BACKGROUND AND AIMS:Fibroblast growth factor (FGF) 1 demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic and obese mice by an uncertain mechanism. This study investigated the therapeutic activity and mechanism of a nonmitogenic FGF1 variant carrying 3 substitutions of heparin-binding sites (FGF1△HBS ) against NAFLD.APPROACH AND RESULTS:FGF1△HBS administration was effective in 9-month-old diabetic mice carrying a homozygous mutation in the leptin receptor gene (db/db) with NAFLD; liver weight, lipid deposition, and inflammation declined and liver injury decreased. FGF1△HBS reduced oxidative stress by stimulating nuclear translocation of nuclear erythroid 2 p45-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1△HBS also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1△HBS . In palmitate-treated hepatic cells, small interfering RNA (siRNA) knockdown of Nrf2 abolished only FGF1△HBS antioxidative actions but not improvement of lipid metabolism. In contrast, AMPK inhibition by pharmacological agent or siRNA abolished FGF1△HBS benefits on both oxidative stress and lipid metabolism that were FGF receptor (FGFR) 4 dependent. Further support of these in vitro findings is that liver-specific AMPK knockout abolished therapeutic effects of FGF1△HBS against high-fat/high-sucrose diet-induced hepatic steatosis. Moreover, FGF1△HBS improved high-fat/high-cholesterol diet-induced steatohepatitis and fibrosis in apolipoprotein E knockout mice.CONCLUSIONS:These findings indicate that FGF1△HBS is effective for preventing and reversing liver steatosis and steatohepatitis and acts by activation of AMPK through hepatocyte FGFR4.

CD45 is a pan-leukocyte marker, and CD45 stain is widely used to determine the extent of inflammatory cell infiltration and its association with tissue injury. In this manuscript, we share a reliable immunohistochemistry (IHC) protocol for CD45 staining in sections of paraffin-embedded mouse kidney. A rat anti-CD45 antibody was used as primary antibody, and a mouse adsorbed biotin-conjugated goat anti-rat IgG was selected as secondary antibody. A horseradish peroxidase (HRP)-linked avidin/ biotin detection system was used to amplify the signal, which was detected with 3,3'-Diaminobenzidine ( DAB). With this protocol, we show that the CD45 antibody recognizes cells of hematolymphoid lineage in bone marrow, as well as monocyte/macrophages in liver and lung tissue. The utility of this protocol in pathology research was indicated by dramatically increased CD45-positive (CD45(+)) cells in the kidneys of a mouse model of diabetes. Double staining for CD45 and injury marker KIM-1 showed accumulated CD45(+) cells around injured tubular cells. CD45 and F4/80 macrophage staining on adjacent tissue sections revealed overlap of CD45(+) cells with other inflammatory cells.

Diabetes-induced oxidative stress contributes to endothelial progenitor cells (EPCs) dysfunction and impairs endothelial regeneration. Thus, we tested whether increasing antioxidant protein metallothionein (MT) in EPCs promotes angiogenesis in a hind limb ischemia (HLI) model in endothelial-specific MT transgenic (JTMT) mice with high fat diet and streptozocin (HFD/STZ)-induced diabetes. Compared with littermate wild type (WT) diabetic mice, JTMT mice had improved blood flow recovery and angiogenesis after HLI. Similarly, transplantation of JTMT bone marrow-derived mononuclear cells (BM-MNCs) stimulated greater blood flow recovery in db/db mice with HLI than did WT BM-MNCs. The improved recovery was associated with augmented EPC function: mobilization and infiltration in JTMT mice; and, enhanced capillary EPC incorporation in db/db mice. Further, cultured JTMT-EPCs had enhanced cell survival, migration, and capillary tube formation in hypoxia/hyperglycemic conditions compared with WT-EPCs. Mechanistically, MT overexpression enhanced hypoxia-inducible factor 1α (HIF-1α), stromal cell-derived factor (SDF-1) and vascular endothelial growth factor (VEGF) expression, and reduced oxidative stress in ischemic tissues. MT’s pro-EPC effects were abrogated by siRNA knockdown of HIF-1α without affecting MT’s anti-oxidant action. These results indicate that endothelial-specific MT overexpression is sufficient to protect against diabetes-induced impairment of angiogenesis by promoting EPC functions though upregulation of HIF-1α/SDF-1/VEGF signaling and reducing oxidative stress. Disclosure K. Wang: None. X. Dai: None. J. Chen: None. F. Shen: None. K.A. Wintergerst: None. P.N. Epstein: None. L. Cai: None. Y. Tan: None. Funding American Diabetes Association (1-11-BS-017, 1-15-BS-018 to L.C.)

In the article cited above, affiliation 1 was mistakenly included for authors Yi Tan and Lu Cai. The author …

Diabetes-induced oxidative stress is one of the major contributors to dysfunction of endothelial progenitor cells (EPCs) and impaired endothelial regeneration. Thus, we tested whether increasing antioxidant protein metallothionein (MT) in EPCs promotes angiogenesis in a hind limb ischemia (HLI) model in endothelial MT transgenic (JTMT) mice with high fat diet and streptozocin-induced diabetes. Compared with littermate wild-type (WT) diabetic mice, JTMT diabetic mice had improved blood flow recovery and angiogenesis after HLI. Similarly, transplantation of JTMT bone marrow-derived mononuclear cells (BM-MNCs) stimulated greater blood flow recovery in db/db mice with HLI than did WT BM-MNCs. The improved recovery was associated with augmented EPC mobilization and angiogenic function. Further, cultured EPCs from diabetic patients exhibited decreased MT expression, increased cell apoptosis and impaired tube formation; while cultured JTMT-EPCs had enhanced cell survival, migration, and tube formation in hypoxia/hyperglycemic conditions compared with WT-EPCs. Mechanistically, MT overexpression enhanced hypoxia-inducible factor 1α (HIF-1α), stromal cell-derived factor (SDF-1) and vascular endothelial growth factor (VEGF) expression, and reduced oxidative stress in ischemic tissues. MT’s pro-EPC effects were abrogated by siRNA knockdown of HIF-1α without affecting MT’s anti-oxidant action. These results indicate that endothelial MT overexpression is sufficient to protect against diabetes-induced impairment of angiogenesis by promoting EPC functions most likely through upregulation of HIF-1α/SDF-1/VEGF signaling and reducing oxidative stress.

Fibroblast growth factor 1 (FGF1) demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic (T2D) and obese mice by uncertain mechanism. This study investigated the therapeutic activity and mechanism of a non-mitagenic FGF1 variant (FGF1ΔHBS) against NAFLD. FGF1ΔHBS administration was effective in 9-month old db/db mice with established NAFLD; liver weight, lipid deposition and inflammation declined and liver function improved. FGF1ΔHBS reduced oxidative stress by nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1ΔHBS also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of AMP-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1ΔHBS. In palmitate treated hepatic cells, siRNA knockdown of Nrf2 abolished only FGF1ΔHBS anti-oxidative actions but not lipid metabolism improvement. In contrast, AMPK inhibition by pharmacological agents or siRNA abolished both FGF1ΔHBS benefits on oxidative stress and lipid metabolism that was FGF receptor 4 (FGFR4) dependent. Further support of these in vitro findings is that liver specific AMPK knockout abolished therapeutic effects of FGF1ΔHBS against high-fat-high-sugar diet-induced hepatic steatosis. These findings indicate that FGF1ΔHBS is effective in late stage NAFLD and acts by activation of AMPK via hepatocyte FGFR4. Disclosure Q. Lin: None. G. Cai: None. Z. Liu: None. J. Li: None. J. Li: None. K.A. Wintergerst: None. P.N. Epstein: None. L. Cai: None. Y. Li: None. Y. Tan: None. Funding American Diabetes Association (1-13-JF-53 to Y.T.), (1-18-IBS-082 to L.C.)

Diabetes-induced oxidative stress is one of the major contributors to dysfunction of endothelial progenitor cells (EPCs) and impaired endothelial regeneration. Thus, we tested whether increasing antioxidant protein metallothionein (MT) in EPCs promotes angiogenesis in a hind limb ischemia (HLI) model in endothelial MT transgenic (JTMT) mice with high-fat diet– and streptozocin-induced diabetes. Compared with littermate wild-type (WT) diabetic mice, JTMT diabetic mice had improved blood flow recovery and angiogenesis after HLI. Similarly, transplantation of JTMT bone marrow–derived mononuclear cells (BM-MNCs) stimulated greater blood flow recovery in db/db mice with HLI than did WT BM-MNCs. The improved recovery was associated with augmented EPC mobilization and angiogenic function. Further, cultured EPCs from patients with diabetes exhibited decreased MT expression, increased cell apoptosis, and impaired tube formation, while cultured JTMT EPCs had enhanced cell survival, migration, and tube formation in hypoxic/hyperglycemic conditions compared with WT EPCs. Mechanistically, MT overexpression enhanced hypoxia-inducible factor 1α (HIF-1α), stromal cell–derived factor (SDF-1), and vascular endothelial growth factor (VEGF) expression and reduced oxidative stress in ischemic tissues. MT’s pro-EPC effects were abrogated by siRNA knockdown of HIF-1α without affecting its antioxidant action. These results indicate that endothelial MT overexpression is sufficient to protect against diabetes-induced impairment of angiogenesis by promoting EPC function, most likely through upregulation of HIF-1α/SDF-1/VEGF signaling and reducing oxidative stress.

Diabetes mellitus associated dysfunction of endothelial progenitor cells (EPCs) may contribute to dysregulation of endothelial regeneration. Whether oxidative protection of EPCs by an antioxidant, such as the protein metallothionein (MT) can have positive angiogenic effects and by what mechanisms remain unclear. Endothelial-specific MT overexpression (JTMT) mice were made diabetic by high fat diet followed by streptozocin administration (HFD/STZ). Diabetic hind limb ischemia (HLI) was established by femoral artery ligation. Bone marrow mononuclear cells (MNCs) collected from JTMT mice were transplanted into db/db mice with HLI. Blood reperfusion was monitored. High glucose and hypoxia conditions in culture were adopted to mimic diabetic ischemia. Compared with wild-type (WT) littermates, JTMT mice were resistant to diabetes-induced impairment in ischemia angiogenesis and blood reperfusion in HFD/STZ diabetes. Similarly, transplantation of MT overexpressing MNCs showed better therapeutic effect on db/db mice with HLI than transplantation of WT MNCs. These changes were accompanied by increased mobilization and infiltration of EPCs in JTMT mice, and enhanced incorporation of EPCs into capillaries in JTMT MNCs transplanted db/db mice. Furthermore, EPCs from JTMT mice exhibited augmented cell survival, tube formation, and migration capacities under diabetic ischemia-like conditions. Mechanistically, the expression of hypoxia-inducible factor 1α (HIF-1α) and the secretion of stromal cell-derived factor (SDF-1) in blood and expression in ischemic tissues were upregulated in JTMT and JTMT-MNC transplanted db/db mice, and in cultured JTMT EPCs, which were accompanied by marked amelioration of oxidative stress. However, MT-mediated elevation of SDF-1 and improvements of function in EPCs were all abrogated by siRNA knockdown of HIF-1α expression without effect on the anti-oxidative capacity of MT. Endothelial-specific MT elevation is sufficient to protect against diabetes mellitus-induced impairment of ischemia angiogenesis by promoting EPC function. The benefits of MT are predominantly mediated by upregulation of the HIF-1α/SDF-1 pathway.

In the article cited above, affiliation 1 was mistakenly included for authors Yi Tan and Lu Cai. The author …

Aims: OVE26 mice (FVB background), genetically overexpressing calmodulin in pancreatic beta cells, develop early onset type 1 diabetes, leading to progressive diabetic nephropathy (DN), with features of established human DN. The role of gender in characteristics of renal lesions has remained unexplored. Methods: Male and female OVE26 mice were compared to age and sex matched wild-type, nondiabetic FVB mice at ages of 4, 12, 24 and 36 weeks. Nephropathy was examined by measuring urine albumin-to-creatinine ratio, histopathology, expression of pathological markers and immunochemistry in the same cohort of mice. Results: Progression of diabetic kidney disease was evident first in the OVE26 glomerulus, initially as mesangial matrix expansion at 4 weeks followed by loss of podocytes, glomerular volume expansion and severe albuminuria at 12 weeks. Tubule dilation and initiation of interstitial fibrosis did not become significant until 24 weeks. T-lymphocyte infiltration into the renal parenchyma appeared at 36 weeks. OVE26 female mice developed more advanced DN than male OVE26 mice, such as more severe albuminuria, greater podocyte loss, additional fibrosis and significantly more inflammatory cell infiltration. The female OVE26 mice had lowest level of plasma estradiol in all 36 weeks old mice, as well as renal estrogen receptors. Conclusions: This demonstration of the role of gender, combined with the detailed characterization of DN progression illustrates the value of OVE26 mice for understanding gender effects on DN and provides the basis for researchers to better select the age and sex of OVE26 mice in future studies of type 1 DN. Research in context: What is already known about this subject? OVE26 mice, genetically overexpressing calmodulin in pancreatic beta cells, develop early onset type 1 diabetes. OVE26 mice are a widely used and valuable rodent model which develop severe, progressive diabetic nephropathy, with features of established human diabetic nephropathy. What is the key question? Does gender play a role in determining characteristics of renal lesions and severity of nephropathy? What are the new findings? Female OVE26 mice had more severe albuminuria, greater podocyte loss. Female OVE26 mice had additional fibrosis and significantly more inflammatory cell infiltration. Diabetes induced reductions in estradiol levels and renal estrogen receptors may be responsible for the female sensitization to DN in OVE26 mice. How might this impact on clinical practice in the foreseeable future? Our findings provide the basis for researchers to better select the age and sex of OVE26 mice in future studies of type 1 DN.

Diabetes mellitus associated dysfunction of endothelial progenitor cells (EPCs) may contribute to dysregulation of endothelial regeneration. Metallothionein (MT) acts as an antioxidant in various pathophysiological processes. Whether MT in EPCs has positive angiogenic effects and the mechanisms involved remain unclear. Endothelial MT overexpression (JTMT) mice were made diabetic by high fat diet followed by STZ administration (HFD/STZ). Diabetic hind limb ischemia model was established by femoral artery ligation. Bone marrow derived mononuclear leucocytes (MNCs) collected from JTMT mice were transplanted into db/db mice with hind limb ischemia. Blood reperfusion was monitored and oxidative stress was measured. High glucose and hypoxia conditions in culture were adopted to mimic diabetic ischemia in vitro. MT improved blood perfusion of ischemic hind limb and promoted angiogenesis in HFD/STZ diabetes. Oxidative stress of ischemic muscles was aggravated under diabetic condition, which was reduced in JTMT mice. Compared with WT mice, the expression of HIF-1α, SDF-1, VEGF and p-Akt were all increased in JTMT mice, along with elevation of plasma SDF-1α and VEGF. Moreover, transplantation of MT overexpressed MNCs showed better therapeutic effect on db/db diabetic mice with hind limb ischemia than control or transplantation of normal MNCs. Transplantation was accompanied by elevated HIF-1α, SDF-1 and Akt signaling. Vitro studies demonstrated that MT overexpression reduced high glucose and hypoxia induced EPC dysfunction and preserved high glucose and hypoxia impaired HIF-1α, SDF-1, VEGF and Akt signaling, all of which were abolished by siRNA knockdown of HIF-1α. In conclusion, elevated MT enhances angiogenic function and therapeutic efficacy of EPCs in diabetic limb ischemia. The benefits of MT are predominantly mediated by a HIF-1α/SDF-1/Akt pathway under diabetic ischemia conditions. Disclosure K. Wang: None. X. Dai: None. J. He: None. C. Yang: None. J. Chen: None. K.A. Wintergerst: None. P.N. Epstein: None. L. Cai: None. Y. Tan: None. Funding American Diabetes Association (1-15-BS-018, 1-18-IBS-082 to L.C.); 1-13-JF-53 (to Y.T.)

Stromal cell-derived factor 1 (SDF-1)-chemokine receptors CXCR4/7 axis plays a vital role in diabetic limb ischemia. Our previous studies demonstrated that elevated CXCR7 improves therapeutic efficacy of endothelial progenitor cells (EPCs) in diabetic limb ischemia. The effects of a specific CXCR7 agonist TC14012 on diabetic hind limb ischemia remains untested. We hypothesize that CXCR7 expression is reduced in diabetic EPCs and TC14012 improve angiogenic function of EPCs in diabetic limb ischemia. Early WT-EPCs were transfected with siRNA against CXCR7, and early db/db-EPCs were infected with CXCR7 lentivirus. Tube formation and capillary density of ischemic gastrocnemius muscle (GS) and soleus muscle (SS) at 4 weeks post-ischemic surgery were adopt for indication of angiogenesis. Diabetic hind limb ischemia (HLI) models were established by femoral artery ligation, followed by administration of TC14012 subcutaneously. Total and cell surface expression of CXCR7 were significantly decreased in EPCs from db/db mice compared with those from WT mice, whereas no significant differences of CXCR4 expression were observed. Tube formation by EPCs from db/db mice was also impaired compared with WT-EPCs. siRNA knockdown of CXCR7 was accompanied by impaired tube formation. Conversely, increasing CXCR7 levels in db/db EPCs completely reversed impaired tube formation function. High glucose (HG) dose dependently decreased the expression of CXCR7 in vitro but not that of CXCR4. HG also impaired tube formation of EPC from WT mice. Tube formation function of HUVEC was impaired after treated with HG (25mM) for 24 hours, which could be prevented by TC14012. Most importantly, TC14012 significantly improved blood flow restoration of ischemic hind limb starting from 2 weeks after ligation and promoted angiogenesis of ischemic GS and SS. Diabetes attenuates CXCR7 expression and impairs angiogenesis of EPCs. TC14012 improves blood reperfusion of diabetic hind limb ischemia. Disclosure K. Wang: None. X. Dai: None. J. Chen: None. P.N. Epstein: None. L. Cai: None. K.A. Wintergerst: None. Y. Qian: None. Y. Tan: None.

Acute glomerulonephritis is characterized by rapid glomerular neutrophil recruitment, proteinuria, and glomerular hypercellularity. The current study tested the hypothesis that the release of neutrophil granule contents plays a role in both the loss of filtration barrier leading to proteinuria and the increase in glomerular cells. Inhibition of neutrophil exocytosis with a peptide inhibitor prevented proteinuria and attenuated podocyte and endothelial cell injury but had no effect on glomerular hypercellularity in an experimental acute glomerulonephritis model in mice. Cultivation of podocytes with neutrophil granule contents disrupted cytoskeletal organization, an in vitro model for podocyte effacement and loss of filtration barrier. Activated, cultured podocytes released cytokines that stimulated neutrophil chemotaxis, primed respiratory burst activity, and stimulated neutrophil exocytosis. We conclude that crosstalk between podocytes and neutrophils contributes to disruption of the glomerular filtration barrier in acute glomerulonephritis. Neutrophil granule products induce podocyte injury but do not participate in the proliferative response of intrinsic glomerular cells.

Background: Exercise promotes metabolic remodeling in the heart, which is associated with physiological cardiac growth; however, it is not known whether or how physical activity–induced changes in cardiac metabolism cause myocardial remodeling. In this study, we tested whether exercise-mediated changes in cardiomyocyte glucose metabolism are important for physiological cardiac growth. Methods: We used radiometric, immunologic, metabolomic, and biochemical assays to measure changes in myocardial glucose metabolism in mice subjected to acute and chronic treadmill exercise. To assess the relevance of changes in glycolytic activity, we determined how cardiac-specific expression of mutant forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase affect cardiac structure, function, metabolism, and gene programs relevant to cardiac remodeling. Metabolomic and transcriptomic screenings were used to identify metabolic pathways and gene sets regulated by glycolytic activity in the heart. Results: Exercise acutely decreased glucose utilization via glycolysis by modulating circulating substrates and reducing phosphofructokinase activity; however, in the recovered state following exercise adaptation, there was an increase in myocardial phosphofructokinase activity and glycolysis. In mice, cardiac-specific expression of a kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgene (GlycoLo mice) lowered glycolytic rate and regulated the expression of genes known to promote cardiac growth. Hearts of GlycoLo mice had larger myocytes, enhanced cardiac function, and higher capillary-to-myocyte ratios. Expression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in the heart (GlycoHi mice) increased glucose utilization and promoted a more pathological form of hypertrophy devoid of transcriptional activation of the physiological cardiac growth program. Modulation of phosphofructokinase activity was sufficient to regulate the glucose–fatty acid cycle in the heart; however, metabolic inflexibility caused by invariantly low or high phosphofructokinase activity caused modest mitochondrial damage. Transcriptomic analyses showed that glycolysis regulates the expression of key genes involved in cardiac metabolism and remodeling. Conclusions: Exercise-induced decreases in glycolytic activity stimulate physiological cardiac remodeling, and metabolic flexibility is important for maintaining mitochondrial health in the heart.

RationalEndothelial progenitor cells (EPCs) respond to SDF‐1 through receptors CXCR7 and CXCR4. Whether SDF‐1 receptors involves in diabetes induced EPCs dysfunction remains unknown.ObjectiveTo determine the role of SDF‐1 receptors in diabetic EPCs dysfunction.Methods and ResultsCXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, while up‐regulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low‐density lipoprotein (ox‐LDL) and high glucose (HG) also reduced CXCR7 expression, impaired tube formation and increased oxidative stress and apoptosis. The damaging effects of ox‐LDL and HG were markedly reduced by SDF‐1 pretreatment in EPCs transduced with CXCR7 lentivirus (CXCR7‐EPCs) but not in EPCs transduced with control lentivirus (Null‐EPCs). Most importantly, CXCR7‐EPCs were superior to Null‐EPCs for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that ox‐LDL and HG inhibited Akt and GSK‐3β phosphorylation, nuclear export of Fyn and nuclear localization of Nrf2, resulting in inhibiting Nrf2 downstream target genes HO‐1, NQO‐1 and catalase, and a corresponding increase in EPC oxidative stress. This destructive cascade could be blocked by SDF‐1 treatment in CXCR7‐EPCs. Furthermore, inhibition of PI3K/Akt prevented SDF‐1/CXCR7‐mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF‐1/CXCR7 on EPC function in vitro and in vivo.ConclusionsElevated expression of CXCR7 enhances EPC resistance to diabetes‐induced oxidative damage and improves therapeutic efficacy of EPCs for treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by the Akt/GSK‐3β/Fyn pathway producing increased activity of Nrf2.Support or Funding InformationThis study was supported in part by a Junior Faculty Award (1–13‐JF‐53) from American Diabetes Association, an Innovative Grant (1‐INO‐2014‐122‐A‐N) from Juvenile Diabetes Research Foundation, NSFC projects (81573435, 81200917, 81200239, 81370917).

In an effort to test our working hypothesis that diabetes‐induced renal glomerular damage may be the result of oxidative insult, JTMT transgenic mice were produced that overexpress the antioxidant protein metallothionein specifically in endothelial cells. JTMT animals were crossed with severely diabetic OVE transgenic mice to determine whether an endothelial specific antioxidant transgene might provide renoprotection from chronic diabetic complications. In double transgenic 150 day‐old OVE‐JTMT mice, diabetic parameters including blood glucose and HbA1c were indistinguishable from age‐matched OVE mice. However, our data indicate that endothelial‐specific metallothionein overexpression in OVE‐JTMT mice reduced a number of nephropathic complications of diabetes including severe albuminuria. In addition, compared to OVE diabetic mice, double transgenic OVE‐JTMT animals showed significant protection to all major components of the glomerular filtration barrier (i.e. podocyte foot process effacement, glomerular basement membrane thickening, and endothelial damage as evidenced by reduced loss of percent area of glomerular luminal capillary endothelial fenestrations). Moreover, when compared to OVE diabetic animals, OVE‐JTMT mice showed reduced glomerular hypertrophy, lowered mesangial cell density and reduced total glomerular cytoproliferation. These results suggest a direct role of oxidative damage to endothelial cells and indicate that their targeted protection can reduce or delay several features of diabetic nephropathy.

Cardiac insulin resistance is a key pathogenic factor for diabetic cardiomyopathy (DCM), but the mechanism remains largely unclear. We found that diabetic hearts exhibited decreased phosphorylation of total Akt and isoform Akt2 but not Akt1 in wild-type (WT) male FVB mice, which was accompanied by attenuation of Akt downstream glucose metabolic signal. All of these signal changes were not observed in metallothionein cardiac-specific transgenic (MT-TG) hearts. Furthermore, insulin-induced glucose metabolic signals were attenuated only in WT diabetic hearts. In addition, diabetic hearts exhibited increased Akt-negative regulator tribbles pseudokinase 3 (TRB3) expression only in WT mice, suggesting that MT may preserve Akt2 function via inhibiting TRB3. Moreover, MT prevented tert-butyl hydroperoxide (tBHP)–reduced insulin-stimulated Akt2 phosphorylation in MT-TG cardiomyocytes, which was abolished by specific silencing of Akt2. Specific silencing of TRB3 blocked tBHP inhibition of insulin-stimulated Akt2 phosphorylation in WT cardiomyocytes, whereas overexpression of TRB3 in MT-TG cardiomyocytes and hearts abolished MT preservation of insulin-stimulated Akt2 signals and MT prevention of DCM. Most importantly, supplementation of Zn to induce MT preserved cardiac Akt2 signals and prevented DCM. These results suggest that diabetes-inhibited cardiac Akt2 function via TRB3 upregulation leads to aberrant cardiac glucose metabolism. MT preservation of cardiac Akt2 function by inhibition of TRB3 prevents DCM.

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

Aims/hypothesis Diabetic nephropathy is the leading cause of end-stage renal disease. Previously we reported that C66, a novel analogue of curcumin with a very high bioavailability, ameliorated diabetic nephropathy in mice, with little known about the mechanism. The present study aimed to define the mechanism by which C66 ameliorates diabetic nephropathy.Methods Our aim was to discover whether C66 acts through the activation of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2), which governs the antioxidant response. Streptozotocin-induced Nrf2 (also known as Nfe2l2)-knockout and wild-type (WT) diabetic mice were treated with C66. To determine whether the actions of C66 on NRF2 are mediated by microRNA (miR)-200a, WT diabetic mice were treated with C66 in the presence or absence of an in vivo miR-200a inhibitor (locked nucleic acid-modified anti-miR-200a [LNA-200a]) for 6 months. To determine whether miR-21 downregulation provided an NRF2-independent basis for C66 protection, Nrf2-knockout diabetic mice were treated with either C66 or an inhibitor of miR-21 (locked nucleic acid-modified anti-miR-21 [LNA-21]).Results Deletion of Nrf2 partially abolished diabetic nephropathy protection by C66, confirming the requirement of NRF2 for this protection. Diabetic mice, but not C66-treated diabetic mice, developed significant albuminuria, renal oxidative damage and fibrosis. C66 upregulated renal miR-200a, inhibited kelch-like ECH-associated protein 1 and induced NRF2 function, effects that were prevented by LNA-200a. However, LNA-200a only partially reduced the protection afforded by C66, suggesting the existence of miR-200a/NRF2-independent mechanisms for C66 protection. C66 was also found to inhibit diabetes induction of miR-21. Both C66 and LNA-21 produced similar reductions in miR-21, albuminuria and renal fibrosis.Conclusions/interpretation The present study indicates that in addition to upregulating NRF2 by increasing miR-200a, C66 also protects against diabetic nephropathy by inhibiting miR-21.