OVE26 Diabetic Mice Research Articles

LRG1 loss effectively restrains glomerular TGF-β signaling to attenuate diabetic kidney disease

Transforming growth factor (TGF)-β signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-β signaling regulators can substantially influence TGF-β's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-β-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-β-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-β signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-β signaling to attenuate DKD.

S-07-6: NEUTROPHIL EXTRACELLULAR TRAPS AS MEDIATORS OF ENDOTHELIAL DAMAGE IN HYPERTENSION AND DIABETES

Objective: Endothelial dysfunction (ED) plays a key role in the pathogenesis of hypertension and diabetes, yet its molecular determinants are poorly understood. Concomitant hypertension and diabetes can synergistically increase damage to the vasculature, with increasing evidence suggesting that resulting immune dysregulation may contribute to endothelial injury. Neutrophil extracellular traps (NETs), networks of chromatin and protein-containing extracellular fibers released to fight infection, are generated from neutrophils following activation of the citrullinating enzyme peptidylarginine deiminase 4 (PAD4). PAD4 has been shown to be upregulated in atherosclerosis, vasculitis, and various inflammatory autoimmune diseases, leading to NET-induced endothelial injury. Whether NETs could contribute to hypertension- and diabetes-induced ED has not been established. The objective of the present study is to investigate the impact of hypertension and diabetes on PAD4 activity and the contribution of NETs to resulting vascular injury. Design and Methods: Promyelocytic HL-60 cells were differentiated to neutrophil-like cells and treated with angiotensin II (10−7 M) and/or high glucose (25 mM) for 24 hours, with or without subsequent NET-inducing PMA stimulation (500 nM). NETs were isolated from the culture medium by differential centrifugation and assessed by Western blot analysis of citrullinated histone H3 (citH3, a primary NET component). Isolated NETs were used to treat Human Umbilical Vein Endothelial Cells (HUVECs) at 0.5–500 ng/ml for 24 hours, after which cell viability (XTT cell assay) and proliferation (BrdU assay) were measured. We also assessed NETosis in mice expressing human renin in the liver (TTRhRen) intercrossed with OVE26 diabetic mice [i.e. hypertensive-diabetic, HD mice], compared with wild-type (WT) FVBN/J mice. We examined kidney levels of citH3, neutrophil elastase (NE), and PAD4 expression by immunohistochemistry. Results: Treatment with high glucose, but not angiotensin II, increased citH3 levels in neutrophil-like cells. NETs isolated from PMA-stimulated cells decreased HUVEC viability by ∼2-fold at 5 ng/ml (P = < 0.0001), and dose dependently decreased proliferation from 0.5–500 ng/ml, with a ∼80% decrease at 500 ng/ml. In HD mice, we observed a significant increase in kidney levels of citH3 (P < 0.0001), NE (P < 0.0001), and PAD4 (P < 0.0001), which was associated with the presence of albuminuria and increased blood pressure levels. Conclusion: These results suggest that NETs are upregulated in hypertension and diabetes and may contribute to endothelial damage and microvascular injury.

The single-cell landscape of kidney immune cells reveals transcriptional heterogeneity in early diabetic kidney disease

The pathogenesis of diabetic kidney disease (DKD) involves multifactorial processes that converge to initiate and advance the disease. Although DKD is not typically classified as an inflammatory glomerular disease, mounting evidence supports the involvement of kidney inflammation as a key contributor in DKD pathogenesis, particularly through macrophages. However, detailed identification and corresponding phenotypic changes of macrophages in DKD remain poorly understood. To capture the gene expression changes in specific macrophage cell subsets in early DKD, we performed single-cell transcriptomic analysis of CD45-enriched kidney immune cells from type 1 diabetic OVE26 mice at two time points during the disease development. We also undertook a focused analysis of mononuclear phagocytes (macrophages and dendritic cells). Our results show increased resident and infiltrating macrophage subsets in the kidneys of mice with diabetes over time, with heightened expression of pro-inflammatory or anti-inflammatory genes in a subset-specific manner. Further analysis of macrophage polarization states in each subset in the kidneys showed changes consistent with the continuum of activation and differentiation states, with gene expression tending to shift toward undifferentiated phenotypes but with increased M1-like inflammatory phenotypes over time. By deconvolution analysis of RNAseq samples and by immunostaining of biopsies from patients with DKD, we further confirmed a differential expression of select genes in specific macrophage subsets essentially recapitulating the studies in mice. Thus, our study provides a comprehensive analysis of macrophage transcriptomic profiles in early DKD that underscores the dynamic macrophage phenotypes in disease progression.

ANGIOTENSIN II INCREASES FORMATION OF NEUTROPHIL EXTRACELLULAR TRAPS: ROLE IN HYPERTENSION AND DIABETES

Objective: Endothelial dysfunction plays a central mechanistic role in the pathogenesis of hypertension and diabetes. However, the molecular determinants of endothelial dysfunction are not fully understood. Peptidylarginine deiminases (PADs) irreversibly convert arginine to citrulline in protein, generating neoantigens that are unrecognizable to the immune system. PAD4, an isoform predominantly expressed in neutrophils is upregulated in autoimmune and coronary artery disease. PAD4 activation, in turn, promotes vascular injury through the formation of neutrophil extracellular traps (NETs), networks of chromatin and protein-containing extracellular fibres released to fight infection. Whether PAD4 activity is altered in hypertension and diabetes and whether this contributes to disease pathogenesis is not known. The objective of the present study was to examine the effect of angiotensin II on formation of neutrophil extracellular traps in vitro and in an animal model of hypertension and diabetes. Design and method: Neutrophil-like cells were generated from differentiation of the HL-60 cell line and then treated with angiotensin II (10-7 M) for 24 hours. NET formation was assessed by Western blot analysis of citrullinated histone H3 (a primary component of NETs). We further assessed NET formation in mice expressing human renin under the control of the transthyretin promoter (TTRhRen) intercrossed with OVE26 diabetic mice [i.e. hypertensive-diabetic, HD mice] and compared with wild-type (WT) FVBN/J mice. We examined levels of citrullinated histone H3, neutrophil elastase, and PAD4 expression in the kidney of HD and WT mice by immunohistochemistry. Results: Treatment of neutrophil-like cells with angiotensin-II increased levels of citrullinated histone H3 by ∼2-fold. Similarly, in HD mice we observed a significant increase in citrullinated histone H3 (P < 0.0001), in neutrophil elastase (P < 0.0001), and in PAD4 protein levels (P < 0.0001) compared with WT. Increased PAD4 activity was associated with the presence of albuminuria, renal interstitial fibrosis and reduced glomerular filtration rate. Conclusions: Taken together these data suggest that angiotensin II induces NET formation leading to renal accumulation in hypertension and diabetes. PAD4 may therefore play a role in target organ injury in hypertension and diabetes.

Protection against diabetic cardiomyopathy is achieved using a combination of sulforaphane and zinc in type 1 diabetic OVE26 mice.

Sulforaphane (SFN) can effectively induce nuclear factor E2–related factor 2 (Nrf2), and zinc (Zn) can effectively induce metallothionein (MT), both of which have been shown to protect against diabetic cardiomyopathy (DCM). However, it is unclear whether combined treatment with SFN and Zn offers better cardiac protection than either one alone. Here, we treated 5‐week‐old OVE mice that spontaneously develop type 1 diabetes with SFN and/or Zn for 18 weeks. Cardiac dysfunction, by echocardiography, and pathological alterations and remodelling, shown by cardiac hypertrophy, fibrosis, inflammation and oxidative damage, examined by histopathology, Western blotting and real‐time PCR, were observed in OVE mice. All these dysfunction and pathological abnormalities seen in OVE mice were attenuated in OVE mice with treatment of either SFN, Zn or SFN/Zn, and the combined treatment with SFN/Zn was better than single treatments at ameliorating DCM. In addition, combined SFN and Zn treatment increased Nrf2 function and MT expression in the heart of OVE mice to a greater extent than SFN or Zn alone. This indicates that the dual activation of Nrf2 and MT by combined treatment with SFN and Zn may be more effective than monotherapy at preventing the development of DCM via complementary, additive mechanisms.

489-P: Loss of Neuropilin 1 in Podocytes Aggravates Diabetic Nephropathy

Podocyte injury is a crucial pathological event in diabetic nephropathy. Neuropilin 1 (NRP1) is a multi-functional transmembrane protein and functions as a co-receptor of extracellular ligands, such as VEGF. Previous studies have shown that NRP1 levels are significantly decreased in the kidney of diabetic patients, and advanced glycation end products down-regulate NRP1 in a mouse podocyte cell line. The function of NRP1 in podocytes has not been clearly understood. We found that NRP1 levels are significantly decreased in the glomeruli of db/db mice, STZ-induced diabetic mice and OVE26 mice. A podocyte-specific NRP1 knockout mouse strain (NRP1-CKO) is generated by crossbreeding NRP1flox/flox mice with Nphs2-Cre transgenic mice. Compared to NRP1flox/flox mice, the NRP1-CKO mice in the absence of diabetes showed increased 24h urine albumin excretion in the age of 2 months and a reduced creatinine clearance at the age of 4 months. Enlarged foot processes of podocytes and flattened podocyte body was found in NRP1-CKO mice, compared to age-matched NRP1flox/flox mice. NRP1-CKO mice showed significantly decreased podocyte numbers per glomerulus and increased apoptosis. We generated diabetic NRP1-CKO mice (NRP1-CKO-OVE26) by crossbreeding the NRP1-CKO mice with OVE26 diabetic mice. NRP1-CKO/OVE26 diabetic mice developed more severe proteinuria, compared to the age- and genetic background-matched NRP1flox/flox/OVE26 mice. As shown by PAS staining, the glomerular structure was disturbed, and glomerular mesangial expansion was more severe in NRP1-CKO/OVE26 mice. Phosphorylation of MAPKP38 was significantly elevated in the NRP1-deficient glomeruli, compared to age-matched NRP1flox/flox glomeruli, suggesting that NRP1 deficiency induces activation of P38 signaling, further confirming podocyte stress and injury in NRP1-CKO mice. These results suggest that NRP1 ablation impairs renal function and exacerbates diabetic nephropathy and NRP1 is essential in the regulation of podocyte function and survival. Disclosure R. Cheng: None. X. He: None. J. Ma: None. Funding Oklahoma Center for the Advancement of Science and Technology; National Institutes of Health

470-P: Combined Use of Sulforaphane and Zinc Provides a Better Protection against Diabetic Cardiomyopathy than Either One Alone in Type 1 Diabetic OVE26 Mice

Diabetic cardiomyopathy (DCM) is the main cause of heart failure in diabetic patients. Oxidative stress is considered to be one of the most important mechanisms. Thus, treatments targeting oxidative stress may represent suitable strategies for the effective prevention of DCM. Nuclear factor-E2-related factor 2 (Nrf2), a transcription factor responsible to oxidative stress, goes to nuclei and binds to antioxidant response elements of promoters of several antioxidant genes. Metallothionein (MT) is a protein that can bind heavy metal ions such as zinc (Zn) and also a potent scavenger of free radicals because of its high thiol contents. Genetically overexpressing either Nrf2 or MT specifically in the heart protects from DCM. In addition, we also showed that induction of MT with Zn or activation of Nrf2 with sulforaphane (SFN) also partially protects from DCM. Here we further explore whether up-regulation of both simultaneously with Zn and SFN combination would provide a better protection against DCM than either alone. OVE26 mouse is one genetic type 1 diabetic model with the diabetes onset at 3 weeks old and survival more than one year without insulin treatment, so it is very good model for diabetic complication study. 5-week old OVE26 mice were confirmed with hyperglycemia and then treated with SFN (0.5 mg/kg) and/or Zn (5 mg/kg) for 18 weeks. 23-week old OVE26 mice showed typical DCM, reflected by cardiac dysfunction and remodeling fibrosis by echocardiography and collagen staining, along significantly oxidative damage, and inflammatory responses using western blotting and real-time PCR. Treatment of OVE26 mice either with SFN or Zn significantly prevented the development of DCM, along with up-regulating Nrf2 function and overexpressing MT protein, respectively, but the best protection from DCM is in the SFN/Zn group. Therefore, combined use of SFN with Zn may provide a better protection against DCM than SFN or Zn alone for diabetic patients. Disclosure J. Wang: None. W. Wang: None. L. Cai: None. Funding American Diabetes Association (1-15-BS-018, 1-18-IBS-082 to L.C.)

SIRT1 Is a Potential Drug Target for Treatment of Diabetic Kidney Disease.

Multiple studies have demonstrated a critical role of Sirtuin-1 (SIRT1) deacetylase in protecting kidney cells from cellular stresses. A protective role of SIRT1 has been reported in both podocytes and renal tubular cells in multiple kidney disease settings, including diabetic kidney disease (DKD). We and others have shown that SIRT1 exerts renoprotective effects in DKD in part through the deacetylation of transcription factors involved in the disease pathogenesis, such as p53, FOXO, RelA/p65NF-κB, STAT3, and PGC1α/PPARγ. Recently we showed that the podocyte-specific overexpression of SIRT1 attenuated proteinuria and kidney injury in an experimental model of DKD, further confirming SIRT1 as a potential target to treat kidney disease. Known agonists of SIRT1 such as resveratrol diminished diabetic kidney injury in several animal models. Similarly, we also showed that puerarin, a Chinese herbal medicine compound, activates SIRT1 to provide renoprotection in mouse models of DKD. However, as these are non-specific SIRT1 agonists, we recently developed a more specific and potent SIRT1 agonist (BF175) that significantly attenuated diabetic kidney injury in type 1 diabetic OVE26 mice. We also previously reported that MS417, a bromodomain inhibitor that disrupts the interaction between the acetyl-residues of NF-κB and bromodomain-containing protein 4 (BRD4) also attenuates DKD. These results suggest that SIRT1 agonists and bromodomain inhibitors could be potential new therapuetic treatments against DKD progression.

Increased podocyte Sirtuin-1 function attenuates diabetic kidney injury

Podocyte injury and loss contribute to the progression of glomerular diseases, including diabetic kidney disease. We previously found that the glomerular expression of Sirtuin-1 (SIRT1) is reduced in human diabetic glomeruli and that the podocyte-specific loss of SIRT1 aggravated albuminuria and worsened kidney disease progression in diabetic mice. SIRT1 encodes an NAD-dependent deacetylase that modifies the activity of key transcriptional regulators affected in diabetic kidneys, including NF-κB, STAT3, p53, FOXO4, and PGC1-α. However, whether the increased glomerular SIRT1 activity is sufficient to ameliorate the pathogenesis of diabetic kidney disease has not been explored. We addressed this by inducible podocyte-specific SIRT1 overexpression in diabetic OVE26 mice. The induction of SIRT1 overexpression in podocytes for six weeks in OVE26 mice with established albuminuria attenuated the progression of diabetic glomerulopathy. To further validate the therapeutic potential of increased SIRT1 activity against diabetic kidney disease, we developed a new, potent and selective SIRT1 agonist, BF175. In cultured podocytes BF175 increased SIRT1-mediated activation of PGC1-α and protected against high glucose-mediated mitochondrial injury. Invivo, administration of BF175 for six weeks in OVE26 mice resulted in a marked reduction in albuminuria and in glomerular injury in a manner similar to podocyte-specific SIRT1 overexpression. Both podocyte-specific SIRT1 overexpression and BT175 treatment attenuated diabetes-induced podocyte loss and reduced oxidative stress in glomeruli of OVE26 mice. Thus, increased SIRT1 activity protects against diabetes-induced podocyte injury and effectively mitigates the progression of diabetic kidney disease.

Differential expression of endoplasmic reticulum stress-response proteins in different renal tubule subtypes of OVE26 diabetic mice.

Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-responseinduced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced proapoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.

Zinc treatment prevents type 1 diabetes-induced hepatic oxidative damage, endoplasmic reticulum stress, and cell death, and even prevents possible steatohepatitis in the OVE26 mouse model: Important role of metallothionein

Whether zinc is able to improve diabetes-induced liver injury remains unknown. Transgenic type 1 diabetic (OVE26) mice develop hyperglycemia at 3 weeks old; therefore therapeutic effect of zinc on diabetes-induced liver injury was investigated in OVE26 mice. Three-month old OVE26 and age-matched wild-type mice were treated by gavage with saline or zinc at 5mg/kg body-weight every other day for 3 months. Hepatic injury was examined by serum alanine aminotransferase (ALT) level with liver histopathological and biochemical changes. OVE26 mice at 6 months old showed significant increases in serum ALT level and hepatic oxidative damage, endoplasmic reticulum stress and associated cell death, mild inflammation, and fibrosis. However, all these hepatic morphological and functional changes were significantly prevented in 3-month zinc-treated OVE26 mice. Mechanistically, zinc treatment significantly increased hepatic metallothionein, a protein with known antioxidant activity, in both wild-type and OVE26 mice. These results suggest that there were significantly functional, structural and biochemical abnormalities in the liver of OVE26 diabetic mice at 6 months old; however, all these changes could be prevented with zinc treatment, which was associated with the upregulation of hepatic metallothionein expression.

A novel mouse model of advanced diabetic kidney disease.

Currently available rodent models exhibit characteristics of early diabetic nephropathy (DN) such as hyperfiltration, mesangial expansion, and albuminuria yet features of late DN (hypertension, GFR decline, tubulointerstitial fibrosis) are absent or require a significant time investment for full phenotype development. Accordingly, the aim of the present study was to develop a mouse model of advanced DN with hypertension superimposed (HD mice). Mice transgenic for human renin cDNA under the control of the transthyretin promoter (TTRhRen) were employed as a model of angiotensin-dependent hypertension. Diabetes was induced in TTRhRen mice through low dose streptozotocin (HD-STZ mice) or by intercrossing with OVE26 diabetic mice (HD-OVE mice). Both HD-STZ and HD-OVE mice displayed more pronounced increases in urinary albumin levels as compared with their diabetic littermates. Additionally, HD mice displayed renal hypertrophy, advanced glomerular scarring and evidence of tubulointerstitial fibrosis. Both HD-OVE and HD-STZ mice showed evidence of GFR decline as FITC-inulin clearance was decreased compared to hyperfiltering STZ and OVE mice. Taken together our results suggest that HD mice represent a robust model of type I DN that recapitulates key features of human disease which may be significant in studying the pathogenesis of DN and in the assessment of putative therapeutics.

High Glucose Forces a Positive Feedback Loop Connecting Akt Kinase and FoxO1 Transcription Factor to Activate mTORC1 Kinase for Mesangial Cell Hypertrophy and Matrix Protein Expression

High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion, which are recognized as cardinal signatures for the development of diabetic nephropathy. How mesangial cells sustain the activated state of Akt is not clearly understood. Here we show Akt-dependent phosphorylation of the transcription factor FoxO1 by high glucose. Phosphorylation-deficient, constitutively active FoxO1 inhibited the high glucose-induced phosphorylation of Akt to suppress the phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased the phosphorylation of Akt, resulting in increased mTORC1 activity similar to high glucose treatment. Notably, FoxO1 regulates high glucose-induced protein synthesis, hypertrophy, and expression of fibronectin and PAI-1. High glucose paves the way for complications of diabetic nephropathy through the production of reactive oxygen species (ROS). We considered whether the FoxO1 target antioxidant enzyme catalase contributes to sustained activation of Akt. High glucose-inactivated FoxO1 decreases the expression of catalase to increase the production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate the inactivation of FoxO1 and PRAS40, resulting in the inhibition of mTORC1 and mesangial cell hypertrophy and fibronectin and PAI-1 expression. Finally, using kidney cortices from type 1 diabetic OVE26 mice, we show that increased FoxO1 phosphorylation is associated with decreased catalase expression and increased fibronectin and PAI-1 expression. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of Akt involving inactivated FoxO1 and a decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression.

Diminished Autophagy Limits Cardiac Injury in Mouse Models of Type 1 Diabetes

Cardiac autophagy is inhibited in type 1 diabetes. However, it remains unknown if the reduced autophagy contributes to the pathogenesis of diabetic cardiomyopathy. We addressed this question using mouse models with gain- and loss-of-autophagy. Autophagic flux was inhibited in diabetic hearts when measured at multiple time points after diabetes induction by streptozotocin as assessed by protein levels of microtubule-associated protein light chain 3 form 2 (LC3-II) or GFP-LC3 puncta in the absence and presence of the lysosome inhibitor bafilomycin A1. Autophagy in diabetic hearts was further reduced in beclin 1- or Atg16-deficient mice but was restored partially or completely by overexpression of beclin 1 to different levels. Surprisingly, diabetes-induced cardiac damage was substantially attenuated in beclin 1- and Atg16-deficient mice as shown by improved cardiac function as well as reduced levels of oxidative stress, interstitial fibrosis, and myocyte apoptosis. In contrast, diabetic cardiac damage was dose-dependently exacerbated by beclin 1 overexpression. The cardioprotective effects of autophagy deficiency were reproduced in OVE26 diabetic mice. These effects were associated with partially restored mitophagy and increased expression and mitochondrial localization of Rab9, an essential regulator of a non-canonical alternative autophagic pathway. Together, these findings demonstrate that the diminished autophagy is an adaptive response that limits cardiac dysfunction in type 1 diabetes, presumably through up-regulation of alternative autophagy and mitophagy.

Therapeutic Effect of MG132 on the Aortic Oxidative Damage and Inflammatory Response in OVE26 Type 1 Diabetic Mice

The present study tested whether MG132 increases vascular nuclear factor E2-related factor-2 (Nrf2) expression and transcription to provide a therapeutic effect on diabetes-induced pathogenic changes in the aorta. To this end, three-month-old OVE26 diabetic and age-matched control mice were intraperitoneally injected with MG-132, 10 μg/kg daily for 3 months. OVE26 transgenic type 1 diabetic mice develop hyperglycemia at 2-3 weeks of age and exhibit albuminuria at 3 months of age with mild increases in TNF-α expression and 3-NT accumulation in the aorta. Diabetes-induced significant increases in the wall thickness and structural derangement of aorta were found in OVE26 mice with significant increases in aortic oxidative and nitrosative damage, inflammation, and remodeling at 6 months of diabetes, but not at 3 months of diabetes. However, these pathological changes seen at the 6 months of diabetes were abolished in OVE26 mice treated with MG-132 for 3 months that were also associated with a significant increase in Nrf2 expression in the aorta as well as transcription of downstream genes. These results suggest that chronic treatment with low-dose MG132 can afford an effective therapy for diabetes-induced pathogenic changes in the aorta, which is associated with the increased Nrf2 expression and transcription.

Therapeutic effect of MG-132 on diabetic cardiomyopathy is associated with its suppression of proteasomal activities: roles of Nrf2 and NF-κB

MG-132, a proteasome inhibitor, can upregulate nuclear factor (NF) erythroid 2-related factor 2 (Nrf2)-mediated antioxidative function and downregulate NF-κB-mediated inflammation. The present study investigated whether through the above two mechanisms MG-132 could provide a therapeutic effect on diabetic cardiomyopathy in the OVE26 type 1 diabetic mouse model. OVE26 mice develop hyperglycemia at 2-3 wk after birth and exhibit albuminuria and cardiac dysfunction at 3 mo of age. Therefore, 3-mo-old OVE26 diabetic and age-matched control mice were intraperitoneally treated with MG-132 at 10 μg/kg daily for 3 mo. Before and after MG-132 treatment, cardiac function was measured by echocardiography, and cardiac tissues were then subjected to pathological and biochemical examination. Diabetic mice showed significant cardiac dysfunction, including increased left ventricular systolic diameter and wall thickness and decreased left ventricular ejection fraction with an increase of the heart weight-to-tibia length ratio. Diabetic hearts exhibited structural derangement and remodeling (fibrosis and hypertrophy). In diabetic mice, there was also increased systemic and cardiac oxidative damage and inflammation. All of these pathogenic changes were reversed by MG-132 treatment. MG-132 treatment significantly increased the cardiac expression of Nrf2 and its downstream antioxidant genes with a significant increase of total antioxidant capacity and also significantly decreased the expression of IκB and the nuclear accumulation and DNA-binding activity of NF-κB in the heart. These results suggest that MG-132 has a therapeutic effect on diabetic cardiomyopathy in OVE26 diabetic mice, possibly through the upregulation of Nrf2-dependent antioxidative function and downregulation of NF-κB-mediated inflammation.

Potential role for Nrf2 activation in the therapeutic effect of MG132 on diabetic nephropathy in OVE26 diabetic mice

Oxidative stress is a major cause of diabetic nephropathy. Upregulation of the key antioxidative transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), was found to prevent the development of diabetic nephropathy. The present study was designed to explore the therapeutic effect of Nrf2 induced by proteasomal inhibitor MG132 at a low dose (10 μg/kg) on diabetic nephropathy. Transgenic type 1 diabetic (OVE26) mice displayed renal dysfunction with albuminuria by 3 mo of age, at which time MG132 treatment was started. After 3-mo treatment with MG132, renal function, morphology, and biochemical changes were examined with real-time PCR, Western blotting, and immunohistochemical examination. Compared with age-matched, nontreated diabetic mice, MG132-treated diabetic mice showed significant improvements in terms of renal structural and functional alterations. These therapeutic effects were associated with increased Nrf2 expression and transcriptional upregulation of Nrf2-regulated antioxidants. Mechanistic study using human renal tubular HK11 cells confirmed the role of Nrf2, as silencing the Nrf2 gene with its specific siRNA abolished MG132 prevention of high-glucose-induced profibrotic response. Furthermore, diabetes was found to significantly increase proteasomal activity in the kidney, an effect that was significantly attenuated by 3 mo of treatment with MG132. These results suggest that MG132 upregulates Nrf2 function via inhibition of diabetes-increased proteasomal activity, which can provide the basis for the therapeutic effect of MG132 on the kidney against diabetes-induced oxidative damage, inflammation, fibrosis, and eventual dysfunction.

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1–7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

Targeted Inhibition of Calpain Reduces Myocardial Hypertrophy and Fibrosis in Mouse Models of Type 1 Diabetes

OBJECTIVERecently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.RESEARCH DESIGN AND METHODSDiabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions.RESULTSCalpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose–induced proliferation and MMP activities were prevented by calpain inhibition.CONCLUSIONSMyocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

Uninephrectomy of Diabetic OVE26 Mice Greatly Accelerates Albuminuria, Fibrosis, Inflammatory Cell Infiltration and Changes in Gene Expression

Background/Aims: OVE26 (OVE) mice provide a valuable model of advanced diabetic nephropathy (DN), but they take 8 months to develop moderate interstitial fibrosis and reduced glomerular filtration rate (GFR). The aim of this project was to produce a more rapid and advanced model of DN. Methods: Uninephrectomy was applied to OVE and FVB mice at 2 months of age. Albuminuria, GFR, glomerulosclerosis, interstitial fibrosis, gene expression and monocyte infiltration were evaluated as a function of diabetes and uninephrectomy. Results: Albuminuria, monocyte infiltration, mesangial matrix expansion and renal fibrosis were greatly accelerated in uninephrectomized mice. DN was more advanced 10 weeks after uninephrectomy than in untreated OVE mice at 8 months of age. Uninephrectomy had almost no effect on these characteristics in non-diabetic mice. Microarray studies indicated that the accelerated fibrosis and cell infiltration in nephrectomized OVE mice were accompanied by corresponding gene expression changes in canonical pathways for fibrosis and inflammation. Conclusion: Uninephrectomy greatly accelerates all features of diabetic renal damage. This procedure provides a 10-week period after surgery to examine very large changes in the pathology of DN. The model may be particularly useful for testing new therapies and for analysis of the progression of albuminuria and fibrosis in DN.