objective of this study is to investigate the effect of dual blockage of renin-angiotensin system (RAS) on renal kallikrein expression and inflammatory response in diabetic nephropathy (DN). Methods. Rats were randomly divided into 5 groups with 10 rats in each group: normal control; DN model induced by high fat and high sucrose diets; and DN treated with either benazepril 10 mg/kg/d, irbesartan 30 mg/kg/d, or both. After 8-week treatment, we examined changes in the kidney histopathology, function and immunohistochemical stain of kallikrein, macrophage marker CD68, and profibrotic markers transforming growth factor- (TGF-) β and α-smooth muscle action (SMA). Results. DN rats showed enlarged kidneys with glomerulosclerosis, interstitial chronic inflammation and fibrosis, and proteinuria. All the pathological damage and functional impairments were improved after the RAS blockades (all P < 0.05). Compared with monotherapy, combined treatment further alleviated the kidney impairments in parallel to increased tubular immunoreactivity for kallikrein and decreased immunopositive cells for CD68, TGF-β, and α-SMA. Conclusion. The renoprotective effects of the dual RAS blockade in diabetic nephropathy may be attributed to improved tubular kallikrein expression and interstitial inflammatory response.

Effect of dietary CLA supplementation on renal inflammation in diabetic mice

Dietary conjugated linoleic acid (CLA) supplementation was studied for diabetic renal inflammation improvement through modulation of inflammatory responses and oxidative stress. ICR mice were administrated alloxan (180 mg/kg, i.p.) once and fed an AIN-93G diet, or diets containing 0.5 or 1% CLA for 8 weeks. Dietary CLA supplementation did not change body weights or ameliorate fasting blood glucose, blood urea nitrogen, and kidney MDA levels. However, dietary CLA supplementation improved plasma creatinine levels and overall inflammatory response in diabetic nephropathy by modulation of phosphorylated inhibitory kappa B and the protein levels of interleukine-1β, inducible nitric oxide synthetase, and the c-reactive protein without regulation of blood glucose levels. CLA supplementation in diabetic mice did not restore the manganese superoxide dismutase antioxidant defense system. CLA supplementation was beneficial in diabetic nephropathy through regulation of inflammatory response and has potential as an antiinflammatory terapeutic in patients with mild diabetes and diabetic complications.

TGFβ and CCN2/CTGF mediate actin related gene expression by differential E2F1/CREB activation

BackgroundCCN2/CTGF is an established effector of TGFβ driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFβ leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function.ResultsFrom a panel of genes regulated by TGFβ and CCN2, we used co-inertia analysis to identify and then experimentally verify a subset of transcription factors, E2F1 and CREB, that regulate an expression fingerprint implicated in altered actin dynamics and cell hypertrophy. Importantly, actin related genes containing E2F1 and CREB binding sites, stratified by expression profile within the dataset. Further analysis of actin and cytoskeletal related genes from patients with diabetic nephropathy suggests recapitulation of this programme during the development of renal disease. The Rho family member Cdc42 was also found uniquely to be activated in cells treated with TGFβ and CCN2; Cdc42 interacting genes were differentially regulated in diabetic nephropathy.ConclusionsTGFβ and CCN2 attenuate CREB and augment E2F1 transcriptional activation with the likely effect of altering actin cytoskeletal and cell growth/hypertrophic gene activity with implications for cell dysfunction in diabetic kidney disease. The cytoskeletal regulator Cdc42 may play a role in this signalling response.

High glucose induces macrophage inflammatory protein-3 in renal proximal tubule cells via a transforming growth factor- 1 dependent mechanism

Hyperglycaemia is a causative factor in the pathogenesis of diabetic nephropathy, known to induce chemokines in the kidney. Macrophage inflammatory protein-3 alpha (MIP-3 alpha) is a CC chemokine that has been reported to attract memory T lymphocytes. Our previous microarray study showed significant increased level of MIP-3 alpha in high glucose-induced transcriptional profile in renal proximal tubule cells. Transforming growth factor-beta1 (TGF-beta1) is a key regulator in inflammation and fibrosis in diabetes mellitus setting. This study aimed to determine the role of TGF beta 1 in high glucose-induced MIP-3 alpha expression. An in vitro model of human proximal tubular cells (HK-2 cells) and an in vivo model of the transgenic (mRen-2)27 diabetic rat, well characterized as a model of human diabetic nephropathy, were used. Small interfering RNA technology was used to silence TGF-beta1 gene in HK-2 cells and subsequent experiments were performed to measure mRNA and protein levels of MIP-3 alpha using real time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to measure the protein level of MIP-3 alpha and CD3 a marker of T lymphocytes in the in vivo model. MIP-3 alpha mRNA and protein expression was increased in HK-2 cells by high glucose and TGF-beta1. MIP-3 alpha was up-regulated in the dilated tubules of diabetic rats compared with non-diabetic control animals and CD3 was found to be present around the dilated tubules expressing MIP-3 alpha. This up-regulation was attenuated in the presence of an angiotensin-converting enzyme (ACE) inhibitor. MIP-3 alpha expression significantly decreased in cells in which the TGF-beta1 gene was silenced using small interfering RNA. Furthermore, exposure to high glucose did not induce MIP-3 alpha expression in TGF-beta1 gene silenced cells compared with wild-type cells. In summary, we have uniquely demonstrated that high glucose increases MIP-3 alpha through a TGF beta 1 dependent pathway, suggesting the centrality of TGF-beta1 in both the inflammatory and previously demonstrated fibrotic responses in diabetic nephropathy.

Oxidative stress and cellular stress response in diabetic nephropathy

Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status, a cellular-adaptive response occurs requiring functional chaperones, antioxidant production, and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes-induced nephropathy and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring advanced glycation end-products (pentosidine), protein oxidation (protein carbonyls [DNPH]), and lipid oxidation (4-hydroxy-2-nonenal [HNE] and F2-isoprostanes) in plasma, lymphocytes, and urine, whereas the lymphocyte levels of the heat shock proteins (Hsps) heme oxygenase-1 (HO-1), Hsp70, and Hsp60 as well as thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. We found increased levels of pentosidine (P < 0.01), DNPH (P < 0.05 and P < 0.01), HNE (P < 0.05 and P < 0.01), and F2-isoprostanes (P < 0.01) in all the samples from type 2 diabetic patients with nephropathy with respect to control group. This was paralleled by a significant induction of cellular HO-1, Hsp60, Hsp70, and TrxR-1 (P < 0.05 and P < 0.01). A significant upregulation of both HO-1 and Hsp70 has been detected also in lymphocytes from type 2 diabetic patients without uraemia. Significant positive correlations between DNPH and Hsp60, as well as between the degree of renal failure and HO-1 or Hsp70, also have been found in diabetic uremic subjects. In conclusion, patients affected by type 2 diabetes complicated with nephropathy are under condition of systemic oxidative stress, and the induction of Hsp and TrxR-1 is a maintained response in counteracting the intracellular pro-oxidant status.