Hedgehog interacting protein activates sodium-glucose cotransporter 2 expression and promotes renal tubular epithelial cell senescence in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipRT-KO), hypothesising that local deficiency of HHIP in the renal tubules would attenuate tubular cell senescence, thereby preventing DKD tubulopathy. METHODS: Low-dose streptozotocin was employed to induce diabetes in both HhipRT-KO and control (Hhipfl/fl) mice. Transgenic mice overexpressing Hhip in renal proximal tubular cells (RPTC) (HhipRPTC-Tg) were used for validation, and primary RPTCs and human RPTCs (HK2) were used for in vitro studies. Kidney morphology/function, tubular senescence and the relevant molecular measurements were assessed. RESULTS: Compared with Hhipfl/fl mice with diabetes, HhipRT-KO mice with diabetes displayed lower blood glucose levels, normalised GFR, ameliorated urinary albumin/creatinine ratio and less severe DKD, including tubulopathy. Sodium-glucose cotransporter 2 (SGLT2) expression was attenuated in RPTCs of HhipRT-KO mice with diabetes compared with Hhipfl/fl mice with diabetes. In parallel, an increased tubular senescence-associated secretory phenotype involving release of inflammatory cytokines (IL-1ß, IL-6 and monocyte chemoattractant protein-1) and activation of senescence markers (p16, p21, p53) in Hhipfl/fl mice with diabetes was attenuated in HhipRT-KO mice with diabetes. In contrast, HhipRPTC-Tg mice had increased tubular senescence, which was inhibited by canagliflozin in primary RPTCs. In HK2 cells, HHIP overexpression or recombinant HHIP increased SGLT2 protein expression and promoted cellular senescence by targeting both ataxia-telangiectasia mutated and ataxia-telangiectasia and Rad3-related-mediated cell arrest. CONCLUSIONS/INTERPRETATION: Tubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.

Angiotensin II type-2-receptor stimulation ameliorates focal and segmental glomerulosclerosis in mice.

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.

AT2R deficiency in mice accelerates podocyte dysfunction in diabetic progeny in a sex-dependent manner.

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.

Angiotensin II up-regulates sodium-glucose co-transporter 2 expression and SGLT2 inhibitor attenuates Ang II-induced hypertensive renal injury in mice.

Clinical trials indicate that sodium/glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) improve kidney function, yet, the molecular regulation of SGLT2 expression is incompletely understood. Here, we investigated the role of the intrarenal renin-angiotensin system (RAS) on SGLT2 expression. In adult non-diabetic participants in the Nephrotic Syndrome Study Network (NEPTUNE, n=163), multivariable linear regression analysis showed SGLT2 mRNA was significantly associated with angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE) mRNA levels (P<0.001). In vitro, angiotensin II (Ang II) dose-dependently stimulated SGLT2 expression in HK-2, human immortalized renal proximal tubular cells (RPTCs); losartan and antioxidants inhibited it. Sglt2 expression was increased in transgenic (Tg) mice specifically overexpressing Agt in their RPTCs, as well as in WT mice with a single subcutaneous injection of Ang II (1.44 mg/kg). Moreover, Ang II (1000 ng/kg/min) infusion via osmotic mini-pump in WT mice for 4 weeks increased systolic blood pressure (SBP), glomerulosclerosis, tubulointerstitial fibrosis, and albuminuria; canaglifozin (Cana, 15 mg/kg/day) reversed these changes, with the exception of SBP. Fractional glucose excretion (FeGlu) was higher in Ang II+Cana than WT+Cana, whereas Sglt2 expression was similar. Our data demonstrate a link between intrarenal RAS and SGLT2 expression and that SGLT2i ameliorates Ang II-induced renal injury independent of SBP.

Increased urinary excretion of hedgehog interacting protein (uHhip) in early diabetic kidney disease.

Glomerular endothelial cell (GEC) dysfunction occurs in diabetic kidney disease (DKD) and generally precedes albuminuria. We recently reported that hedgehog interacting protein (Hhip), highly expressed in GECs, contributes to DKD development in diabetic mice. Here, we hypothesized that urinary Hhip (uHhip) could identify early DKD; we tested uHhip in mice and humans with diabetes (DM). In both type 1 (Akita) and type 2 (db/db) DM mice, uHhip is elevated prior to the development of albuminuria, while non-DM controls excrete minimal amount of uHhip. In 87 type 2 DM patients and 39 healthy controls, the uHhip/creatinine (Cr) ratio provides a significant discrimination between non-DM and DM groups; 0 [0-69.5] in non-DM, 9.9 [1.7-39.5] in normoalbuminuric DM, 167.7 [95.7-558.7] in microalbuminuric DM, and 207.9 [0-957.2] in macroalbuminuric DM (median [IQR] ng/mmol, P < 0.0001). The log-uHhip/Cr is positively correlated with urine albumin/Cr ratio (UACR) (spearman correlation coefficient 0.47, P < 0.0001). The log-uHhip/Cr is also associated with eGFR, pulse pressure, and urinary cytokines (IL-1ß, IL-6, IL-8, and TGFß1) independent of UACR. By immunostaining, Hhip is localized in glomeruli and tubules, and is increased in human DM kidneys compared with non-DM kidneys. TGFß1 shares the similar staining pattern as Hhip in human DM kidneys. Thus, uHhip appears to be a novel indicator of diabetic GEC injury and is elevated in early DKD before the development of microalbuminuria in mice and humans. Clinical value for detecting early DKD warrants further investigation.

Tubular Deficiency of Heterogeneous Nuclear Ribonucleoprotein F Elevates Systolic Blood Pressure and Induces Glycosuria in Mice.

We reported previously that overexpression of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in renal proximal tubular cells (RPTCs) suppresses angiotensinogen (Agt) expression, and attenuates systemic hypertension and renal injury in diabetic Hnrnpf-transgenic (Tg) mice. We thus hypothesized that deletion of Hnrnpf in the renal proximal tubules (RPT) of mice would worsen systemic hypertension and kidney injury, perhaps revealing novel mechanism(s). Tubule-specific Hnrnpf knockout (KO) mice were generated by crossbreeding Pax8-Cre mice with floxed Hnrnpf mice on a C57BL/6 background. Both male and female KO mice exhibited elevated systolic blood pressure, increased urinary albumin/creatinine ratio, tubulo-interstitial fibrosis and glycosuria without changes in blood glucose or glomerular filtration rate compared with control littermates. However, glycosuria disappeared in male KO mice at the age of 12 weeks, while female KO mice had persistent glycosuria. Agt expression was elevated, whereas sodium-glucose co-transporter 2 (Sglt2) expression was down-regulated in RPTs of both male and female KO mice as compared to control littermates. In vitro, KO of HNRNPF in human RPTCs (HK-2) by CRISPR gRNA up-regulated AGT and down-regulated SGLT2 expression. The Sglt2 inhibitor canagliflozin treatment had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice but induced glycosuria. Our results demonstrate that Hnrnpf plays a role in the development of hypertension and glycosuria through modulation of renal Agt and Sglt2 expression in mice, respectively.

Hedgehog Interacting Protein (Hhip) Regulates Insulin Secretion in Mice Fed High Fat Diets.

Hedgehog interacting protein (Hhip) is essential for islet formation and beta-cell proliferation during pancreatic development; abnormally elevated Hhip expression has been linked to human pancreatitis. Here, we investigate the role of Hhip in modulating insulin secretion in adult Hhip mice (Hhip +/- vs. Hhip+/+) fed high fat diets (HFD). Both sexes of HFD-Hhip +/+ mice developed impaired glucose intolerance, that was only ameliorated in male HFD-Hhip +/- mice that had high levels of circulating plasma insulin, but not in female HFD-Hhip +/- mice. HFD stimulated Hhip gene expression, mainly in beta cells. Male HFD-Hhip +/+ mice had more large islets in which insulin content was reduced; islet architecture was disordered; and markers of oxidative stress (8-OHdG and Nox 2) were increased. In contrast, male HFD-Hhip +/- mice had more small islets with increased beta cell proliferation, enhanced GSIS, less oxidative stress and preserved islet integrity. In vitro, recombinant Hhip increased Nox2 and NADPH activity and decreased insulin-positive beta cells. siRNA-Hhip increased GSIS and abolished the stimulation of sodium palmitate (PA)-BSA on Nox2 gene expression. We conclude that pancreatic Hhip gene inhibits insulin secretion by altering islet integrity and promoting Nox2 gene expression in beta cells in response to HDF-mediated beta cell dysfunction, a novel finding.

Hedgehog Interacting Protein Promotes Fibrosis and Apoptosis in Glomerular Endothelial Cells in Murine Diabetes.

We investigated whether renal hedgehog interacting protein (Hhip) expression contributes to the progression of diabetic nephropathy (DN) and studied its related mechanism(s) in vivo and in vitro. Here, we show that Hhip expression is highly elevated in glomerular endothelial cells of adult type 1 diabetic (T1D) Akita and T2D db/db mouse kidneys as compared to non-diabetic control littermates. Hyperglycemia enhances reactive oxygen species (ROS) generation via NADPH oxidase 4 (Nox4) activation and stimulates renal Hhip gene expression, and that elevated renal Hhip gene expression subsequently activates the TGFß1- Smad2/3 cascade and promotes endothelial to mesenchymal transition associated with endothelial cell fibrosis/apoptosis in vivo and in vitro. Furthermore, kidneys of low-dose streptozotocin-induced diabetic heterozygous Hhip deficient (Hhip+/-) mice displayed a normal albumin/creatinine ratio with fewer features of DN (glomerulosclerosis/fibrosis and podocyte apoptosis/loss) and less evidence of renal compensation (glomerular hypertrophy and hyperfiltration) as compared to diabetic wild type controls (Hhip+/+). Thus, our studies demonstrated that renal Hhip expression is associated with nephropathy development in diabetes and that hyperglycemia-induced renal Hhip expression may mediate glomerular endothelial fibrosis and apoptosis in diabetes, a novel finding.

AT2 R deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein (Hhip) gene expression.

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.

Overexpression of angiotensinogen downregulates aquaporin 1 expression via modulation of Nrf2-HO-1 pathway in renal proximal tubular cells of transgenic mice.

INTRODUCTION: We aimed to examine the regulation of aquaporin 1 expression in an angiotensinogen transgenic mouse model, focusing on underlying mechanisms. METHODS: Male transgenic mice overexpressing rat angiotensinogen in their renal proximal tubular cells (RPTCs) and rat immortalised RPTCs stably transfected with rat angiotensinogen cDNA were used. RESULTS: Angiotensinogen-transgenic mice developed hypertension and nephropathy, changes that were either partially or completely attenuated by treatment with losartan or dual renin-angiotensin system blockade (losartan and perindopril), respectively, while hydralazine prevented hypertension but not nephropathy. Decreased expression of aquaporin 1 and heme oxygenase-1 and increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and sodium-hydrogen exchanger 3 were observed in RPTCs of angiotensinogen-transgenic mice and in angiotensinogen-transfected immortalised RPTCs. These parameters were normalised by dual renin-angiotensin system blockade. Both in vivo and in vitro studies identified a novel mechanism in which angiotensinogen overexpression in RPTCs enhances the cytosolic accumulation of Nrf2 via the phosphorylation of pGSK3ß Y216. Consequently, lower intranuclear Nrf2 levels are less efficient to trigger heme oxygenase-1 expression as a defence mechanism, which subsequently diminishes aquaporin 1 expression in RPTCs. CONCLUSIONS: Angiotensinogen-mediated downregulation of aquaporin 1 and Nrf2 signalling may play an important role in intrarenal renin-angiotensin system-induced hypertension and kidney injury.

Post-weaning high-fat diet accelerates kidney injury, but not hypertension programmed by maternal diabetes.

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.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-ß1-induced kidney injury in a mouse model of diabetes.

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.

Maternal diabetes modulates kidney formation in murine progeny: the role of hedgehog interacting protein (HHIP).

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.

Heterogeneous nuclear ribonucleoprotein F suppresses angiotensinogen gene expression and attenuates hypertension and kidney injury in diabetic mice.

We investigated the impact of heterogeneous nuclear ribonucleoprotein F (hnRNP F) overexpression on angiotensinogen (Agt) gene expression, hypertension, and renal proximal tubular cell (RPTC) injury in high-glucose milieu both in vivo and in vitro. Diabetic Akita transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs were created, and the effects on systemic hypertension, Agt gene expression, renal hypertrophy, and interstitial fibrosis were studied. We also examined immortalized rat RPTCs stably transfected with control plasmid or plasmid containing hnRNP F cDNA in vitro. The results showed that hnRNP F overexpression attenuated systemic hypertension, suppressed Agt and transforming growth factor-ß1 (TGF-ß1) gene expression, and reduced urinary Agt and angiotensin II levels, renal hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg mice. In vitro, hnRNP F overexpression prevented the high-glucose stimulation of Agt and TGF-ß1 mRNA expression and cellular hypertrophy in RPTCs. These data suggest that hnRNP F plays a modulatory role and can ameliorate hypertension, renal hypertrophy, and interstitial fibrosis in diabetes. The underlying mechanism is mediated, at least in part, via the suppression of intrarenal Agt gene expression in vivo. hnRNP F may be a potential target in the treatment of hypertension and kidney injury in diabetes.

Catalase prevents maternal diabetes-induced perinatal programming via the Nrf2-HO-1 defense system.

We investigated whether overexpression of catalase (CAT) in renal proximal tubular cells (RPTCs) could prevent the programming of hypertension and kidney disease in the offspring of dams with maternal diabetes. Male offspring of nondiabetic and diabetic dams from two transgenic (Tg) lines (Hoxb7-green fluorescent protein [GFP]-Tg [controls] and Hoxb7/CAT-GFP-Tg, which overexpress CAT in RPTCs) were studied from the prenatal period into adulthood. Nephrogenesis, systolic blood pressure, renal hyperfiltration, kidney injury, and reactive oxygen species (ROS) generation were assessed. Gene expression of transforming growth factor-ß1 (TGF-ß1), nuclear factor erythroid 2p45-related factor-2 (Nrf2), and heme oxygenase-1 (HO-1) was tested in both in vitro and in vivo studies. Renal dysmorphogenesis was observed in offspring of Hoxb7-GFP-Tg dams with severe maternal diabetes; the affected male offspring displayed higher renal ROS generation and developed hypertension and renal hyperfiltration as well as renal injury with heightened TGF-ß1 expression in adulthood. These changes were ameliorated in male offspring of diabetic Hoxb7/CAT-GFP-Tg dams via the Nrf2-HO-1 defense system. CAT promoted Nrf2 nuclear translocation and HO-1 gene expression, seen in both in vitro and in vivo studies. In conclusion, CAT overexpression in the RPTCs ameliorated maternal diabetes-induced perinatal programming, mediated, at least in part, by triggering the Nrf2-HO-1 defense system.

Angiotensin II type II receptor deficiency accelerates the development of nephropathy in type I diabetes via oxidative stress and ACE2.

Since the functional role(s) of angiotensin II (Ang II) type II receptor (AT(2)R) in type I diabetes is unknown, we hypothesized that AT(2)R is involved in decreasing the effects of type I diabetes on the kidneys. We induced diabetes with low-dose streptozotocin (STZ) in both AT(2)R knockout (AT(2)RKO) and wild-type (WT) male mice aged 12 weeks and followed them for 4 weeks. Three subgroups nondiabetic, diabetic, and insulin-treated diabetic (Rx insulin implant) were studied. Systolic blood pressure (SBP), physiological parameters, glomerular filtration rate (GFR), renal morphology, gene expression, and apoptosis were assessed. After 4 weeks of diabetes, compared to WT controls, AT(2)RKO mice clearly developed features of early diabetic nephropathy (DN), such as renal hypertrophy, tubular apoptosis, and progressive extracellular matrix (ECM) protein accumulation as well as increased GFR. AT(2)RKO mice presented hypertension unaffected by diabetes. Renal oxidative stress (measured as heme oxygenase 1 (HO-1) gene expression and reactive oxygen species (ROS) generation) and intrarenal renin angiotensin system components, such as angiotensinogen (Agt), AT(1)R, and angiotensin-converting enzyme (ACE) gene expression, were augmented whereas angiotensin-converting enzyme2 (ACE2) gene expression was decreased in renal proximal tubules (RPTs) of AT(2)RKO mice. The renal changes noted above were significantly enhanced in diabetic AT(2)RKO mice but partially attenuated in insulin-treated diabetic WT and AT(2)RKO mice. In conclusion, AT(2)R deficiency accelerates the development of DN, which appears to be mediated, at least in part, via heightened oxidative stress and ACE/ACE2 ratio in RPTs.

High glucose promotes nascent nephron apoptosis via NF-kappaB and p53 pathways.

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.

Maternal diabetes programs hypertension and kidney injury in offspring.

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.