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 TGFβ1 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 TGFβ1 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.d iabetes during pregnancy, whether gestational or pregestational diabetes (type 1 or type 2), results in offspring at high risk of developing hypertension, cardiovascular disease, and chronic kidney disease in adult life. This phenomenon, termed perinatal programming, in which intrauterine events are associated with later adverse changes, has attracted much attention (1-3). Substantial epidemiologic data have also suggested that the offspring whose mothers were diabetic during pregnancy are susceptible to metabolic disturbances induced by postnatal overnutrition, as seen with high-fat diet (HFD) or with increased caloric intake in early life (1)(2)(3)(4).Women who have diabetes during pregnancy and/or are obese and hyperinsulinemic are at risk of delivering macrosomic newborns (high birth weight), and both shortand long-term outcomes of macrosomic neonates are influenced by postnatal overnutrition (1-4). In high-birth-weight neonates, subsequent growth in infancy and risk of becoming obese or diabetic are directly and linearly linked-e.g., the higher the birth weight, the greater the risk of overweight and metabolic disturbances later in life (1-4). In contrast, pregnant women with severe, uncontrolled diabetes or diabetic complications, such as diabetic nephropathy and/or retinopathy, are at high risk of having a microsomic fetus (i.e., a fetus with intrauterine growth restriction (IUGR)) (5,6). Such infants may be markedly small for dates; but many develop excessive weight gain and increased fat deposition in early infancy, a proxy for neonatal overnutrition (7,8). However, the long-term outcome of IUGR offspring who experience overnutrition in early life is incomplet...

O-linked N-acetylglucosamine (O-GlcNAc) protein modification has been implicated in the regulation of signaling pathways, cell function, and gene expression. Glutamine:fructose-6-phosphate amidotransferase-1 (GFAT-1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway (HBP), which generates the sugar nucleotide UDP-GlcNAc, where this nucleotide acts as the donor for O-GlcNAc modification. In this study, we determined whether GFAT-1 regulates adipogenesis in adipocytes. 3T3-L1 preadipocytes were differentiated using medium containing high glucose, insulin, dexamethasone, and isobutylmethylxanthine. Cells were harvested 4, 8, and 12 h and 1, 2, 3, 4, 6, and 8 days after the initiation of differentiation. Global level of O-GlcNAc modification increased 4 h after induction and persisted for 8 days of observation. GFAT-1 mRNA and protein expression was also upregulated beginning 4 h after induction. Pharmacological inhibition of GFAT-1 or GFAT-1 siRNA treatment blocked the increase in O-GlcNAcylation and the formation of lipid droplets in adipocytes. GFAT-1 may regulate the expression of C/EBPβ, PPARγ, SREBP-1, fatty acid synthase, S3-12, perilipin, or adipophilin during adipogenesis. Our results suggest that GFAT-1 plays a critical role in modulating adipogenesis via the regulation of protein O-GlcNAcylation in adipocytes.

Angiotensin II type 2 receptor (AT R) deficiency in AT 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 AT R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT RKO mice were used to assess glomerulogenesis, while wild-type and AT RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT 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 TGFβ1-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 AT 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.

Clinical trials indicate that sodium-glucose co-transporter 2 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 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 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.

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 TGFβ1- 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.