Insulin Resistance In Podocytes Research Articles

Dendrobium officinale polysaccharide decreases podocyte injury in diabetic nephropathy by regulating IRS-1/AKT signal and promoting mitophagy.

High glucose (HG) caused oxidative stress and mitochondrial dysfunction, resulting in insulin resistance in podocytes, a key mechanism of diabetic nephropathy. Dendrobium officinale polysaccharide (DOP) was able to improve insulin resistance and antioxidant capability. The purpose of this study is to explore the mechanism by which DOP decreases the podocyte injury induced by HG. MPC5 cells were treated with HG, DOP, and IRS-1/2 inhibitor NT157. Afterwards, glucose consumption, generations of ROS and MDA were measured using the detection kits. Mitophagy was monitored using both MtphagTracyker and LysoTracker. The mitochondrial membrane potential was evaluated by JC-1 staining. DOP was also used in a mouse model of diabetes, with the measurements of urine albumin, blood creatinine and blood urea nitrogen. Treatment with DOP suppressed the HG-induced reduction of glucose consumption, the phosphorylation of IRS-1 (phospho Y632), AKT (phospho Ser473 and Thr308) and Nephrin. In addition, HG-induced augment of ROS and MDA, formation of γ-H2A.X foci and translocation of AKT to nucleus were inhibited by DOP. DOP enhanced mitophagy, which was associated with decreased mitochondrial membrane potential and ROS production. DOP conferred protective effect on podocyte in the diabetic mouse by reducing the albumin/creatinine ratio and blood urea nitrogen, and restoring Nephrin expression in podocytes. DOP alleviates HG-induced podocyte injuryby regulating IRS-1/AKT signal and promoting mitophagy.

Pit 1 transporter (SLC20A1) as a key factor in the NPP1-mediated inhibition of insulin signaling in human podocytes.

Podocytes are crucially involved in blood filtration in the glomerulus. Their proper function relies on efficient insulin responsiveness. The insulin resistance of podocytes, defined as a reduction of cell sensitivity to this hormone, is the earliest pathomechanism of microalbuminuria that is observed in metabolic syndrome and diabetic nephropathy. In many tissues, this alteration is mediated by the phosphate homeostasis-controlling enzyme nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). By binding to the insulin receptor (IR), NPP1 inhibits downstream cellular signaling. Our previous research found that hyperglycemic conditions affect another protein that is involved in phosphate balance, type III sodium-dependent phosphate transporter 1 (Pit 1). In the present study, we evaluated the insulin resistance of podocytes after 24 h of incubation under hyperinsulinemic conditions. Thereafter, insulin signaling was inhibited. The formation of NPP1/IR complexes was observed at that time. A novel finding in the present study was our observation of an interaction between NPP1 and Pit 1 after the 24 h stimulation of podocytes with insulin. After downregulation of the SLC20A1 gene, which encodes Pit 1, we established insulin resistance in podocytes that were cultured under native conditions, manifested as a lack of intracellular insulin signaling and the inhibition of glucose uptake via the glucose transporter type 4. These findings suggest that Pit 1 might be a major factor that participates in the NPP1-mediated inhibition of insulin signaling.

"Multiomics" Analyses Combined with Systems Pharmacology Reveal the Renoprotection of Mangiferin Monosodium Salt in Rats with Diabetic Nephropathy: Focus on Improvements in Renal Ferroptosis, Renal Inflammation, and Podocyte Insulin Resistance.

We explored the protection of mangiferin monosodium salt (MGM) on kidney injury in rats with streptozotocin (STZ)-induced diabetic nephropathy (DN) by "multiomics" analysis combined with systems pharmacology, with a specific focus on ferroptosis, inflammation, and podocyte insulin resistance (IR) signaling events in kidneys. MGM treatment afforded renoprotective effects on rats with STZ-induced DN by alleviating systemic IR-induced renal inflammation and podocyte IR. These mechanisms were correlated mainly with the MGM treatment-induced inhibition of the mitogen-activated protein kinase/nuclear factor-kappa B axis and activation of the phosphorylated insulin receptor substrate 1(Tyr608)/phosphorylated phosphatidylinositol 3-kinase/phosphorylated protein kinase B axis in the kidneys of DN rats. MGM had an ameliorative function in renal ferroptosis in rats with STZ-induced DN by upregulating mevalonate-mediated antioxidant capacities (glutathione peroxidase 4 and ferroptosis suppressor protein 1/coenzyme Q10 axis) and weakening acyl-CoA synthetase long-chain family member 4-mediated proferroptotic generation of lipid drivers in kidneys. MGM may be a promising alternative strategy for the treatment of DN.

Reduction of DUSP4 contributes to podocytes oxidative stress, insulin resistance and diabetic nephropathy

Podocytes are insulin-sensitive cells, and their loss is critical in diabetic nephropathy (DN) progression that could lead to end-stage kidney disease. We have previously shown that decreased DUSP4 expression caused elevated JNK phosphorylation in the diabetic kidney and worsened DN characteristics. Yet, the role of DUSP4 in diabetic podocyte insulin resistance and the progression of DN remains unclear. Here, we report that HG-exposed podocytes exhibited reduced DUSP4 expression, increased phosphorylation of JNK and serine 307 of IRS1 as well as Nox4 expression, while decreasing insulin signaling actions. DUSP4 overexpression, JNK and Nox1/4 inhibition prevented HG-induced serine 307 phosphorylation of IRS1 and restored insulin actions. Diabetic mice showed renal dysfunction and insulin resistance, characteristics that were exacerbated in diabetic DUSP4 deficient mice due to Nox1/4 upregulation. Thus, our results demonstrated that diabetes-induced reduction of DUSP4 leads to JNK activation and elevated Nox4 expression, which contributes to podocyte dysfunction, insulin resistance and progression of DN.

PTEN-induced kinase 1 deficiency alters albumin permeability and insulin signaling in podocytes.

Alterations of insulin signaling in diabetes are associated with podocyte injury, proteinuria, and renal failure. Insulin stimulates glucose transport to cells and regulates other intracellular processes that are linked to cellular bioenergetics, such as autophagy, gluconeogenesis, fatty acid metabolism, and mitochondrial homeostasis. The dysfunction of mitochondrial dynamics, including mitochondrial fusion, fission, and mitophagy, has been observed in high glucose-treated podocytes and renal cells from patients with diabetes. Previous studies showed that prolonged hyperglycemia is associated with the development of insulin resistance in podocytes, and high glucose-treated podocytes exhibit an increase in mitochondrial fission and decrease in markers of mitophagy. In the present study, we found that deficiency of the main mitophagy protein PTEN-induced kinase 1 (PINK1) significantly increased albumin permeability and hampered glucose uptake to podocytes. We suggest that PINK1 inhibition impairs the insulin signaling pathway, in which lower levels of phosphorylated Akt and membrane fractions of the insulin receptor and glucose transporter-4 were observed. Moreover, PINK1-depleted podocytes exhibited lower podocin and nephrin expression, thus identifying a potential mechanism whereby albumin leakage increases under hyperglycemic conditions when mitophagy is inhibited. In conclusion, we found that PINK1 plays an essential role in insulin signaling and the maintenance of proper permeability in podocytes. Therefore, PINK1 may be a potential therapeutic target for the treatment or prevention of diabetic nephropathy.

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.

GPR43 deficiency protects against podocyte insulin resistance in diabetic nephropathy through the restoration of AMPKα activity.

Rationale: Albuminuria is an early clinical feature in the progression of diabetic nephropathy (DN). Podocyte insulin resistance is a main cause of podocyte injury, playing crucial roles by contributing to albuminuria in early DN. G protein-coupled receptor 43 (GPR43) is a metabolite sensor modulating the cell signalling pathways to maintain metabolic homeostasis. However, the roles of GPR43 in podocyte insulin resistance and its potential mechanisms in the development of DN are unclear.Methods: The experiments were conducted by using kidney tissues from biopsied DN patients, streptozotocin (STZ) induced diabetic mice with or without global GPR43 gene knockout, diabetic rats treated with broad-spectrum oral antibiotics or fecal microbiota transplantation, and cell culture model of podocytes. Renal pathological injuries were evaluated by periodic acid-schiff staining and transmission electron microscopy. The expression of GPR43 with other podocyte insulin resistance related molecules was checked by immunofluorescent staining, real-time PCR, and Western blotting. Serum acetate level was examined by gas chromatographic analysis. The distribution of gut microbiota was measured by 16S ribosomal DNA sequencing with faeces.Results: Our results demonstrated that GPR43 expression was increased in kidney samples of DN patients, diabetic animal models, and high glucose-stimulated podocytes. Interestingly, deletion of GPR43 alleviated albuminuria and renal injury in diabetic mice. Pharmacological inhibition and knockdown of GPR43 expression in podocytes increased insulin-induced Akt phosphorylation through the restoration of adenosine 5'-monophosphate-activated protein kinase α (AMPKα) activity. This effect was associated with the suppression of AMPKα activity through post-transcriptional phosphorylation via the protein kinase C-phospholipase C (PKC-PLC) pathway. Antibiotic treatment-mediated gut microbiota depletion, and faecal microbiota transplantation from the healthy donor controls substantially improved podocyte insulin sensitivity and attenuated glomerular injury in diabetic rats accompanied by the downregulation of the GPR43 expression and a decrease in the level of serum acetate.Conclusion: These findings suggested that dysbiosis of gut microbiota-modulated GPR43 activation contributed to albuminuria in DN, which could be mediated by podocyte insulin resistance through the inhibition of AMPKα activity.

Saturated fatty acids induce insulin resistance in podocytes through inhibition of IRS1 via activation of both IKK\u03b2 and mTORC1

Diabetic nephropathy (DN), a microvascular complication of diabetes, is the leading cause of end-stage renal disease worldwide. Multiple studies have shown that podocyte dysfunction is a central event in the progression of the disease. Beside chronic hyperglycemia, dyslipidemia can induce insulin resistance and dysfunction in podocytes. However, the exact mechanisms of free fatty acid (FFA)-induced podocyte insulin unresponsiveness are poorly understood. We used a type 2 diabetic mouse model (db/db) and mouse podocytes exposed to palmitic acid for 24 h followed by an insulin stimulation. Renal function and pathology were evaluated at 25 weeks of age to confirm the DN development. Our results demonstrate that saturated FFA activated the serine/threonine kinases IκB kinase (IKK)β/IκBα and mTORC1/S6K1, but not protein kinase C and c-jun N-terminal kinase, in podocytes and glomeruli of db/db mice. Activation of both kinases promoted serine 307 phosphorylation of IRS1, a residue known to provoke IRS1 inhibition. Using IKK, mTORC1 and ceramide production inhibitors, we were able to blunt IRS1 serine 307 phosphorylation and restore insulin stimulation of Akt. In conclusion, our results indicate that FFA and diabetes contribute to insulin resistance through the activation of IKKβ and S6K1 leading to podocyte dysfunction and DN.

Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor

Aims/hypothesisPodocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy.MethodsConditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs.ResultsPodocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-α and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton.Conclusions/interpretationTogether, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.

Pathogenesis and treatment of insulin resistance in chronic kidney disease and interventional effects of Chinese herbal medicine

The kidney is the target organ of insulin with abundant insulin receptors. Thereinto,the renal intrinsic cells including glomerular podocytes,endothelial cells,mesangial cells,renal tubular epitheliums and collecting duct epithelial cells are all highly sensitive to insulin as the effector cells. Furthermore,the structural and functional abnormalities of these cells are closely related to insulin and its receptors activity. It is reported that the chronic kidney disease（CKD）patients have systemic or renal insulin resistance（IR）. IR is not only the pathogenic factor of CKD but also one of the mechanisms of CKD progression. The pathogenic factors of IR in the CKD patients include the systemic factors and the local factors in muscles and fat cells. The pathogenesis of IR is related to glomeruli,proximal tubules,collecting ducts and corresponding renal intrinsic cells such as podocytes,mesangial cells,renal tubular epitheliums and collecting duct epithelial cells. IR-related signaling pathways include insulin receptor substrate（IRS）/phosphatidylinositol 3 kinase（PI3K）/serine threonine kinase（Akt）pathway,adenosine monophosphate activated protein kinase（AMPK）pathway,glucose transporter4（GLUT4）pathway,nuclear factor（NF）-κB pathway and mitogen activated protein kinase（MAPK）pathway. Among them,IRS1/PI3K/Akt2 is the main signaling pathway of IR in podocytes of glomeruli, thus intervening its activity can improve podocyte injury. In clinic,some classical oral hypoglycemic agents and diuretic including metformin,rosiglitazone,glibenclamide,thiazolidinedione and spironolactone,as well as some extracts from Chinese herbal medicines including astragalus polysaccharides,quercetin,puerarin,emodin,berberine,curcumin and geniposide can both affect insulin and its receptor activity,and regulate IR-related signaling pathways,thereby ameliorating IR and CKD progression. Overall,the pharmacological studies based on IR-related signaling pathways in the renal intrinsic cells of CKD will become one of the developmental directions in the future.

Persistent Insulin Resistance in Podocytes Caused by Epigenetic Changes of SHP-1 in Diabetes.

Poor glycemic control profoundly affects protein expression and the cell signaling action that contributes to glycemic memory and irreversible progression of diabetic nephropathy (DN). We demonstrate that SHP-1 is elevated in podocytes of diabetic mice, causing insulin unresponsiveness and DN. Thus, sustained SHP-1 expression caused by hyperglycemia despite systemic glucose normalization could contribute to the glycemic memory effect in DN. Microalbuminuria, glomerular filtration rate, mesangial cell expansion, and collagen type IV and transforming growth factor-β expression were significantly increased in diabetic Ins2+/C96Y mice compared with nondiabetic Ins2+/+ mice and remained elevated despite glucose normalization with insulin implants. A persistent increase of SHP-1 expression in podocytes despite normalization of systemic glucose levels was associated with sustained inhibition of the insulin signaling pathways. In cultured podocytes, high glucose levels increased mRNA, protein expression, and phosphatase activity of SHP-1, which remained elevated despite glucose concentration returning to normal, causing persistent insulin receptor-β inhibition. Histone posttranslational modification analysis showed that the promoter region of SHP-1 was enriched with H3K4me1 and H3K9/14ac in diabetic glomeruli and podocytes, which remained elevated despite glucose level normalization. Hyperglycemia induces SHP-1 promoter epigenetic modifications, causing its persistent expression and activity and leading to insulin resistance, podocyte dysfunction, and DN.

Diet-induced obesity and kidney disease – In search of a susceptible mouse model

Obesity and metabolic syndrome are independent risk factors for chronic kidney disease, even without diabetes or hyperglycemia. Here, we compare two mouse models that are susceptible to diet-induced obesity: the relatively renal injury resistant C57BL/6J strain and the DBA2/J strain which is more sensitive to renal injury. Our studies focused on characterizing the effects of high fat diet feeding on renal oxidative stress, albuminuria, fibrosis and podocyte loss/insulin resistance. While the C57BL/6J strain does not develop significant pathological changes in the kidney, at least on lard based diets within the time frame investigated, it does show increased renal iNOS and nitrotyrosine levels and elevated mitochondrial respiration which may be indicative of mitochondrial lipid overfueling. Restricting the high fat diet to decrease adiposity decreased the levels of cellular oxidative stress markers, indicating that adiposity-related proinflammatory changes such as increased iNOS levels may trigger similar responses in the kidney. Mitochondrial respiration remained higher, suggesting that eating excess lipids, despite normal adiposity may still lead to renal mitochondrial overfueling. In comparison, DBA/2J mice developed albuminuria on similar diets, signs of fibrosis, oxidative stress, early signs of podocyte loss (evaluated by the markers podocin and WT-1) and podocyte insulin resistance (unable to phosphorylate their glomerular Akt when insulin was given). To summarize, while the C57BL/6J strain is not particularly susceptible to renal disease, changes in its mitochondrial lipid handling combined with the easy availability of transgenic technology may be an advantage to design new knockout models related to mitochondrial lipid metabolism. The DBA/2J model could serve as a basis for studying podocyte insulin resistance and identifying early renal markers in obesity before more severe kidney disease develops. Based on our observations, we encourage further critical evaluation of mouse models for obesity related chronic kidney disease.

NOD2 promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy

An increasing number of clinical and animal model studies indicate that activation of the innate immune system and inflammatory mechanisms are important in the pathogenesis of diabetic nephropathy. Nucleotide-binding oligomerization domain containing 2 (NOD2), a member of the NOD-like receptor family, plays an important role in innate immune response. Here we explore the contribution of NOD2 to the pathogenesis of diabetic nephropathy and found that it was upregulated in kidney biopsies from diabetic patients and high-fat diet/streptozotocin-induced diabetic mice. Further, NOD2 deficiency ameliorated renal injury in diabetic mice. In vitro, NOD2 induced proinflammatory response and impaired insulin signaling and insulin-induced glucose uptake in podocytes. Moreover, podocytes treated with high glucose, advanced glycation end-products, tumor necrosis factor-α, or transforming growth factor-β (common detrimental factors in diabetic nephropathy) significantly increased NOD2 expression. NOD2 knockout diabetic mice were protected from the hyperglycemia-induced reduction in nephrin expression. Further, knockdown of NOD2 expression attenuated high glucose-induced nephrin downregulation in vitro, supporting an essential role of NOD2 in mediating hyperglycemia-induced podocyte dysfunction. Thus, NOD2 is one of the critical components of a signal transduction pathway that links renal injury to inflammation and podocyte insulin resistance in diabetic nephropathy.

Insulin directly stimulates VEGF-A production in the glomerular podocyte

Podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Recently, it has become clear that to achieve this, they need to be insulin sensitive and produce an optimal amount of VEGF-A. In other tissues, insulin has been shown to regulate VEGF-A release, but this has not been previously examined in the podocyte. Using in vitro and in vivo approaches, in the present study, we now show that insulin regulates VEGF-A in the podocyte in both mice and humans via the insulin receptor (IR). Insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte-specific IR knockout mice), podocyte VEGF-A production was impaired. Importantly, in vivo, this occurs before the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.

Expression of membrane-bound NPP-type ecto-phosphodiesterases in rat podocytes cultured at normal and high glucose concentrations

The ecto-nucleotide pyrophosphatase/phosphodiesterase family (E-NPPs) contains two membrane-bound members: E-NPP1 and E-NPP3. These enzymes mediate hydrolysis of extracellular nucleotides and their abnormal expression may affect intracellular signal transduction pathways, leading to cellular dysfunction, e.g., insulin resistance. Podocytes are insulin-dependent glomerular epithelial cells that regulate the glomerular filtration rate. Pathology of podocytes is a hallmark of diabetic nephropathy. Here, we investigated the expressions of E-NPP1 and E-NPP3 and activity of E-NPP enzymes in rat podocytes cultured with 5 mM (NG) or 30 mM glucose (HG). Insulin resistance was determined by measuring changes in [1,2- 3H]-deoxy- d-glucose uptake in response to insulin. mRNAs of E-NPP1 and E-NPP3 were detected within podocytes. The E-NPP expressions were confirmed at the protein level using western blot and immunofluorescence techniques. At NG, insulin (300 nM, 3 min) increased glucose uptake 1.5-fold; however, this effect was abolished at HG. The protein expressions of E-NPP1 and E-NPP3 were not affected at HG. The E-NPP activities were 24.68 ± 0.72 and 26.51 ± 1.55 nmol/min/mg protein at NG and HG, respectively. In conclusion, ecto-nucleotide pyrophosphatase/phosphodiesterase 1 and 3 are expressed on podocytes, but changes in expression of these enzymes are most likely not involved in etiology of insulin resistance in podocytes.

Saturated fatty acids induce insulin resistance in human podocytes: implications for diabetic nephropathy.

Cellular insulin resistance is the hallmark of type 2 diabetes and predominantly affects adipose and muscle cells. The saturated free fatty acid palmitate is elevated in insulin-resistant states and may directly contribute to cellular insulin resistance. A spectrum of renal disease is associated with increased markers of insulin resistance, although direct causal mechanisms are not known. In the kidney, glomerular podocytes are novel insulin-sensitive cells that have the ability to rapidly transport glucose. In this study, we tested the hypothesis that palmitate would induce insulin resistance in podocytes. Conditionally immortalized human podocytes were cultured for up to 24 h with 375-750 muM palmitate. Functional effects on glucose uptake and ceramide production were measured. Gene expression was investigated using a focused gene array, and protein signalling and trafficking were studied with Western blotting and immunofluorescence. We found that palmitate blocked insulin-stimulated glucose uptake in human podocytes. This was associated with increased ceramide production, and use of the ceramide inhibitors myriocin and fumonisin B1 partially recovered the insulin sensitivity. At the level of transcription, palmitate downregulated genes associated with several pathways involved in insulin signalling. At the protein level, phosphorylation of the insulin receptor, IRS1 and PKB was reduced and there was impaired translocation of GLUT4 to the cell surface. This is the first study to demonstrate a direct effect of saturated fatty acids on podocyte function. These findings may represent a novel link between systemic insulin resistance and the development of nephropathy.