Deletion of the gene for adiponectin accelerates diabetic nephropathy in the Ins2 (+/C96Y) mouse.

AIMS/HYPOTHESIS: Diabetic nephropathy is one of the most common forms of chronic kidney disease. The role of adiponectin in the development of diabetic nephropathy has not been elucidated, and the aim of the present study was to investigate the hypothesis that deletion of the gene for adiponectin would accelerate diabetic nephropathy in the Akita mouse. METHODS: We followed four groups of mice from 4 weeks to 16 weeks of age (n ≥ 10 in each group): wild-type (WT) (Ins2 (+/+) Adipoq(+/+)) mice; APN(-/-) (Ins2(+/+) Adipoq(-/-)) mice; Akita (Ins2(+/C96Y) Adipoq(+/+)) mice and Akita/APN(-/-) (Ins2(+/C96Y) Adipoq(-/-)) mice. The mice were then killed and diabetic kidney injury was assessed. In vitro experiments were performed in primary mesangial cells. RESULTS: Mice from both diabetic groups exhibited increased glomerular adiponectin receptor 1 (adipoR1) expression, kidney hypertrophy, glomerular enlargement, increased albuminuria and tissue oxidative stress compared with the WT control. Deletion of the adiponectin gene had no effect on glycaemia. However, Akita/APN(-/-) mice exhibited a greater extent of renal hypertrophy. In vitro, adiponectin attenuated high-glucose-induced phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K). A higher level of fibrosis was observed in the tubulointerstitial and glomerular compartments of the Akita/APN(-/-) mice and adiponectin was found to inhibit TGFß-induced Smad2 and Smad3 phosphorylation in vitro. There was an exaggerated inflammatory response in the Akita/APN(-/-) mice. Adiponectin also inhibited high-glucose-induced activation of nuclear factor κB (NFκB) in mesangial cells. CONCLUSIONS/INTERPRETATION: Our data suggest that adiponectin is an important determinant of the kidney response to high glucose in vivo and in vitro.

Adiponectin attenuates angiotensin II-induced oxidative stress in renal tubular cells through AMPK and cAMP-Epac signal transduction pathways.

Obesity is a risk factor for chronic kidney disease (CKD) progression. Circulating levels of adiponectin, an adipokine, decrease with obesity and play a protective role in the cardiovascular system. We hypothesized that adiponectin might also protect the kidney. Because activation of the renin-angiotensin system (RAS) is a contributor to CKD progression, we tested our hypothesis by studying the interactions between adiponectin and angiotensin II (ANG II) in renal tubular cells. Primary human renal proximal tubule cells expressed both adiponectin receptor 1 and 2 (adipoR1 and R2). ANG II-induced NADPH oxidase activation and oxidative stress were attenuated by adiponectin and dependent on adipoR1. Activation of AMPK with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) mimicked, while inhibition of AMPK with compound C abrogated, the effect of adiponectin on ANG II-induced activation of NADPH oxidase. Similarly, the effect of adiponectin was recapitulated by the stable cAMP analogs 4-chlorophenylthio (pCPT)-cAMP and dibutyryl (db)-cAMP and blocked by the adenylate cyclase inhibitor SQ22536. Adiponectin did not activate PKA in renal tubular cells, and the specific PKA inhibitor myristoylated PKI (14-22) amide failed to block the inhibitory effect of adiponectin on ANG II-induced NADPH oxidase activation. In contrast, the specific Epac activator 8-(4-chlorophenylthio)-2'-O-methyl (8-CPT-2-O-Me)-cAMP blocked ANG II-induced activation of NADPH oxidase, an effect that was reversed by coincubation with the AMPK inhibitor compound C. Finally, adiponectin attenuated ANG II-induced NF-κB activation and fibronectin protein expression. These in vitro findings support the hypothesis that adiponectin may attenuate the deleterious effects of ANG II in the kidney and play a protective role in CKD.

Loss of TIMP3 selectively exacerbates diabetic nephropathy.

Diabetic nephropathy is the most common cause of end-stage renal disease. Polymorphism in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene, and the ECM-bound inhibitor of matrix metalloproteinases (MMPs), has been linked to diabetic nephropathy in humans. To elucidate the mechanism, we generated double mutant mice in which the TIMP3 gene was deleted in the genetic diabetic Akita mouse background. The aggravation of diabetic injury occurred in the absence of worsening of hypertension or hyperglycemia. In fact, myocardial TIMP3 levels were not affected in Akita hearts, and cardiac diastolic and systolic function remained unchanged in the double mutant mice. However, TIMP3 levels increased in Akita kidneys and deletion of TIMP3 exacerbated the diabetic renal injury in the Akita mouse, characterized by increased albuminuria, mesangial matrix expansion, and kidney hypertrophy. The progression of diabetic renal injury was accompanied by the upregulation of fibrotic and inflammatory markers, increased production of reactive oxygen species and NADPH oxidase activity, and elevated activity of TNF-α-converting enzyme (TACE) in the TIMP3(-/-)/Akita kidneys. Moreover, while the elevated phospho-Akt (S473 and T308) and phospho-ERK1/2 in the Akita mice was not detected in the TIMP3(-/-)/Akita kidneys, PKCß1 (but not PKCα) was markedly elevated in the double mutant kidneys. Our data provide definitive evidence for a critical and selective role of TIMP3 in diabetic renal injury consistent with gene expression findings from human diabetic kidneys.

Angiotensin-(1-7)-induced activation of ERK1/2 is cAMP/protein kinase A-dependent in glomerular mesangial cells.

The renin-angiotensin system (RAS) plays an important role in renal physiology and kidney injury. Although the cellular effects of the RAS activation are generally attributed to angiotensin II (ANG II), the recent identification of angiotensin-converting enzyme 2 has shifted the focus to other peptides including Ang-(1-7). The G protein-coupled receptor for Ang-(1-7), mas, is expressed by mesangial cells (MC) but the signal transduction pathways activated by Ang-(1-7) in MC have not been fully elucidated. Accordingly, we studied the effect of Ang-(1-7) on extracellular signal-related kinase (ERK)1/2 activation in rat MC. Ang-(1-7)-induced ERK1/2 phosphorylation in MC is time- and concentration-dependent. Pretreatment of MC with the mas receptor antagonist A-779 but not the AT(1) antagonist losartan or the AT(2) antagonist PD123319 abrogated ERK1/2 activation. Neither pretreatment with the NADPH oxidase inhibitors diphenyleneiodonium and apocynin nor pretreatment with the epidermal growth factor (EGF) receptor antagonists AG1478 and PD158780 attenuated Ang-(1-7)-induced activation of ERK1/2. Even though each of these compounds abolished ANG II-induced activation of ERK1/2. Ang-(1-7) increased intracellular cAMP levels and activated protein kinase A (PKA) and inhibition of either adenylyl cyclase or PKA activity attenuated Ang-(1-7)-induced ERK1/2 activation. In conclusion, Ang-(1-7)-induced activation of ERK1/2 is cAMP/PKA-dependent in MC, but independent of NADPH oxidase and the EGF receptor.

Enhanced susceptibility to biomechanical stress in ACE2 null mice is prevented by loss of the p47(phox) NADPH oxidase subunit.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is an important negative regulator of the renin-angiotensin system. Loss of ACE2 enhances the susceptibility to heart disease but the mechanism remains elusive. We hypothesized that ACE2 deficiency activates the NADPH oxidase system in pressure overload-induced heart failure. METHODS AND RESULTS: Using the aortic constriction model, we subjected wild-type (Ace2(+/y)), ACE2 knockout (ACE2KO, Ace2(-/y)), p47(phox) knockout (p47(phox)KO, p47(phox-)(/-)), and ACE2/p47(phox) double KO mice to pressure overload. We examined changes in peptide levels, NADPH oxidase activity, gene expression, matrix metalloproteinases (MMP) activity, pathological signalling, and heart function. Loss of ACE2 resulted in enhanced susceptibility to biomechanical stress leading to eccentric remodelling, increased pathological hypertrophy, and worsening of systolic performance. Myocardial angiotensin II (Ang II) levels were increased, whereas Ang 1-7 levels were lowered. Activation of Ang II-stimulated signalling pathways in the ACE2-deficient myocardium was associated with increased expression and phosphorylation of p47(phox), NADPH oxidase activity, and superoxide generation, leading to enhanced MMP-mediated degradation of the extracellular matrix. Additional loss of p47(phox) in the ACE2KO mice normalized the increased NADPH oxidase activity, superoxide production, and systolic dysfunction following pressure overload. Ang 1-7 supplementation suppressed the increased NADPH oxidase and rescued the early dilated cardiomyopathy in pressure-overloaded ACE2KO mice. CONCLUSION: In the absence of ACE2, biomechanical stress triggers activation of the myocardial NAPDH oxidase system with a critical role of the p47(phox) subunit. Increased production of superoxide, activation of MMP, and pathological signalling leads to severe adverse myocardial remodelling and dysfunction in ACE2KO mice.

Human recombinant ACE2 reduces the progression of diabetic nephropathy.

OBJECTIVE: Diabetic nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function accelerates diabetic kidney injury, whereas renal ACE2 is downregulated in diabetic nephropathy. We examined the ability of human recombinant ACE2 (hrACE2) to slow the progression of diabetic kidney injury. RESEARCH DESIGN AND METHODS: Male 12-week-old diabetic Akita mice (Ins2(WT/C96Y)) and control C57BL/6J mice (Ins2(WT/WT)) were injected daily with placebo or with rhACE2 (2 mg/kg, i.p.) for 4 weeks. Albumin excretion, gene expression, histomorphometry, NADPH oxidase activity, and peptide levels were examined. The effect of hrACE2 on high glucose and angiotensin II (ANG II)-induced changes was also examined in cultured mesangial cells. RESULTS: Treatment with hrACE2 increased plasma ACE2 activity, normalized blood pressure, and reduced the urinary albumin excretion in Akita Ins2(WT/C96Y) mice in association with a decreased glomerular mesangial matrix expansion and normalization of increased alpha-smooth muscle actin and collagen III expression. Human recombinant ACE2 increased ANG 1-7 levels, lowered ANG II levels, and reduced NADPH oxidase activity. mRNA levels for p47(phox) and NOX2 and protein levels for protein kinase Calpha (PKCalpha) and PKCbeta1 were also normalized by treatment with hrACE2. In vitro, hrACE2 attenuated both high glucose and ANG II-induced oxidative stress and NADPH oxidase activity. CONCLUSIONS: Treatment with hrACE2 attenuates diabetic kidney injury in the Akita mouse in association with a reduction in blood pressure and a decrease in NADPH oxidase activity. In vitro studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1-7 signaling.