Losartan and metabolite EXP3179 activate endothelial function without lowering blood pressure in AT2 receptor KO mice.

BACKGROUND: We have documented profound release of nitric oxide (NO) and endothelium-derived hyperpolarization factor (EDHF) by angiotensin II (ANGII) receptor 1 (AT1) blocker (ARB) losartan and its unique metabolite EXP3179, a pleiotropic effect that may help rationalize the protective properties of ARBs. Since blood pressure (BP) lowering by ARBs likely require an ANGII-dependent switch from AT1 to ANGII receptor 2 (AT2) signaling, a receptor known to stimulate endothelial NO release, we investigated the contribution of AT1 and AT2 to losartan and EXP3179's endothelial function-activating properties. EXPERIMENTAL APPROACH: Two AT1 ligands were used in an attempt to block the AT1-dependent endothelium-enhancing effects of EXP3179. AT2-null mice were used to evaluate the acute ex vivo and chronic in vivo effects of EXP3179 (20µM) and losartan (0.6 g/l), respectively, on endothelial function, BP and aortic stiffness. KEY RESULTS: Ex vivo blockade of AT1 receptors did not attenuate EXP3179's effects on NO and EDHF-dependent endothelial function activation. We observed significant reductions in PE-induced contractility with EXP3179 in both WT and AT2 knockout (KO) aortic rings. In vivo, a 1-month chronic treatment with losartan did not affect pulse wave velocity (PWV) but decreased PE-induced contraction by 74.9 % in WT (p < 0.0001) and 47.3 % in AT2 KO (p < 0.05). Presence of AT2 was critical to losartan's BP lowering activity. CONCLUSION: In contrast to BP lowering, the endothelial function-enhancing effects of losartan and EXP3179 are mostly independent of the classic ANGII/AT1/AT2 pathway, which sheds light on ARB pleiotropism.

Apelin prevents diabetes-induced poor collateral vessel formation and blood flow reperfusion in ischemic limb.

Introduction: Peripheral arterial disease (PAD) is a major risk factor for lower-extremity amputation in diabetic patients. Unfortunately, previous clinical studies investigating therapeutic angiogenesis using the vascular endothelial growth factor (VEGF) have shown disappointing results in diabetic patients, which evokes the necessity for novel therapeutic agents. The apelinergic system (APJ receptor/apelin) is highly upregulated under hypoxic condition and acts as an activator of angiogenesis. Apelin treatment improves revascularization in nondiabetic models of ischemia, however, its role on angiogenesis in diabetic conditions remains poorly investigated. This study explored the impact of Pyr-apelin-13 in endothelial cell function and diabetic mouse model of hindlimb ischemia. Methods: Nondiabetic and diabetic mice underwent femoral artery ligation to induce limb ischemia. Diabetic mice were implanted subcutaneously with osmotic pumps delivering Pyr-apelin-13 for 28 days. Blood flow reperfusion was measured for 4 weeks post-surgery and exercise willingness was assessed with voluntary wheels. In vitro, bovine aortic endothelial cells (BAECs) were exposed to normal (NG) or high glucose (HG) levels and hypoxia. Cell migration, proliferation and tube formation assays were performed following either VEGF or Pyr-apelin-13 stimulation. Results and Discussion: Following limb ischemia, blood flow reperfusion, functional recovery of the limb and vascular density were improved in diabetic mice receiving Pyr-apelin-13 compared to untreated diabetic mice. In cultured BAECs, exposure to HG concentrations and hypoxia reduced VEGF proangiogenic actions, whereas apelin proangiogenic effects remained unaltered. Pyr-apelin-13 induced its proangiogenic actions through Akt/AMPK/eNOS and RhoA/ROCK signaling pathways under both NG or HG concentrations and hypoxia exposure. Our results identified the apelinergic system as a potential therapeutic target for angiogenic therapy in diabetic patients with PAD.

Deletion of protein tyrosine phosphatase SHP-1 restores SUMOylation of podocin and reverses the progression of diabetic kidney disease.

Both clinical and experimental data suggest that podocyte injury is involved in the onset and progression of diabetic kidney disease (DKD). Although the mechanisms underlying the development of podocyte loss are not completely understood, critical structural proteins such as podocin play a major role in podocyte survival and function. We have reported that the protein tyrosine phosphatase SHP-1 expression increased in podocytes of diabetic mice and glomeruli of patients with diabetes. However, the in vivo contribution of SHP-1 in podocytes is unknown. Conditional podocyte-specific SHP-1-deficient mice (Podo-SHP-1-/-) were generated to evaluate the impact of SHP-1 deletion at four weeks of age (early) prior to the onset of diabetes and after 20 weeks (late) of diabetes (DM; Ins2+/C96Y) on kidney function (albuminuria and glomerular filtration rate) and kidney pathology. Ablation of the SHP-1 gene specifically in podocytes prevented and even reversed the elevated albumin/creatinine ratio, glomerular filtration rate progression, mesangial cell expansion, glomerular hypertrophy, glomerular basement membrane thickening and podocyte foot process effacement induced by diabetes. Moreover, podocyte-specific deletion of SHP-1 at an early and late stage prevented diabetes-induced expression of collagen IV, fibronectin, transforming growth factor-ß, transforming protein RhoA, and serine/threonine kinase ROCK1, whereas it restored nephrin, podocin and cation channel TRPC6 expression. Mass spectrometry analysis revealed that SHP-1 reduced SUMO2 post-translational modification of podocin while podocyte-specific deletion of SHP-1 preserved slit diaphragm protein complexes in the diabetic context. Thus, our data uncovered a new role of SHP-1 in the regulation of cytoskeleton dynamics and slit diaphragm protein expression/stability, and its inhibition preserved podocyte function preventing DKD progression.

Enhanced SHP-1 Expression in Podocyturia Is Associated with Kidney Dysfunction in Patients with Diabetes.

Background: Diabetic kidney disease (DKD) remains the leading cause of end stage kidney disease worldwide. Despite significant advances in kidney care, there is a need to improve noninvasive techniques to predict the progression of kidney disease better for patients with diabetes. After injury, podocytes are shed in urine and may be used as a biologic tool. We previously reported that SHP-1 is upregulated in the kidney of diabetic mice, leading to podocyte dysfunction and loss. Our objective was to evaluate the expression levels of SHP-1 in urinary podocytes and kidney tissues of patients with diabetes. Methods: In this prospective study, patients with and without diabetes were recruited for the quantification of SHP-1 in kidney tissues, urinary podocytes, and peripheral blood monocytes. Immunochemistry and mass spectrometry techniques were applied for kidney tissues. Urinary podocytes were counted, and expression of SHP-1 and podocyte markers were measured by quantitative PCR. Results: A total of 66 participants (diabetic n=48, nondiabetic n=18) were included in the analyses. Diabetes was associated with increased SHP-1 expression in kidney tissues (P=0.03). Nephrin and podocin mRNA was not significantly increased in urinary podocytes from patients with diabetes compared with those without diabetes, whereas levels of SHP-1 mRNA expression significantly correlated with HbA1c and estimated glomerular filtration rate (eGFR). Additionally, follow-up (up to 2 years post recruitment) evaluation indicated that SHP-1 mRNA expression continued to increase with eGFR decline. Conclusions: Levels of SHP-1 in urinary podocytes may serve as an additional marker of glomerular disease progression in this population.

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.

Transcriptomic modulation in response to high-intensity interval training in monocytes of older women with type 2 diabetes.

PURPOSE: Type 2 diabetes is associated with a higher risk of cardiovascular diseases, lowering the quality of life and increasing mortality rates of affected individuals. Circulating monocytes are tightly involved in the atherosclerosis process leading to cardiovascular diseases (CVD), and their inflammatory profile can be modified by exercise. The objective was to exploratory identify genes associated with CVD that could be regulated by high-intensity interval training (HIIT) in monocytes of type 2 diabetes patients. METHODS: Next-generation RNA sequencing (RNA-seq) analyses were conducted on isolated circulating monocytes (CD14+) of six women aged 60 and over with type 2 diabetes who completed a 12-week supervised HIIT intervention on a treadmill. RESULTS: Following the intervention, a reduction of resting diastolic blood pressure was observed. Concomitant with this result, 56 genes were found to be downregulated following HIIT intervention in isolated monocytes. A large proportion of the regulated genes was involved in cellular adhesion, migration and differentiation into an "atherosclerosis-specific" macrophage phenotype. CONCLUSION: The downregulation of transcripts in monocytes globally suggests a favorable cardiovascular effect of the HIIT in older women with type 2 diabetes. In the context of precision medicine and personalized exercise prescription, shedding light on the fundamental mechanisms underlying HIIT effects on the gene profile of immune cells is essential to develop efficient nonpharmacological strategies to prevent CVD in high-risk population.

Diabetes Impaired Ischemia-Induced PDGF (Platelet-Derived Growth Factor) Signaling Actions and Vessel Formation Through the Activation of Scr Homology 2-Containing Phosphatase-1.

Objective: Critical limb ischemia is a major complication of diabetes characterized by insufficient collateral vessel development and proper growth factor signaling unresponsiveness. Although mainly deactivated by hypoxia, phosphatases are important players in the deregulation of proangiogenetic pathways. Previously, SHP-1 (Scr homology 2-containing phosphatase-1) was found to be associated with the downregulation of growth factor actions in the diabetic muscle. Thus, we aimed to gain further understanding of the impact of SHP-1 on smooth muscle cell (SMC) function under hypoxic and diabetic conditions. Approach and Results: Despite being inactivated under hypoxic conditions, high glucose level exposure sustained SHP-1 phosphatase activity in SMC and increased its interaction with PDGFR (platelet-derived growth factor receptor)-ß, thus reducing PDGF proangiogenic actions. Overexpression of an inactive form of SHP-1 fully restored PDGF-induced proliferation, migration, and signaling pathways in SMC exposed to high glucose and hypoxia. Nondiabetic and diabetic mice with deletion of SHP-1 specifically in SMC were generated. Ligation of the femoral artery was performed, and blood flow was measured for 4 weeks. Blood flow reperfusion, vascular density and maturation, and limb survival were all improved while vascular apoptosis was attenuated in diabetic SMC-specific SHP-1 null mice as compared to diabetic mice. Conclusions: Diabetes and high glucose level exposure maintained SHP-1 activity preventing hypoxia-induced PDGF actions in SMC. Specific deletion of SHP-1 in SMC partially restored blood flow reperfusion in the diabetic ischemic limb. Therefore, local modulation of SHP-1 activity in SMC could represent a potential therapeutic avenue to improve the proangiogenic properties of SMC under ischemia and diabetes.

Fish oil replacement prevents, while docosahexaenoic acid-derived protectin DX mitigates end-stage-renal-disease in atherosclerotic diabetic mice.

Diabetic nephropathy (DN) remains the major cause of end-stage renal disease (ESRD). We used high-fat/high-sucrose (HFHS)-fed LDLr-/- /ApoB100/100 mice with transgenic overexpression of IGFII in pancreatic ß-cells (LRKOB100/IGFII) as a model of ESRD to test whether dietary long chain omega-3 polyunsaturated fatty acids LCω3FA-rich fish oil (FO) could prevent ESRD development. We further evaluated the potential of docosahexaenoic acid (DHA)-derived pro-resolving lipid mediators, 17-hydroxy-DHA (17-HDHA) and Protectin DX (PDX), to reverse established ESRD damage. HFHS-fed vehicle-treated LRKOB100/IGFII mice developed severe kidney dysfunction leading to ESRD, as revealed by advanced glomerular fibrosis and mesangial expansion along with reduced percent survival. The kidney failure outcome was associated with cardiac dysfunction, revealed by reduced heart rate and prolonged diastolic and systolic time. Dietary FO prevented kidney damage, lean mass loss, cardiac dysfunction, and death. 17-HDHA reduced podocyte foot process effacement while PDX treatment alleviated kidney fibrosis and mesangial expansion as compared to vehicle treatment. Only PDX therapy was effective at preserving the heart function and survival rate. These results show that dietary LCω3FA intake can prevent ESRD and cardiac dysfunction in LRKOB100/IGFII diabetic mice. Our data further reveals that PDX can protect against renal failure and cardiac dysfunction, offering a potential new therapeutic strategy against ESRD.

Endothelial deletion of PKCδ prevents VEGF inhibition and restores blood flow reperfusion in diabetic ischemic limb.

AIMS: Peripheral artery disease is a complication of diabetes leading to critical hindlimb ischemia. Diabetes-induced inhibition of VEGF actions is associated with the activation of protein kinase Cδ (PKCδ). We aim to specifically investigate the role of PKCδ in endothelial cell (EC) function and VEGF signaling. METHODS: Nondiabetic and diabetic mice, with (ec-Prkcd-/-) or without (ec-Prkcdf/f) endothelial deletion of PKCδ, underwent femoral artery ligation. Blood flow reperfusion was assessed up to 4 weeks post-surgery. Capillary density, EC apoptosis and VEGF signaling were evaluated in the ischemic muscle. Src homology region 2 domain-containing phosphatase-1 (SHP-1) phosphatase activity was assessed in vitro using primary ECs. RESULTS: Ischemic muscle of diabetic ec-Prkcdf/f mice exhibited reduced blood flow reperfusion and capillary density while apoptosis increased as compared to nondiabetic ec-Prkcdf/f mice. In contrast, blood flow reperfusion and capillary density were significantly improved in diabetic ec-Prkcd-/- mice. VEGF signaling pathway was restored in diabetic ec-Prkcd-/- mice. The deletion of PKCδ in ECs prevented diabetes-induced VEGF unresponsiveness through a reduction of SHP-1 phosphatase activity. CONCLUSIONS: Our data provide new highlights in mechanisms by which PKCδ activation in EC contributed to poor collateral vessel formation, thus, offering novel therapeutic targets to improve angiogenesis in the diabetic limb.

Saturated fatty acids induce insulin resistance in podocytes through inhibition of IRS1 via activation of both IKKß 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)ß/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 IKKß and S6K1 leading to podocyte dysfunction and DN.

Ablation of angiotensin type 2 receptor prevents endothelial nitric oxide synthase glutathionylation and nitration in ischaemic abductor muscle of diabetic mice.

Peripheral artery disease is a severe complication of diabetes. We have reported that the deletion of angiotensin type 2 receptor in diabetic mice promoted vascular angiogenesis in the ischaemic muscle 4 weeks following ischaemia. However, the angiotensin type 2 receptor deletion beneficial effects occurred 2 weeks post surgery suggesting that angiotensin type 2 receptor may regulate other pro-angiogenic signalling pathways during the early phases of ischaemia. Nondiabetic and diabetic angiotensin type 2 receptor-deficient mice (Agtr2-/Y) underwent femoral artery ligation after 2 months of diabetes. Blood perfusion was measured every week up to 2 weeks post surgery. Expression of vascular endothelial growth factor, vascular endothelial growth factor receptor and endothelial nitric oxide synthase expression and activity were evaluated. Blood flow reperfusion in the ischaemic muscle of diabetic Agtr2+/Y mice was recovered at 35% as compared to a 68% recovery in diabetic Agtr2-/Y mice. The expression of vascular endothelial growth factor and its receptors was diminished in diabetic Agtr2+/Y mice, an observation not seen in diabetic Agtr2-/Y mice. Interestingly, Agtr2-/Y mice were protected from diabetes-induced glutathionylation, nitration and decreased endothelial nitric oxide synthase expression, which correlated with reduced endothelial cell death and enhanced vascular density in diabetic ischaemic muscle. In conclusion, our results suggest that the deletion of angiotensin type 2 receptor promotes blood flow reperfusion in diabetes by favouring endothelial cell survival and function.

Calcifediol Decreases Interleukin-6 Secretion by Cultured Human Trophoblasts From GDM Pregnancies.

Gestational diabetes mellitus (GDM) is often characterized by low maternal calcifediol (25OHD) and high inflammation levels. This study aimed to determine whether placental protein expressions of CYP27B1, vitamin D receptor (VDR), and CYP24A1 are impaired in GDM and to investigate the effect of a 25OHD treatment on IL-6 secretion by GDM trophoblasts compared with normoglycemic (NG) trophoblasts. Placental tissue samples were harvested to determine protein expression of CYP27B1, VDR, and CYP24A1 by immunoblots. Isolated trophoblasts were stimulated with 25OHD concentrations (25 to 2000 nM) once a day for 3 days and IL-6 secretion was quantified (ELISA). We recruited 17 NG women, 19 women with GDM treated with diet and exercise alone (GDM-d) and 9 women with GDM who necessitated insulin therapy (GDM-i). Protein expressions of CYP27B1 and VDR were significantly higher in placental tissue from GDM-d women compared with NG women (both P = 0.02), whereas no differences were detected between GDM-i and NG placental tissues. In cultured trophoblasts (two groups; n = 5 NG and n = 5 GDM-d), exposure to increasing 25OHD concentrations significantly decreased IL-6 secretion in the GDM-d group only (P = 0.006). After treatment with 25OHD (2000 nM), IL-6 secretion was lower in the GDM-d group compared with the NG group (P = 0.03). Our results suggest an upregulation of the VDR-1,25(OH)2D complex bioavailability in GDM-d placentas, possibly reflecting a compensatory mechanism aiming to ensure that vitamin D can exert its genomic and nongenomic effects in the target cells of the placental-fetal unit. Our findings support an anti-inflammatory effect of vitamin D at the feto-maternal interface in GDM-d pregnancies.

Diabetes-Induced DUSP4 Reduction Promotes Podocyte Dysfunction and Progression of Diabetic Nephropathy.

Diabetic nephropathy (DN) remains the leading cause of end-stage renal disease. Hyperglycemia-induced podocyte dysfunction is a major contributor of renal function impairment in DN. Previous studies showed that activation of mitogen-activated protein kinase (MAPK) in diabetes promotes podocyte dysfunction and cell death. Dual specificity phosphatases (DUSPs) are a family of phosphatases mainly responsible for MAPK inhibition. In this study, we demonstrated that diabetes and high glucose exposure decreased DUSP4 expression in cultured podocytes and glomeruli. Diabetes-induced DUSP4 reduction enhanced p38 and c-Jun N-terminal kinase (JNK) activity and podocyte dysfunction. The overexpression of DUSP4 prevented the activation of p38, JNK, caspase 3/7 activity, and NADPH oxidase 4 expression induced by high glucose level exposure. Deletion of DUSP4 exacerbated albuminuria and increased mesangial expansion and glomerular fibrosis in diabetic mice. These morphological changes were associated with profound podocyte foot process effacement, cell death, and sustained p38 and JNK activation. Moreover, inhibition of protein kinase C-δ prevented DUSP4 expression decline and p38/JNK activation in the podocytes and renal cortex of diabetic mice. Analysis of DUSP4 expression in the renal cortex of patients with diabetes revealed that decreased DUSP4 mRNA expression correlated with reduced estimated glomerular filtration rate (<60 mL/min/1.73 m2). Thus, this study demonstrates that preserving DUSP4 expression could protect against podocyte dysfunction and preserve glomerular function in DN.

Combination of high-fat/high-fructose diet and low-dose streptozotocin to model long-term type-2 diabetes complications.

The epidemic of type 2 diabetes mellitus (T2DM) is fueled by added fructose consumption. Here, we thus combined high-fat/high-fructose diet, with multiple low-dose injections of streptozotocin (HF/HF/Stz) to emulate the long-term complications of T2DM. HF/HF/Stz rats, monitored over 56 weeks, exhibited metabolic dysfunctions associated with the different stages of the T2DM disease progression in humans: an early prediabetic phase characterized by an hyperinsulinemic period with modest dysglycemia, followed by a late stage of T2DM with frank hyperglycemia, normalization of insulinemia, marked dyslipidemia, hepatic fibrosis and pancreatic ß-cell failure. Histopathological analyses combined to [18F]-FDG PET imaging further demonstrated the presence of several end-organ long-term complications, including reduction in myocardial glucose utilization, renal dysfunction as well as microvascular neuropathy and retinopathy. We also provide for the first time a comprehensive µ-PET whole brain imaging of the changes in glucose metabolic activity within discrete cerebral regions in HF/HF/Stz diabetic rats. Altogether, we developed and characterized a unique non-genetic preclinical model of T2DM adapted to the current diet and lifestyle that recapitulates the major metabolic features of the disease progression, from insulin resistance to pancreatic ß-cell dysfunction, and closely mimicking the target-organ damage occurring in type 2 diabetic patients at advanced stages.

Deletion of AT2 Receptor Prevents SHP-1-Induced VEGF Inhibition and Improves Blood Flow Reperfusion in Diabetic Ischemic Hindlimb.

OBJECTIVE: Ischemia caused by narrowing of femoral artery is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes mellitus. We have previously reported that the inhibition of the angiogenic response to VEGF (vascular endothelial growth factor) in diabetic mice was associated with the increased expression of SHP-1 (SH2 domain-containing phosphatase 1), a protein that can be activated by the AT2 (angiotensin II type 2) receptor. Deletion of AT2 receptor has been shown to promote angiogenesis within the ischemic muscle. However, the relative impact of AT2 receptor in diabetic condition remains unknown. APPROACH AND RESULTS: Nondiabetic and diabetic AT2 null (Atgr2-/Y) mice underwent femoral artery ligation after 2 months of diabetes mellitus. Blood perfusion was measured every week ≤4 weeks post-surgery. Expression of the VEGF, SHP-1, and renin-angiotensin pathways was evaluated. Blood flow in the ischemic muscle of diabetic Atgr2-/Y mice recovered faster and ≤80% after 4 weeks compared with 51% recovery in diabetic control littermates. Diabetic Atgr2-/Y had reduced apoptotic endothelial cells and elevated small vessel formation compared with diabetic Atgr2+/Y mice, as well as increased SHP-1 expression and reduced VEGF receptor activity. In endothelial cells, high glucose levels and AT2 agonist treatment did not change SHP-1, VEGF, and VEGF receptor expression. However, the activity of SHP-1 and its association with the VEGF receptors were increased, causing inhibition of the VEGF action in endothelial cell proliferation and migration. CONCLUSIONS: Our results suggest that the deletion of AT2 receptor reduced SHP-1 activity and restored VEGF actions, leading to an increased blood flow reperfusion after ischemia in diabetes mellitus.

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-ß 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-ß 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.

Increased SHP-1 protein expression by high glucose levels reduces nephrin phosphorylation in podocytes.

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr(1127) and Tyr(1152) are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2(+/C96Y)) compared with control littermate mice (Ins2(+/+)), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.

PKCδ impaired vessel formation and angiogenic factor expression in diabetic ischemic limbs.

Decreased collateral vessel formation in diabetic peripheral limbs is characterized by abnormalities of the angiogenic response to ischemia. Hyperglycemia is known to activate protein kinase C (PKC), affecting the expression and activity of growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The current study investigates the role of PKCδ in diabetes-induced poor collateral vessel formation and inhibition of angiogenic factors expression and actions. Ischemic adductor muscles of diabetic Prkcd(+/+) mice exhibited reduced blood reperfusion, vascular density, and number of small vessels compared with nondiabetic Prkcd(+/+) mice. By contrast, diabetic Prkcd(-/-) mice showed significant increased blood flow, capillary density, and number of capillaries. Although expression of various PKC isoforms was unchanged, activation of PKCδ was increased in diabetic Prkcd(+/+) mice. VEGF and PDGF mRNA and protein expression were decreased in the muscles of diabetic Prkcd(+/+) mice and were normalized in diabetic Prkcd(-/-) mice. Furthermore, phosphorylation of VEGF receptor 2 (VEGFR2) and PDGF receptor-ß (PDGFR-ß) were blunted in diabetic Prkcd(+/+) mice but elevated in diabetic Prkcd(-/-) mice. The inhibition of VEGFR2 and PDGFR-ß activity was associated with increased SHP-1 expression. In conclusion, our data have uncovered the mechanisms by which PKCδ activation induced poor collateral vessel formation, offering potential novel targets to regulate angiogenesis therapeutically in diabetic patients.

Expression of SHP-1 induced by hyperglycemia prevents insulin actions in podocytes.

Renal podocyte apoptosis is an early event of diabetic nephropathy progression. Insulin action is critical for podocyte survival. Previous studies demonstrated that Src homology-2 domain-containing phosphatase-1 (SHP-1) is elevated in renal cortex of type 1 diabetic mice; we hypothesized that hyperglycemia-induced SHP-1 expression may affect insulin actions in podocytes. Type 1 diabetic Akita mice (Ins2(+/C96Y)) developed elevated foot process effacement and podocyte apoptosis compared with control littermate mice (Ins2(+/+)). In contrast to Ins2(+/+) mice, insulin-stimulated protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) phosphorylation were remarkably reduced in renal podocytes of Akita mice. This renal insulin resistance was associated with elevated SHP-1 expression in the glomeruli. Cultured podocytes exposed to high glucose concentration (HG; 25 mM) for 96 h exhibited high levels of apoptotic markers and caspase-3/7 enzymatic activity. HG exposure raised mRNA and protein levels of SHP-1 and reduced the insulin-signaling pathway in podocytes. Overexpression of dominant-negative SHP-1 in podocytes prevented HG effects and restored insulin actions. Elevated SHP-1 expression induced by high glucose levels was directly associated with insulin receptor-ß in vitro and in vivo to prevent insulin-stimulated Akt and ERK phosphorylation. In conclusion, our results showed that high levels of SHP-1 expression in glomeruli cause insulin resistance and podocyte loss, thereby contributing to diabetic nephropathy.