RESUMO
Podocyte damage and loss are the early event in the development of focal segmental glomerulosclerosis (FSGS). Podocytes express angiotensin II type-2-receptor (AT2R), which may play a key role in maintaining kidney integrity and function. Here, we examined the effects of AT2R deletion and AT2R agonist compound 21 (C21) on the evolution of FSGS. FSGS was induced by adriamycin (ADR) injection in both male wild-type (WT) and AT2R knockout (KO) mice. C21 was administered to WT-FSGS mice either one day before or 7 days after ADR (Pre-C21 or Post-C21), using two doses of C21 at either 0.3 (low dose, LD) or 1.0 (high dose, HD) mg/kg/day. ADR-induced FSGS was more severe in AT2RKO mice compared with WT-FSGS mice, and included profound podocyte loss, glomerular fibrosis, and albuminuria. Glomerular cathepsin L expression increased more in AT2RKO-FSGS than in WT-FSGS mice. C21 treatment ameliorated podocyte injury, most significantly in the Pre C21-HD group, and inhibited glomerular cathepsin L expression. In vitro, Agtr2 knock-down in mouse podocyte cell line given ADR confirmed the in vivo data. Mechanistically, C21 inhibited cathepsin L expression, which protected synaptopodin from destruction and stabilized actin cytoskeleton. C21 also prevented podocyte apoptosis. In conclusion, AT2R activation by C21 ameliorated ADR-induced podocyte injury in mice by the inhibition of glomerular cathepsin L leading to the maintenance of podocyte integrity and prevention of podocyte apoptosis.
Assuntos
Glomerulosclerose Segmentar e Focal , Nefropatias , Podócitos , Receptor Tipo 2 de Angiotensina/metabolismo , Angiotensina II/metabolismo , Animais , Catepsina L/metabolismo , Catepsina L/farmacologia , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Imidazóis , Nefropatias/metabolismo , Masculino , Camundongos , Camundongos Knockout , Podócitos/metabolismo , Sulfonamidas , TiofenosRESUMO
AIMS/HYPOTHESIS: The angiotensin II receptor type 2 (AT2R) may be a potential therapeutic target for the treatment of hypertension and chronic kidney disease (CKD). The expression and function of AT2R in the vasculature and kidney appear sexually dimorphic. We hypothesised that Agtr2 knockout dams (AT2RKO) with gestational diabetes would program their offspring for subsequent hypertension and CKD in a sex-dependent manner. METHODS: Age- and sex-matched offspring of non-diabetic and diabetic dams of wild-type (WT) and AT2RKO mice were followed from 4 to 20 weeks of age and were monitored for development of hypertension and nephropathy; a mouse podocyte cell line (mPOD) was also studied. RESULTS: Body weight was progressively lower in female compared with male offspring throughout the lifespan. Female but not male offspring from diabetic AT2RKO dams developed insulin resistance. Compared with the offspring of non-diabetic dams, the progeny of diabetic dams had developed more hypertension and nephropathy (apparent glomerulosclerosis with podocyte loss) at 20 weeks of age; this programming was more pronounced in the offspring of AT2RKO diabetic dams, particularly female AT2RKO progeny. Female AT2RKO offspring had lower basal ACE2 glomerular expression, resulting in podocyte loss. The aberrant ACE2/ACE ratio was far more diminished in glomeruli of female progeny of diabetic AT2RKO dams than in male progeny. Knock-down of Agtr2 in mPODs confirmed the in vivo data. CONCLUSIONS/INTERPRETATION: AT2R deficiency accelerated kidney programming in female progeny of diabetic dams, possibly due to loss of protective effects of ACE2 expression in the kidney.
Assuntos
Diabetes Mellitus , Resistência à Insulina , Nefropatias , Podócitos , Animais , Feminino , Rim , Masculino , CamundongosRESUMO
AIMS/HYPOTHESIS: We previously reported that renal tubule-specific deletion of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) results in upregulation of renal angiotensinogen (Agt) and downregulation of sodium-glucose co-transporter 2 (Sglt2) in HnrnpfRT knockout (KO) mice. Non-diabetic HnrnpfRT KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated Sglt2 expression. Here, we investigated the effect of renal tubular Hnrnpf deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes. METHODS: Akita HnrnpfRT KO mice were generated through crossbreeding tubule-specific (Pax8)-Cre mice with Akita floxed-Hnrnpf mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita HnrnpfRT KO mice were studied up to the age of 24 weeks (n = 8/group). RESULTS: Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita HnrnpfRT KO mice than Akita mice. Compared with Akita mice, Akita HnrnpfRT KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita HnrnpfRT KO mice. Treatment of Akita HnrnpfRT KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal Agt expression was elevated in Akita mice and further increased in Akita HnrnpfRT KO mice. In contrast, Sglt2 expression was increased in Akita and decreased in Akita HnrnpfRT KO mice. CONCLUSIONS/INTERPRETATION: The renoprotective effect of Sglt2 downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.
Assuntos
Injúria Renal Aguda/prevenção & controle , Diabetes Mellitus Tipo 1/prevenção & controle , Modelos Animais de Doenças , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/fisiologia , Túbulos Renais/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Angiotensinogênio , Animais , Glicemia/metabolismo , Pressão Sanguínea , Western Blotting , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Regulação para Baixo , Taxa de Filtração Glomerular/fisiologia , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antagonistas de Receptores Purinérgicos P1/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Teofilina/análogos & derivados , Teofilina/farmacologiaRESUMO
Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFß1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Proteínas de Transporte/metabolismo , Expressão Gênica/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Glicoproteínas de Membrana/metabolismo , Podócitos/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Adulto , Animais , Proteínas de Transporte/genética , Feminino , Glomerulosclerose Segmentar e Focal/genética , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos Knockout , Pessoa de Meia-Idade , Receptor Tipo 2 de Angiotensina/deficiênciaRESUMO
BACKGROUND: The aim of this study was to establish the underlying mechanisms by which a post-weaning high-fat diet (HFD) accelerates the perinatal programming of kidney injury occurring in the offspring of diabetic mothers. METHODS: Male mice, offspring of nondiabetic and diabetic dams were fed with normal diet (ND) or HFD from 4 to 20 wk of age. Rat renal proximal tubular cells were used in vitro. RESULTS: On ND, the offspring of dams with severe maternal diabetes had an intrauterine growth restriction (IUGR) phenotype and developed mild hypertension and evidence of kidney injury in adulthood. Exposing the IUGR offspring to HFD resulted in rapid weight gain, catch-up growth, and later to profound kidney injury with activation of renal TGFß1 and collagen type IV expression, increased oxidative stress, and enhanced renal lipid deposition, but not systemic hypertension. Given our data, we speculate that HFD or free fatty acids may accelerate the process of perinatal programming of kidney injury, via increased CD36 and fatty acid-binding protein 4 expression, which may target reactive oxygen species, nuclear factor-kappa B, and TGFß1 signaling in vivo and in vitro. CONCLUSION: Early postnatal exposure to overnutrition with a HFD increases the risk of development of kidney injury, but not hypertension, in IUGR offspring of dams with maternal diabetes.
Assuntos
Diabetes Gestacional/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Hipertensão/fisiopatologia , Rim/fisiopatologia , Animais , Apoptose , Peso Corporal , Antígenos CD36/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Retardo do Crescimento Fetal/fisiopatologia , Teste de Tolerância a Glucose , Hipertensão/complicações , Rim/lesões , Nefropatias/complicações , Nefropatias/imunologia , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Fenótipo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Ratos , DesmameRESUMO
AIMS/HYPOTHESIS: We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-ß1 signalling, TGF-ß1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. METHODS: Adult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. RESULTS: Overexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-ß1, TGF-ß receptor II [Tgf-ßrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1-7 receptor [MasR]) expression, and normalised urinary Ang 1-7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-ß1 and Tgf-ßrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-ß1 signalling and TGF-ß1 inhibition of Ace-2 gene expression. CONCLUSIONS/INTERPRETATION: These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-ß1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Peptidil Dipeptidase A/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Enzima de Conversão de Angiotensina 2 , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Expressão Gênica/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Transgênicos , Peptidil Dipeptidase A/genéticaRESUMO
We investigated the relationship between Ang-(1-7) [angiotensin-(1-7)] action, sHTN (systolic hypertension), oxidative stress, kidney injury, ACE2 (angiotensin-converting enzyme-2) and MasR [Ang-(1-7) receptor] expression in Type 1 diabetic Akita mice. Ang-(1-7) was administered daily [500 µg/kg of BW (body weight) per day, subcutaneously] to male Akita mice from 14 weeks of age with or without co-administration of an antagonist of the MasR, A779 (10 mg/kg of BW per day). The animals were killed at 20 weeks of age. Age-matched WT (wild-type) mice served as controls. Ang-(1-7) administration prevented sHTN and attenuated kidney injury (reduced urinary albumin/creatinine ratio, glomerular hyperfiltration, renal hypertrophy and fibrosis, and tubular apoptosis) without affecting blood glucose levels in Akita mice. Ang-(1-7) also attenuated renal oxidative stress and the expression of oxidative stress-inducible proteins (NADPH oxidase 4, nuclear factor erythroid 2-related factor 2, haem oxygenase 1), pro-hypertensive proteins (angiotensinogen, angiotensin-converting enzyme, sodium/hydrogen exchanger 3) and profibrotic proteins (transforming growth factor-ß1 and collagen IV), and increased the expression of anti-hypertensive proteins (ACE2 and MasR) in Akita mouse kidneys. These effects were reversed by A779. Our data suggest that Ang-(1-7) plays a protective role in sHTN and RPTC (renal proximal tubular cell) injury in diabetes, at least in part, through decreasing renal oxidative stress-mediated signalling and normalizing ACE2 and MasR expression.
Assuntos
Angiotensina I/farmacologia , Diabetes Mellitus Tipo 1/complicações , Fibrose/prevenção & controle , Hipertensão/prevenção & controle , Nefropatias/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Análise de Variância , Angiotensina I/administração & dosagem , Angiotensina I/uso terapêutico , Angiotensina I/urina , Angiotensina II/análogos & derivados , Enzima de Conversão de Angiotensina 2 , Animais , Glicemia , Western Blotting , Fibrose/etiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Histológicas , Hipertensão/etiologia , Imuno-Histoquímica , Injeções Subcutâneas , Nefropatias/etiologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/uso terapêutico , Fragmentos de Peptídeos/urina , Peptidil Dipeptidase A/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/metabolismoRESUMO
AIMS/HYPOTHESIS: We hypothesised that maternal diabetes impairs kidney formation in offspring via augmented expression of hedgehog interacting protein (HHIP). Our gene-array results were performed in neonatal kidneys from our murine model of maternal diabetes and indicated that Hhip expression was significantly modulated by maternal diabetes. METHODS: We systematically examined the functional role of HHIP in kidney formation in our murine maternal diabetes model and elucidated the potential mechanisms related to dysnephrogenesis in vitro. RESULTS: The kidneys of the offspring of diabetic dams, compared with those of the offspring of control non-diabetic dams, showed retardation of development--small kidneys and less ureteric bud (UB) branching morphogenesis. Augmented HHIP expression was observed in the offspring of diabetic dams, initially localised to differentiated metanephric mesenchyme and UB epithelium and subsequently in maturing glomerular endothelial and tubulointerstitial cells. The heightened HHIP targeting TGF-ß1 signalling was associated with dysmorphogenesis. In vitro, HHIP overexpression decreased sonic hedgehog and paired box gene 2 proteins (SHH and PAX2, respectively) and increased transcriptional nuclear factor-kappa B (NFκB, p50/p65), phosphorylation of p53, and TGF-ß1 expression. In contrast, overexpression of PAX2 inhibited HHIP and NFκB and activated SHH, N-myc and p27(Kip1) expression. Moreover, high glucose stimulated HHIP expression, and then targeted TGF-ß1 signalling. Thus, PAX2, via a negative autocrine feedback mechanism, attenuated the stimulatory effect of high glucose on HHIP expression. CONCLUSIONS/INTERPRETATION: Maternal diabetes modulates kidney formation in young progeny mediated, at least in part, via augmented HHIP expression.
Assuntos
Proteínas de Transporte/metabolismo , Diabetes Gestacional/metabolismo , Diabetes Gestacional/fisiopatologia , Rim/embriologia , Rim/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Feminino , Camundongos , GravidezRESUMO
ANGPTL8, expressed mainly in the liver and adipose tissue, regulates the activity of lipoprotein lipase (LPL) present in the extracellular space and triglyceride (TG) metabolism through its interaction with ANGPTL3 and ANGPTL4. Whether intracellular ANGPTL8 can also exert effects in tissues where it is expressed is uncertain. ANGPTL8 expression was low in preadipocytes and much increased during differentiation. To better understand the role of intracellular ANGPTL8 in adipocytes and assess whether it may play a role in adipocyte differentiation, we knocked down its expression in normal mouse subcutaneous preadipocytes. ANGPTL8 knockdown reduced adipocyte differentiation, cellular TG accumulation and also isoproterenol-stimulated lipolysis at day 7 of differentiation. RNA-Seq analysis of ANGPTL8 siRNA or control siRNA transfected SC preadipocytes on days 0, 2, 4 and 7 of differentiation showed that ANGPTL8 knockdown impeded the early (day 2) expression of adipogenic and insulin signaling genes, PPARγ, as well as genes related to extracellular matrix and NF-κB signaling. Insulin mediated Akt phosphorylation was reduced at an early stage during adipocyte differentiation. This study based on normal primary cells shows that ANGPTL8 has intracellular actions in addition to effects in the extracellular space, like modulating LPL activity. Preadipocyte ANGPTL8 expression modulates their differentiation possibly via changes in insulin signaling gene expression.
Assuntos
Adipogenia , Insulina , Camundongos , Animais , Diferenciação Celular/genética , Adipogenia/genética , Transdução de Sinais , RNA Interferente Pequeno , Proteína 8 Semelhante a AngiopoietinaRESUMO
We investigated the relationship among oxidative stress, hypertension, renal injury, and angiotensin-converting enzyme-2 (ACE2) expression in type 1 diabetic Akita mice. Blood glucose, blood pressure, and albuminuria were monitored for up to 5 mo in adult male Akita and Akita catalase (Cat) transgenic (Tg) mice specifically overexpressing Cat, a key antioxidant enzyme in their renal proximal tubular cells (RPTCs). Same-age non-Akita littermates and Cat-Tg mice served as controls. In separate studies, adult male Akita mice (14 wk) were treated with ANG 1-7 (500 µg·kg⻹·day⻹ sc) ± A-779, an antagonist of the Mas receptor (10 mg·kg⻹·day⻹ sc), and euthanized at the age of 18 wk. The left kidneys were processed for histology and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess protein and gene expression. Urinary angiotensinogen (AGT), angiotensin II (ANG II), and ANG 1-7 were quantified by specific ELISAs. Overexpression of Cat attenuated renal oxidative stress; prevented hypertension; normalized RPTC ACE2 expression and urinary ANG 1-7 levels (both were low in Akita mice); ameliorated glomerular filtration rate, albuminuria, kidney hypertrophy, tubulointerstitial fibrosis, and tubular apoptosis; and suppressed profibrotic and proapoptotic gene expression in RPTCs of Akita Cat-Tg mice compared with Akita mice. Furthermore, daily administration of ANG 1-7 normalized systemic hypertension in Akita mice, which was reversed by A-779. These data demonstrate that Cat overexpression prevents hypertension and progression of nephropathy and highlight the importance of intrarenal oxidative stress and ACE2 expression contributing to hypertension and renal injury in diabetes.
Assuntos
Catalase/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Hipertensão/prevenção & controle , Túbulos Renais/patologia , Estresse Oxidativo , Peptidil Dipeptidase A/metabolismo , Albuminúria , Angiotensina I/administração & dosagem , Angiotensina I/urina , Enzima de Conversão de Angiotensina 2 , Angiotensinogênio/urina , Animais , Apoptose , Catalase/genética , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/prevenção & controle , Fibrose , Hipertensão/etiologia , Rim/patologia , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/urinaRESUMO
The role(s) of nuclear factor erythroid 2-related factor 2 (NRF2) in diabetic kidney disease (DKD) is/are controversial. We hypothesized that Nrf2 deficiency in type 2 diabetes (T2D) db/db mice (db/dbNrf2 knockout (KO)) attenuates DKD progression through the down-regulation of angiotensinogen (AGT), sodium-glucose cotransporter-2 (SGLT2), scavenger receptor CD36, and fatty -acid-binding protein 4 (FABP4), and lipid accumulation in renal proximal tubular cells (RPTCs). Db/dbNrf2 KO mice were studied at 16 weeks of age. Human RPTCs (HK2) with NRF2 KO via CRISPR-Cas9 genome editing and kidneys from patients with or without T2D were examined. Compared with db/db mice, db/dbNrf2 KO mice had lower systolic blood pressure, fasting blood glucose, kidney hypertrophy, glomerular filtration rate, urinary albumin/creatinine ratio, tubular lipid droplet accumulation, and decreased expression of AGT, SGLT2, CD36, and FABP4 in RPTCs. Male and female mice had similar results. NRF2 KO attenuated the stimulatory effect of the Nrf2 activator, oltipraz, on AGT, SGLT2, and CD36 expression and high-glucose/free fatty acid (FFA)-stimulated lipid accumulation in HK2. Kidneys from T2D patients exhibited markedly higher levels of CD36 and FABP4 in RPTCs than kidneys from non-diabetic patients. These data suggest that NRF2 exacerbates DKD through the stimulation of AGT, SGLT2, CD36, and FABP4 expression and lipid accumulation in RPTCs of T2D.
RESUMO
We investigated the effects of dual renin-angiotensin system (RAS) blockade on angiotensin-converting enzyme-2 (Ace2) expression, hypertension, and renal proximal tubular cell (RPTC) apoptosis in type 1 diabetic Akita angiotensinogen (Agt)-transgenic (Tg) mice that specifically overexpress Agt in their RPTCs. Adult (11 wk old) male Akita and Akita Agt-Tg mice were treated with two RAS blockers (ANG II receptor type 1 blocker losartan, 30 mg·kg(-1)·day(-1)) and angiotensin-converting enzyme (ACE) inhibitor perindopril (4 mg·kg(-1)·day(-1)) in drinking water. Same-age non-Akita littermates and Agt-Tg mice served as controls. Blood pressure, blood glucose, and albuminuria were monitored weekly. The animals were euthanized at age 16 wk. The left kidneys were processed for immunohistochemistry and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess gene and protein expression. Urinary ANG II and ANG 1-7 were quantified by ELISA. RAS blockade normalized renal Ace2 expression and urinary ANG 1-7 levels (both of which were low in untreated Akita and Akita Agt-Tg), prevented hypertension, albuminuria, tubulointerstitial fibrosis and tubular apoptosis, and inhibited profibrotic and proapoptotic gene expression in RPTCs of Akita and Akita Agt-Tg mice compared with non-Akita controls. Our results demonstrate the effectiveness of RAS blockade in preventing intrarenal RAS activation, hypertension, and nephropathy progression in diabetes and support the important role of intrarenal Ace2 expression in modulating hypertension and renal injury in diabetes.
Assuntos
Angiotensinogênio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/etiologia , Túbulos Renais Proximais/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Enzima de Conversão de Angiotensina 2 , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Angiotensinogênio/genética , Animais , Apoptose , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Expressão Gênica , Hipertensão/etiologia , Rim/metabolismo , Rim/patologia , Losartan/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nefroesclerose/metabolismo , Peptidil Dipeptidase A/metabolismo , Perindopril/farmacologia , Ratos , Sistema Renina-Angiotensina/efeitos dos fármacos , TransgenesRESUMO
OBJECTIVE: Glycerol-3-phosphate (Gro3P) phosphatase (G3PP) hydrolyzes Gro3P to glycerol that exits the cell, thereby operating a "glycerol shunt", a metabolic pathway that we identified recently in mammalian cells. We have investigated the role of G3PP and the glycerol shunt in the regulation of glucose metabolism and lipogenesis in mouse liver. METHODS: We generated hepatocyte-specific G3PP-KO mice (LKO), by injecting AAV8-TBG-iCre to male G3PPfl/fl mice. Controls received AAV8-TBG-eGFP. Both groups were fed chow diet for 10 weeks. Hyperglycemia (16-20 mM) was induced by glucose infusion for 55 h. Hepatocytes were isolated from normoglycemic mice for ex vivo studies and targeted metabolomics were measured in mice liver after glucose infusion. RESULTS: LKO mice showed no change in body weight, food intake, fed and fasted glycemia but had increased fed plasma triglycerides. Hepatic glucose production from glycerol was increased in fasted LKO mice. LKO mouse hepatocytes displayed reduced glycerol production, elevated triglyceride and lactate production at high glucose concentration. Hyperglycemia in LKO mice led to increased liver weight and accumulation of triglycerides, glycogen and cholesterol together with elevated levels of Gro3P, dihydroxyacetone phosphate, acetyl-CoA and some Krebs cycle intermediates in liver. Hyperglycemic LKO mouse liver showed elevated expression of proinflammatory cytokines and M1-macrophage markers accompanied by increased plasma triglycerides, LDL/VLDL, urea and uric acid and myocardial triglycerides. CONCLUSIONS: The glycerol shunt orchestrated by G3PP acts as a glucose excess detoxification pathway in hepatocytes by preventing metabolic disturbances that contribute to enhanced liver fat, glycogen storage, inflammation and lipid build-up in the heart. We propose G3PP as a novel therapeutic target for hepatic disorders linked to nutrient excess.
Assuntos
Glicerol , Hiperglicemia , Monoéster Fosfórico Hidrolases , Animais , Masculino , Camundongos , Glucose/metabolismo , Glicerol/metabolismo , Glicogênio/metabolismo , Hiperglicemia/metabolismo , Fígado/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Triglicerídeos/metabolismoRESUMO
A hyperglycemic environment in utero reduces kidney size and nephron number due to nascent nephron apoptosis. However, the underlying mechanisms are incompletely understood. The present study investigated whether the nascent nephron apoptosis promoted by high glucose is mediated via the transcription factor NF-κB and p53 signaling pathways. Neonatal mouse kidneys from the offspring of nondiabetic, diabetic, and insulin-treated diabetic dams were used for in vivo studies, and MK4 cells, an embryonic metanephric mesenchymal (MM) cell line, were used for in vitro studies. Neonatal kidneys of the offspring of diabetic mothers exhibited an increased number of apoptotic cells and reactive oxygen species (ROS) generation, enhanced NF-κB activation, and nuclear translocation of its subunits (p50 and p65 subunits) as well as phosphorylation (Ser 15) of p53 compared with kidneys of offspring of nondiabetic mothers. Insulin treatment of diabetic dams normalized these parameters in the offspring. In vitro, high-glucose (25 mM) induced ROS generation and significantly increased MK4 cell apoptosis and caspase-3 activity via activation of NF-κB pathway, with p53 phosphorylation and nuclear translocation compared with normal glucose (5 mM). These changes in a high-glucose milieu were prevented by transient transfection of small interfering RNAs for dominant negative IκBα or IKK or p53. Our data demonstrate that high glucose-induced nascent nephron apoptosis is mediated, at least in part, via ROS generation and the activation of NF-κB and p53 pathways.
Assuntos
Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/fisiopatologia , Glucose/farmacologia , NF-kappa B/fisiologia , Néfrons/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia , Animais , Linhagem Celular , Feminino , Glucose/administração & dosagem , Insulina/uso terapêutico , Rim/anormalidades , Camundongos , Subunidade p50 de NF-kappa B/biossíntese , Néfrons/anormalidades , Gravidez , Gravidez em Diabéticas/tratamento farmacológico , Gravidez em Diabéticas/fisiopatologia , Fator de Transcrição RelA/biossínteseRESUMO
Apoptosis of tubular epithelial cells contributes to the tubular atrophy that accompanies diabetic nephropathy. Reactive oxygen species (ROS) promote tubular apoptosis, but the mechanisms by which this occurs are incompletely understood. Here, we sought proapoptotic genes that ROS differentially upregulate in renal proximal tubular cells of diabetic (db/db) mice. We performed microarray analysis using total RNA from freshly isolated renal proximal tubules of nondiabetic, diabetic, and diabetic transgenic mice overexpressing catalase in the proximal tubule (thereby attenuating ROS). We observed greater expression of caspase-12 in the proximal tubules of the diabetic mice compared with the nondiabetic and diabetic transgenic mice. Quantitative PCR and immunohistochemistry confirmed the enhanced expression of caspase-12, as well as members of the endoplasmic reticulum stress-induced apoptotic pathway. Ex vivo, albumin induced caspase-12 activity and expression (protein and mRNA) and mRNA expression of the CCAT/enhancer-binding protein homologous protein in freshly isolated wild-type proximal tubules but not in catalase-overexpressing proximal tubules. In vitro, albumin stimulated activity of both caspase-12 and caspase-3 as well as expression of caspase-12 and CCAT/enhancer-binding protein homologous protein in a human proximal tubule cell line (HK-2). The free radical scavenger tiron inhibited these effects. Furthermore, knockdown of caspase-12 with small interfering RNA reduced albumin-induced apoptosis in HK-2 cells. Taken together, these studies demonstrate that albuminuria may induce tubular apoptosis through generation of ROS and the subsequent expression and activation of endoplasmic reticulum stress genes in the diabetic kidney.
Assuntos
Apoptose , Caspase 12/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Espécies Reativas de Oxigênio/metabolismo , Albuminas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 12/genética , Linhagem Celular , Modelos Animais de Doenças , Retículo Endoplasmático/fisiologia , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , RNA Interferente Pequeno/farmacologia , Fator de Transcrição CHOP/metabolismoRESUMO
We investigated the impact of nuclear factor erythroid 2-related factor 2 (Nrf2) overexpression in renal proximal tubular cells (RPTCs) on blood glucose, kidney injury, and sodium-glucose cotransporter 2 (Sglt2) expression in diabetic Akita Nrf2 -/-/Nrf2RPTC transgenic (Tg) mice. Immortalized human RPTCs (HK2) stably transfected with plasmid containing the SGLT2 promoter and human kidneys from patients with diabetes were also studied. Nrf2 overexpression was associated with increased blood glucose, glomerular filtration rate, urinary albumin-to-creatinine ratio, tubulointerstitial fibrosis, and Sglt2 expression in Akita Nrf2 -/-/Nrf2RPTC Tg mice compared with their Akita Nrf2 -/- littermates. In vitro, oltipraz or transfection of NRF2 cDNA stimulated SGLT2 expression and SGLT2 promoter activity in HK2, and these effects were inhibited by trigonelline or NRF2 siRNA. The deletion of the NRF2-responsive element (NRF2-RE) in the SGLT2 promoter abolished the stimulatory effect of oltipraz on SGLT2 promoter activity. NRF2 binding to the NRF2-RE of the SGLT2 promoter was confirmed by gel mobility shift assay and chromatin immunoprecipitation assays. Kidneys from patients with diabetes exhibited higher levels of NRF2 and SGLT2 in the RPTCs than kidneys from patients without diabetes. These results suggest a link by which NRF2 mediates hyperglycemia stimulation of SGLT2 expression and exacerbates blood glucose and kidney injury in diabetes.
Assuntos
Nefropatias Diabéticas/patologia , Hiperglicemia/patologia , Fator 2 Relacionado a NF-E2/genética , Transportador 2 de Glucose-Sódio/genética , Animais , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Progressão da Doença , Feminino , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transportador 2 de Glucose-Sódio/metabolismo , Regulação para Cima/genéticaRESUMO
Transgenic mice that overexpress angiotensinogen, the sole precursor of angiotensins, in their renal proximal tubular cells develop hypertension, albuminuria, and tubular apoptosis. These pathological changes are due to enhanced generation of reactive oxygen species in the proximal tubule cells. Here, we determined whether overexpression of catalase to decrease oxidant injury in the proximal tubular cells could reverse these abnormalities. Double-transgenic mice specifically overexpressing angiotensinogen and catalase in their renal proximal tubular cells were created by cross-breeding the single transgenics. Non-transgenic littermates served as controls. Overexpression of catalase prevented hypertension, albuminuria, tubulointerstitial fibrosis, and tubular apoptosis in the angiotensinogen transgenic mice. Furthermore, the double transgenics had lower reactive oxygen species generation and reduced pro-fibrotic and apoptotic gene expression in the renal proximal tubular cells. Renal angiotensin converting enzyme-2 expression and urinary angiotensin 1-7 levels were downregulated in the single but normal in the double-transgenic mice. Thus, we suggest that the intrarenal renin-angiotensin system and reactive oxygen species generation have an important role in the development of hypertension and renal injury.
Assuntos
Angiotensinogênio/genética , Catalase/administração & dosagem , Hipertensão/prevenção & controle , Túbulos Renais Proximais/patologia , Animais , Apoptose/efeitos dos fármacos , Catalase/genética , Catalase/uso terapêutico , Expressão Gênica , Hipertensão/etiologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Camundongos , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Sistema Renina-Angiotensina/fisiologiaRESUMO
We investigated whether maternal diabetes programs the offspring to develop hypertension and kidney injury in adulthood and examined potential underlying mechanisms. In a murine model we studied the offspring of three groups of dams (non-diabetic, diabetic, and diabetic treated with insulin). Mean systolic blood pressure in the offspring was monitored from 8 to 20 weeks. Body and kidney weights in the offspring of diabetic mothers were significantly lower than in offspring of non-diabetic mothers. Offspring of diabetic mothers developed hypertension, microalbuminuria, and glucose intolerance. Increased accumulation of extracellular matrix proteins in the glomeruli and marked upregulation of angiotensinogen, angiotensin II type 1 receptor, angiotensin-converting enzyme, transforming growth factor beta-1 (TGF-beta1), and plasminogen activator inhibitor-1 (PAI-1) gene expression were evident in the renal cortex of hypertensive offspring of diabetic mothers. By contrast, angiotensin-converting enzyme-2 (ACE2) gene expression was lower in the hypertensive offspring of diabetic mothers than in that of non-diabetic mothers. These changes were prevented in the offspring of insulin-treated diabetic mothers. These data indicate that maternal diabetes induces perinatal programming of hypertension, renal injury, and glucose intolerance in the offspring and suggest a central role for the activation of the intrarenal renin-angiotensin system and TGF-beta1 gene expression in this process.
Assuntos
Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Regulação da Expressão Gênica no Desenvolvimento , Hipertensão/genética , Gravidez em Diabéticas/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Enzima de Conversão de Angiotensina 2 , Animais , Pressão Sanguínea/genética , Peso Corporal , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Intolerância à Glucose/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Insulina/farmacologia , Rim/crescimento & desenvolvimento , Rim/patologia , Camundongos , Tamanho do Órgão , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Gravidez , Gravidez em Diabéticas/metabolismo , Gravidez em Diabéticas/fisiopatologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
AIMS: SGLT2 inhibitors have been proposed as an adjunct to insulin therapy for glycemic control in type 1 diabetes (T1D) patients. However, concern has been raised due to an increase in renin-angiotensin-system (RAS) activity reported in a clinical trial in which an SGLT2 inhibitor was added while insulin dose was reduced in T1D patients. We previously reported that insulin inhibits intrarenal angiotensinogen (Agt) gene transcription and RAS activation. We hypothesized that insulin, rather than SGLT2 inhibition might regulate the intrarenal RAS. METHODS: We compared RAS activity in non-diabetic wild type mice, Akita mice (T1D model) and Akita mice treated with insulin or the SGLT2 inhibitor canagliflozin. RESULTS: Treatment of Akita mice with insulin or canagliflozin produced similar reductions in blood glucose, whereas insulin, but not canagliflozin, reduced elevated systolic blood pressure. Akita mice exhibited increased renal Agt mRNA/protein expression, which was attenuated by insulin, but not by canagliflozin. Furthermore, insulin was more effective than canagliflozin in lowering kidney weight and albuminuria. CONCLUSIONS: Insulin, but not canagliflozin, lowers intrarenal RAS activity in Akita mice. Our findings can be of potential clinical importance, especially for T1D patients who are not on RAS inhibitors at the time of adding SGLT2 inhibitors.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Sistema Renina-Angiotensina/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Animais , Diabetes Mellitus Tipo 1/sangue , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Masculino , Camundongos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologiaRESUMO
Enhanced energy expenditure in brown (BAT) and white adipose tissues (WAT) can be therapeutic against metabolic diseases. We examined the thermogenic role of adipose α/ß-hydrolase domain 6 (ABHD6), which hydrolyzes monoacylglycerol (MAG), by employing adipose-specific ABHD6-KO mice. Control and KO mice showed similar phenotypes at room temperature and thermoneutral conditions. However, KO mice were resistant to hypothermia, which can be accounted for by the simultaneously increased lipolysis and lipogenesis of the thermogenic glycerolipid/free fatty acid (GL/FFA) cycle in visceral fat, despite unaltered uncoupling protein 1 expression. Upon cold stress, nuclear 2-MAG levels increased in visceral WAT of the KO mice. Evidence is provided that 2-MAG causes activation of PPARα in white adipocytes, leading to elevated expression and activity of GL/FFA cycle enzymes. In the ABHD6-ablated BAT, glucose and oxidative metabolism were elevated upon cold induction, without changes in GL/FFA cycle and lipid turnover. Moreover, response to in vivo ß3-adrenergic stimulation was comparable between KO and control mice. Our data reveal a MAG/PPARα/GL/FFA cycling metabolic signaling network in visceral adipose tissue, which contributes to cold tolerance, and that adipose ABHD6 is a negative modulator of adaptive thermogenesis.