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Insulin facilitates renal sodium reabsorption and attenuates gluconeogenesis. Sex differences in this regulation have not been well characterized. Using tetracycline-inducible Cre-lox recombination, we knocked out (KO) the insulin receptor (InsR) from the renal tubule in adult male (M) and female (F) mice (C57Bl6 background) with a paired box 8 (PAX8) promoter. Body weights were not affected by the KO, but mean kidney weights were reduced in the KO mice (13 and 3%, in M and F, respectively, relative to wild-type (WT) mice). A microscopic analysis revealed 25 and 19% reductions in the proximal tubule (PT) and cortical collecting duct cell heights, respectively, in KOMs relative to WTMs. The reductions were 5 and 11% for KOFs. Western blotting of renal cortex homogenates showed decreased protein levels for the β and γ subunits of the epithelial sodium channel (ENaC) and the sodium-potassium-2-chloride cotransporter type 2 (NKCC2) in both sexes of KO mice; however, α-ENaC was upregulated in KOMs and downregulated in KOFs. Both sexes of KO mice cleared exogenously administered glucose faster than the WT mice and had lower semi-fasted, anesthetized blood glucose levels. However, KOMs (but not KOFs) demonstrated evidence of enhanced renal gluconeogenesis, including higher levels of renal glucose-6-phosphatase, the PT’s production of glucose, post-prandial blood glucose, and plasma insulin, whereas KOFs exhibited downregulation of renal high-capacity sodium glucose cotransporter (SGLT2) and upregulation of SGLT1; these changes appeared to be absent in the KOM. Overall, these findings suggest a sex-differential reliance on intact renal tubular InsR signaling which may be translationally important in type 2 diabetes, obesity, or insulin resistance when renal insulin signaling is reduced.

Hyperandrogenemia (HA) is a hallmark of polycystic ovary syndrome (PCOS) and is an integral element of non-alcoholic fatty liver disease (NALFD) in females. Administering low-dose dihydrotestosterone (DHT) induced a normal weight PCOS-like female mouse model displaying NAFLD. The molecular mechanism of HA-induced NAFLD has not been fully determined. We hypothesized that DHT would regulate hepatic lipid metabolism via increased SREBP1 expression leading to NAFLD. We extracted liver from control and low-dose DHT female mice; and performed histological and biochemical lipid profiles, Western blot, immunoprecipitation, chromatin immunoprecipitation, and real-time quantitative PCR analyses. DHT lowered the 65 kD form of cytosolic SREBP1 in the liver compared to controls. However, DHT did not alter the levels of SREBP2 in the liver. DHT mice displayed increased SCAP protein expression and SCAP-SREBP1 binding compared to controls. DHT mice exhibited increased AR binding to intron-8 of SCAP leading to increased SCAP mRNA compared to controls. FAS mRNA and protein expression was increased in the liver of DHT mice compared to controls. p-ACC levels were unaltered in the liver. Other lipid metabolism pathways were examined in the liver, but no changes were observed. Our findings support evidence that DHT increased de novo lipogenic proteins resulting in increased hepatic lipid content via regulation of SREBP1 in the liver. We show that in the presence of DHT, the SCAP-SREBP1 interaction was elevated leading to increased nuclear SREBP1 resulting in increased de novo lipogenesis. We propose that the mechanism of action may be increased AR binding to an ARE in SCAP intron-8.

Diabetes is a worldwide epidemic that is increasing rapidly to become the seventh leading cause of death in the world. The increased incidence of this disease mirrors a similar uptick in obesity and metabolic syndrome, and, collectively, these conditions can cause deleterious effects on a number of organ systems including the renal and cardiovascular systems. Historically, treatment of type 2 diabetes has focused on decreasing hyperglycemia and glycated hemoglobin levels. However, it now is appreciated that there is more to the puzzle. Emerging evidence has indicated that newer classes of diabetes drugs, sodium-glucose co-transporter 2 inhibitors and glucagon-like peptide 1-receptor agonists, improve cardiovascular and renal function, while appropriately managing hyperglycemia. In this review, we highlight the recent clinical and preclinical studies that have shed light on sodium-glucose co-transporter 2 inhibitors and glucagon-like peptide 1-receptor agonists and their ability to stabilize blood glucose levels while offering whole-body protection in diabetic and nondiabetic patient populations.

Renal ischemia can lead to irreversible damage to the kidney including atrophy and fibrosis. Young females generally experience attenuated pathology due to the protective actions of ovarian steroids, in particular 17β-Estradiol (E2); however, the mechanisms of action remain murky.

miRNAs provide fine tuning of gene expression via inhibition of translation. miR-451 has a modulatory role in cell cycling via downregulation of mechanistic target of rapamycin. We aimed to test whether chronic systemic inhibition of miR-451 would enhance renal fibrosis (associated with deranged autophagy). Adult TallyHo/Jng mice (obese insulin resistant) were randomized to two treatment groups to receive either miR-451 inhibition [via a locked nucleic acid construct] or a similar scrambled locked nucleic acid control for 8 wk. All mice were fed a high-fat diet (60% kcal from fat) ad libitum and humanely euthanized after 12 wk. Kidneys and blood were collected for analysis. Renal expression of miR-451 was sixfold lower in inhibitor-treated mice compared with control mice. miR-451 inhibition increased kidney weight and collagen and glycogen deposition. Blood chemistry revealed significantly higher Na+ and anion gap (relative metabolic acidosis) in inhibitor-treated mice. Western blot analysis and immunohistochemistry of the kidney revealed that the inhibitor increased markers of renal injury and fibrosis, e.g., kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, transforming growth factor-β, 14-3-3 protein-ζ, mechanistic target of rapamycin, AMP-activated protein kinase-α, calcium-binding protein 39, matrix metallopeptidase-9, and the autophagy receptor sequestosome 1. In contrast, the inhibitor reduced the epithelial cell integrity marker collagen type IV and the autophagy markers microtubule-associated protein 1A/1B light chain 3B and beclin-1. Taken together, these results support a protective role for miR-451 in reducing renal fibrosis by enhancing autophagy in obese mice.

Much excitement exists over the cardioprotective and life-extending effects of caloric restriction (CR). This review integrates population studies with experimental animal research to address the positive and negative impact of mild and severe CR on cardiovascular physiology and pathophysiology, with a particular focus on the renin-angiotensin system (RAS). We also highlight the gaps in knowledge and areas ripe for future physiological research.

Peroxisome proliferator activated receptor alpha (PPAR-α) deletion has been shown to increase blood pressure (BP). We hypothesized that the BP increase in PPAR-α KO mice was mediated by increased expression and activity of basolateral Na(+)/K(+) ATPase (NKA) pump. To address this hypothesis, we treated wild-type (WT) and PPAR-α knockout (KO) mice with a slow-pressor dose of angiotensin II (400 ng/kg·min) for 12 days by osmotic minipump. Radiotelemetry showed no significant differences in baseline mean arterial pressure (MAP) between WT and PPAR-α KO mice; however, by day 12 of infusion, MAP was significantly higher in PPAR-α KO mice (156±16) compared to WT mice (138±11 mmHg). NKA activity and protein expression (α1 subunit) were significantly higher in PPAR-α KO mice compared to WT mice. There was no significant difference in NKA mRNA levels. Angiotensin II further increased the expression and activity of the NKA in both genotypes along with the water channel, aquaporin 1 (Aqp1). In contrast, angiotensin II decreased the expression (64-97% reduction in band density) of sodium-hydrogen exchanger-3 (NHE3), NHE regulatory factor-1 (NHERF1, Slc9a3r1), sodium-potassium-2-chloride cotransporter (NKCC2), and epithelial sodium channel (ENaC) β- and γ- subunits in the renal cortex of both WT and PPAR-α KO mice, with no difference between genotypes. The sodium-chloride cotransporter (NCC) was also decreased by angiotensin II, but significantly more in PPAR-α KO (59% WT versus 77% KO reduction from their respective vehicle-treated mice). Our results suggest that PPAR-α attenuates angiotensin II-mediated increased blood pressure potentially via reducing expression and activity of the NKA.

Background/aims Metabolic syndrome and type 2 diabetes are associated with some degree of acidosis. Acidosis has also been shown to upregulate renal gluconeogenesis. Whether impaired insulin or insulin-like-growth factor 1 receptor (IGF1) signaling alter this relationship is not known. Our aim was to determine the effects of deletion of insulin and IGF1 receptors (Insr and Igf1r) from renal proximal tubule (PT) on the gluconeogenic response to acidosis. Methods We developed a mouse model with PT-targeted dual knockout (KO) of the Insr/Igf1r by driving Cre-recombinase with the gamma-glutamyl transferase (gGT) promoter. Male and female mice were maintained as control or acidotic by treatment with NH4Cl in the drinking water for 1-week. Results Acidosis in both genotypes increased renal expression of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1-bisphosphatase (FBP1), but not glucose-6-phosphatase catalytic subunit (G6PC), which showed significantly lower expression in the KO regardless of treatment. Several differences between KO and WT suggested a protective role for insulin/IGF1 receptor signaling in maintaining relative euglycemia in the face of acidosis. First, the increase in FBP1 with acid was greater in the KO (significant interactive term). Secondly, proximal-tubule-associated FOXO1 and AKT overall protein levels were suppressed by acid loading in the KO, but not in the WT. Robust intact insulin signaling would be needed to reduce gluconeogenesis in PT. Third, phosphorylated FOXO1 (pS256) levels were markedly reduced by acid loading in the KO PT, but not in the WT. This reduction would support greater gluconeogenesis. Fourth, the sodium-glucose cotransporter (SGLT1) was increased by acid loading in the KO kidney, but not the WT. While this would not necessarily affect gluconeogenesis, it could result in increased circulatory glucose via renal reabsorption. Reduced susceptibility to glucose-homeostatic dysregulation in the WT could potentially relate to the sharp (over 50%) reduction in renal levels of sirtuin-1 (SIRT1), which deacetylates and regulates transcription of a number of genes. This reduction was absent in the KO. Conclusion Insulin resistance of the kidney may increase whole-body glucose instability a major risk factor for morbidity in diabetes. High dietary acid loads provide a dilemma for the kidney, as ammoniagenesis liberates α-ketoglutarate, which is a substrate for gluconeogenesis. We demonstrate an important role for insulin and/or IGF1 receptor signaling in the PT to facilitate this process and reduce excursions in blood glucose. Thus, medications and lifestyle changes that improve renal insulin sensitivity may also provide added benefit in type 2 diabetes especially when coupled with metabolic acidosis.

The contribution of proximal tubules (PT) to albumin uptake is now well recognized, however, its regulation is understudied area. There are reports suggesting that insulin resistance is associated with the development of albuminuria in nondiabetic individuals. We have previously reported reduced insulin receptor (IR) expression in renal-tubular-epithelial cells, including PT in various models of insulin resistance. However, the effect of a physiological fall in insulin levels and the role for IR in PT in tubular albumin uptake is not clear. To address these gaps in our understanding, we estimated urine excretion and renal uptake of albumin in fasted and fed C57Bl/6 mice injected with fluorescein isothiocyanate (FITC)-albumin (5 µg/mL/kg body weight, intraperitoneal, n = 6 per group). In addition, we compared spot urine analysis from 33 clinically healthy humans after overnight fasting (when insulin levels are lower than in the fed state) and then at 2 hours after 75 g oral glucose challenge (postprandial). Fasted mice had attenuated renal uptake of FITC-albumin and higher excretion in urine, relative to fed mice ( P = 0.04). Moreover, a significant drop in urine albumin-to-creatinine ratio (ACR) and urine albumin concentration (UAC) was observed in the postprandial state in these subjects ( P = 0.001 and P = 0.017, for ACR and UAC, respectively). The drop was negatively associated with postprandial blood glucose levels (ρ = -0.36, P = 0.03 for ΔUAC and ρ = -0.34, P = 0.05 for ΔACR). To test the role of IR in PT, we analyzed 24-hour urine albumin excretion in male mice with targeted deletion of IR from PT (insulin receptor knockout [IRKO]) and their wild-type (WT) littermates ( n = 7 per group). IRKO mice had significantly higher 24-hour urine albumin excretion relative to WT. Moreover, kidneys from KO mice revealed reduced expression of megalin and cubulin proteins in the PT relative to the WT. We also demonstrated insulin (100 nM) induced albumin internalization in human proximal tubule cells (hPT) and this effect of insulin was attenuated in hydroxy-2-naphthalenylmethylphosphonic acid (100 µM), a tyrosine kinase inhibitor, pretreated hPT. Our findings revealed albumin excretion was attenuated by glucose administration to fasting individuals implying a regulatory role for insulin in PT albumin reabsorption. Thus albuminuria associated with insulin resistance/diabetes may relate not only to glomerular dysfunction but also to impairment in insulin-mediated reabsorption.

Micro-RNAs (miRs) encapsulated inside urinary exosomes (uEs) have the potential as early biomarkers. Previously, we reported that a rise in uE miR-451 predicted albuminuria in diabetic rats; however, whether the rise was protective or detrimental, and occurred in response to injury or general hyperglycemia, was unknown. To address this, we studied both human and rat models of renal disease. In humans, uE miR-451 was approximately twofold higher in subjects with early-stage chronic kidney disease (CKD; serum creatinine < 2.0 mg/dl; n = 28), as compared to age-matched healthy controls (n = 23), and had a significant negative correlation with estimated glomerular filtration rate (eGFR) (r2 = −0.10, p = 0.01). Subgroup analysis of CKD subjects showed that those without diabetes had slightly (∼30%) but significantly higher uE miR-451 as compared to those with diabetes, with no differences in albumin excretion, eGFR, serum sodium, and potassium. Using human proximal tubule (hPT) cells, we found that locked nucleic acid (LNA) inhibition of miR-451 resulted in a significant increase in the messenger RNA (mRNA) expression of kidney-injury-associated miR-451 targets, e.g., CAB39, TBX1, and YWHAZ, as compared to treatment with a control LNA. Moreover, hPT cells and their secreted exosomes showed an increase in miR-451 in response to mechanical injury but not high glucose (20 versus 5 mM). For further proof of concept, in diabetic rats, we showed that atorvastatin (AT), a treatment proven to attenuate renal injury without affecting systemic glucose levels, reduced uE miR-451 with the concomitant restoration of renal miR-451. These data elucidate the stimuli for renal miR-451 expression and exosomal release and support its role as a therapeutic target and early biomarker for renal injury in humans.

Sodium-glucose co-transporters (SGLTs) serve to reabsorb glucose in the kidney. Recently, these transporters, mainly SGLT2, have emerged as new therapeutic targets for patients with diabetes and kidney disease; by inhibiting glucose reabsorption, they promote glycosuria, weight loss, and improve glucose tolerance. They have also been linked to cardiac protection and mitigation of liver injury. However, to date, the mechanism(s) by which SGLT2 inhibition promotes systemic improvements is not fully appreciated. Using an obese TallyHo mouse model which recapitulates the human condition of diabetes and nonalcoholic fatty liver disease (NAFLD), we sought to determine how modulation of renal glucose handling impacts liver structure and function. Apart from an attenuation of hyperglycemia, Empagliflozin was found to decrease circulating triglycerides and lipid accumulation in the liver in male TallyHo mice. This correlated with lowered hepatic cholesterol esters. Using in vivo MRI analysis, we further determined that the reduction in hepatic steatosis in male TallyHo mice was associated with an increase in nuchal white fat indicative of “healthy adipose expansion”. Notably, this whitening of the adipose came at the expense of brown adipose tissue. Collectively, these data indicate that the modulation of renal glucose handling has systemic effects and may be useful as a treatment option for NAFLD and steatohepatitis.

P2Y2, a G protein-coupled receptor (R), is expressed in all organs involved in the development of obesity and insulin resistance. To explore the role of it in diet-induced obesity, we fed male P2Y2-R whole body knockout (KO) and wild type (WT) mice (B6D2 genetic background) with regular diet (CNT; 10% calories as fat) or high-fat diet (HFD; 60% calories as fat) with free access to food and water for 16 weeks, and euthanized them. Adjusted for body weights (BW), KO mice consumed modestly, but significantly more HFD vs. WT mice, and excreted well-formed feces with no taint of fat or oil. Starting from the 2nd week, HFD-WT mice displayed significantly higher BW with terminal mean difference of 22% vs. HFD-KO mice. Terminal weights of white adipose tissue (WAT) were significantly lower in the HFD-KO vs. HFD-WT mice. The expression of P2Y2-R mRNA in WAT was increased by 2-fold in HFD-fed WT mice. Serum insulin, leptin and adiponectin levels were significantly elevated in the HFD-WT mice, but not in the HFD-KO mice. When induced in vitro, preadipocytes derived from KO mice fed regular diet did not differentiate and mature as robustly as those from the WT mice, as assessed by cellular expansion and accumulation of lipid droplets. Blockade of P2Y2-R by AR-C118925 in preadipocytes derived from WT mice prevented differentiation and maturation. Under basal conditions, KO mice had significantly higher serum triglycerides and showed slightly impaired lipid tolerance as compared to the WT mice. HFD-fed KO mice had significantly better glucose tolerance (GTT) as compared to HFD-fed WT mice. Whole body insulin sensitivity and mRNA expression of insulin receptor, IRS-1 and GLUT4 in WAT was significantly higher in HFD-fed KO mice vs. HFD-fed WT mice. On the contrary, the expression of pro-inflammatory molecules MCP-1, CCR2, CD68, and F4/80 were significantly higher in the WAT of HFD-fed WT vs. HFD-fed KO mice. These data suggest that P2Y2-R plays a significant role in the development of diet-induced obesity by promoting adipogenesis and inflammation, and altering the production of adipokines and lipids and their metabolism in adipose tissue, and thereby facilitates HFD-induced insulin resistance.

Insulin has many varied actions in the proximal tubule. Two distinct activities include upregulation of sodium/bicarbonate reabsorption and downregulation of gluconeogenesis. The inability to perform these 2 tasks simultaneously under fed and fasted conditions can lead to hyper- or hypoglycemia, acidosis, and/or impaired extracellular fluid regulation. Nakamura and colleagues illuminate our understanding of this process, which appears to be managed in part by recruitment of different insulin receptor substrates under different physiological conditions.

Diabetic nephropathy (DN) is a severe complication of diabetes mellitus and a common cause of end stage renal disease (ESRD). Novel effective therapies are needed to combat disease progression. Several studies suggest a renal protective role of microRNA-451 (miR-451) in ameliorating progression of DN. However, the role of miR-451 in DN is not understood. Defects in the autophagy process has been associated with DN. The current study aimed to elucidate the role of miR-451 in the development of DN in a mouse model of insulin resistance. Male TALLYHO/Jng mice were placed on a high-fat diet (60% kCal) and divided into two treatment groups. Mice were injected (intraperitoneal) once a week for 8-weeks with locked nucleic acid (LNA) miR-451 inhibitor or LNA-scramble (2mg/kg/bw; n=8/treatment). Mice were humanely euthanized after 12 weeks and kidneys harvested. LNA-miR-451 inhibitor significantly reduced renal expression of miR-451 by 6-fold. Collagen deposition was higher in inhibitor treated mice, as revealed by Masson’s Trichrome. Period Acid Schiff did not reveal significant glomerular damage between the two groups. Kidney weight (normalized to body weight) was significantly higher in the inhibitor treated mice. Western blotting analyses of cortex homogenates revealed increased expression of markers of renal injury, KIM1 and NGAL (p Disclosure M.B. Fluitt: None. N. Shivapurkar: None. L. Li: None. C.M. Ecelbarger: None. Funding American Diabetes Association (1-18-PMF-002 to M.B.F.); National Institutes of Health (TL1TR001431)

Previously, we localized ADP-activated P2Y12 receptor (R) in rodent kidney and showed that its blockade by clopidogrel bisulfate (CLPD) attenuates lithium (Li)-induced nephrogenic diabetes insipidus (NDI). Here, we evaluated the effect of prasugrel (PRSG) administration on Li-induced NDI in mice. Both CLPD and PRSG belong to the thienopyridine class of ADP receptor antagonists. Groups of age-matched adult male B6D2 mice (N = 5/group) were fed either regular rodent chow (CNT), or with added LiCl (40 mmol/kg chow) or PRSG in drinking water (10 mg/kg bw/day) or a combination of LiCl and PRSG for 14 days and then euthanized. Water intake and urine output were determined and blood and kidney tissues were collected and analyzed. PRSG administration completely suppressed Li-induced polydipsia and polyuria and significantly prevented Li-induced decreases in AQP2 protein abundance in renal cortex and medulla. However, PRSG either alone or in combination with Li did not have a significant effect on the protein abundances of NKCC2 or NCC in the cortex and/or medulla. Immunofluorescence microscopy revealed that PRSG administration prevented Li-induced alterations in cellular disposition of AQP2 protein in medullary collecting ducts. Serum Li, Na, and osmolality were not affected by the administration of PRSG. Similar to CLPD, PRSG administration had no effect on Li-induced increase in urinary Na excretion. However, unlike CLPD, PRSG did not augment Li-induced increase in urinary arginine vasopressin (AVP) excretion. Taken together, these data suggest that the pharmacological inhibition of P2Y12-R by the thienopyridine group of drugs may potentially offer therapeutic benefits in Li-induced NDI.

Insulin therapy is often needed to overcome insulin receptor resistance in type 2 diabetes, however, the impact of providing additional insulin to already hyperinsulinemic subjects is not clear. We infused male TALLYHO/Jng (TH) mice (insulin resistant) with insulin (50 U/kg&middot, bw/d) or vehicle (control) by osmotic minipump for 14 days. One group of insulin-infused mice was switched to 4% NaCl diet (high-sodium diet, HSD) in the second week. Blood chemistry revealed a significantly higher anion gap and blood sodium concentrations with insulin infusion, i.e., relative metabolic acidosis. Systolic BP and heart rate were slightly (~5 mm Hg) higher in insulin-infused versus control mice. HSD resulted in a modest and transient rise in mean arterial blood pressure (BP), relative to control or insulin-infused, normal-NaCl-fed mice. In kidney, insulin infusion: (1) increased total and phosphorylated (serine-1177) endothelial nitric oxide synthase (eNOS) band densities, (2) reduced band density of the uncoupled form of eNOS, and (3) increased renal homogenate nitric oxide synthase (NOS) activity. Despite this, plasma and urine levels of nitrates plus nitrites (NOx) fell with insulin infusion, by day 14 (40&ndash, 50%) suggesting worsening of resistance. Overall, insulin infusion ramps up the cellular means in kidney to increase vasodilatory and natriuretic NO, but in the long term may be associated with worsening of insulin receptor resistance.

The renal collecting duct and other postmacula densa sites are the primary tubular regions for fine-tuning of electrolyte homeostasis in the body. A role for the mechanistic target of rapamycin (mTOR), a serine-threonine kinase, has recently been appreciated in this regulation. mTOR exists in two distinct multiprotein functional complexes, i.e., mTORC1 and mTORC2. Upregulation of mTORC1, by growth factors and amino acids, is associated with cell cycle regulation and hypertrophic changes. In contrast, mTORC2 has been demonstrated to have a role in regulating Na+ and K+ reabsorptive processes, including those downstream of insulin and serum- and glucocorticoid-regulated kinase (SGK). In addition, mTORC2 can upregulate mTORC1. A number of elegant in vitro and in vivo studies using cell systems and genetically modified mice have revealed mechanisms underlying activation of the epithelial Na+ channel (ENaC) and the renal outer medullary K+ channel (ROMK) by mTORC2. Overall, mTOR in its systematic integration of phosphorylative signaling facilitates the delicate balance of whole body electrolyte homeostasis in the face of changes in metabolic status. Thus, inappropriate regulation of renal mTOR has the potential to result in electrolyte disturbances, such as acidosis/alkalosis, hyponatremia, and hypertension. The goal of this minireview is to highlight the physiological role of mTOR in its complexes in regulating electrolyte homeostasis in the aldosterone-sensitive distal nephron.

High fructose intake has been associated with increased incidences of renal disease and hypertension, among other pathologies. Most fructose is cleared by the portal system and metabolized in the liver; however, systemic levels of fructose can rise with increased consumption. We tested whether there were sex differences in the renal responses to a high-fructose diet in mice. Two-month-old male and female C57BL6/129/SV mice ( n = 6 mice per sex per treatment) were randomized to receive control or high-fructose (65% by weight) diets as pelleted chow ad libitum for 3 mo. Fructose feeding did not significantly affect body weight but led to a 19% and 10% increase in kidney weight in male and female mice, respectively. In male mice, fructose increased the expression (∼50%) of renal cortical proteins involved in metabolism, including glucose transporter 5 (facilitative fructose transporter), ketohexokinase, and the insulin receptor (β-subunit). Female mice had lower basal levels of glucose transporter 5, which were unresponsive to fructose. However, female mice had increased urine volume and plasma K+ and decreased plasma Na+ with fructose, whereas male mice were less affected. Likewise, female mice showed a two- to threefold reduction in the expression Na+-K+-2Cl− cotransporter 2 in the thick ascending limb and aquaporin-2 in the collecting duct with fructose relative to female control mice, whereas male mice had no change. Overall, our results support greater proximal metabolism of fructose in male animals and greater distal tubule/collecting duct (electrolyte homeostasis) alterations in female animals. These sex differences may be important determinants of the specific nature of pathologies that develop in association with high fructose consumption.

The metabolic syndrome (MetS) is associated with a rise in blood pressure (BP) however the mechanisms underlying this association are not known. Nivar and colleagues utilize a number of innovative approaches to evaluate the role of the epithelial sodium channel (ENaC) of the renal collecting duct in the sodium retention and hypertension associated with MetS. C57Bl/6 male, mice fed a high-fat diet for 12 weeks developed several features of MetS including: hyperinsulinemia, hypercholesterolemia, obesity, and elevated BP. They also showed salt-sensitive hypertension and sodium retention in the early time course of switch from normal to a high NaCl diet. Nonetheless, they found no evidence of over-activity of ENaC, using both perfused tubules and in vivo (sensitivity to benzamil) approaches. The authors concluded alternative, perhaps upstream sites of sodium retention. The studies are important in that they highlight the complexity of metabolic syndrome, and shed some new light in a controversial area. They do support renal sodium retention may be at the core, and elucidation of these upstream pathways is in need of additional study.

Arginine vasopressin (AVP) is the predominant hormone regulating urine concentration. Its primary role is to increase water reabsorption from the connecting tubule through the collecting duct. AVP is produced in the hypothalamus, stored in the posterior pituitary, and released as a result of stimulation of osmoreceptors or baroreceptors. Normal circulating levels of AVP approximate 1 pM in humans and rodents. There are three subtypes of the vasopressin receptor (V1a, V1b, and V2). In general, V1 receptors are coupled to phosphoinositide-3-kinase (PI-3K) signaling and calcium release, while V2 receptors are coupled both to the generation of cyclic AMP and mobilization of intracellular calcium. In addition to water, AVP regulates renal chloride, bicarbonate, and potassium reabsorption via specific transporters, exchangers, and channels. Vasopressin actions in the kidney have also been shown to regulate glomerular filtration rate, renal blood flow, nitric oxide generation, and blood pressure in human animal model systems.

The epithelial sodium channel (ENaC) is one of the central effectors involved in regulation of salt and water homeostasis in the kidney. To study mechanisms of ENaC regulation, we generated knockout mice lacking the insulin receptor (InsR KO) specifically in the collecting duct principal cells. Single-channel analysis in freshly isolated split-open tubules demonstrated that the InsR-KO mice have significantly lower ENaC activity compared to their wild-type (C57BL/6J) littermates when animals were fed either normal or sodium-deficient diets. Immunohistochemical and Western blot assays demonstrated no significant changes in expression of ENaC subunits in InsR-KO mice compared to wild-type littermates. Insulin treatment caused greater ENaC activity in split-open tubules isolated from wild-type mice but did not have this effect in the InsR-KO mice. Thus, these results suggest that insulin increases ENaC activity via its own receptor affecting the channel open probability. To further determine the mechanism of the action of insulin on ENaC, we used mouse mpkCCDc14 principal cells. Insulin significantly augmented amiloride-sensitive transepithelial flux in these cells. Pretreatment of the mpkCCDc14 cells with phosphatidylinositol 3-kinase (LY294002; 10 μM) or mTOR (PP242; 100 nM) inhibitors precluded this effect. This study provides new information about the importance of insulin receptors expressed in collecting duct principal cells for ENaC activity.—Pavlov, T. S., Ilatovskaya, D. V., Levchenko, V., Li, L., Ecelbarger, C. M., Staruschenko, A. Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct.

To elucidate the role of the insulin receptor (IR) in collecting duct (CD), we bred mice with IR selectively deleted from CD principal cells using an aquaporin-2 promoter to drive Cre-recombinase expression. Young, adult male knockout (KO) mice had altered plasma and electrolyte homeostasis under high- (HS) and low-sodium (LS) diets, relative to wild-type (WT) littermates. One week of LS feeding led to a significant reduction in urine potassium (K+) and sodium (Na+) excretion in KO, and a reduction in the ratio of Na+to chloride (Cl−) in plasma, relative to WT. HS diet (1 wk) increased plasma K+and reduced urine Na+to Cl−ratio in the KO. Furthermore, KO mice had a significantly ( P = 0.025) blunted natriuretic response to benzamil, an epithelial sodium channel (ENaC) antagonist. Western blotting of cortex homogenates revealed modestly, but significantly (∼15%), lower band density for the β-subunit of ENaC in the KO vs. WT mice, with no differences for the α- or γ-subunits. Moreover, blood pressure (BP), measured by radiotelemetry, was significantly lower in KO vs. WT mice under basal conditions (mmHg): 112 ± 5 (WT), 104 ± 2 (KO), P = 0.023. Chronic insulin infusion reduced heart rate in the WT, but not in the KO, and modestly reduced BP in the WT only. Overall, these results support a fundamental role for insulin through its classic receptor in the modulation of electrolyte homeostasis and BP.

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre‐lox recombination, using a γ‐glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein‐to‐DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30–50% (P

Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors have been shown to improve diabetic nephropathy. However, whether they provide protection via Histone deacetylases (HDAC) inhibition is not clear. We conducted a comparative evaluation of Atorvastatin (AT) versus the non-statin cholesterol-lowering drug, Ezetimibe (EZT) on severity of diabetic nephropathy. Streptozotocin-treated male Wistar rats were fed a cholesterol-supplemented diet and gavaged daily with vehicle, AT or EZT. Control rats received normal diet and gavaged vehicle (n = 8–9/group). Diabetes increased blood glucose, urine albumin-to-creatinine ratio (ACR), kidney pathology and HDAC activity, and reduced renal E-cadherin levels. Both AT and EZT reduced circulating cholesterol, attenuated renal pathology, and did not lower blood glucose. However, AT was significantly more effective than EZT at reducing kidney pathology and HDAC activity. Chromatin immunoprecipitation revealed a significantly higher association of acetylated H3 and H4 with the E-cadherin promoter in kidneys from AT-, relative to EZT- or vehicle-treated rats. Moreover, we demonstrated a direct effect of AT, but not EZT, on HDAC-inhibition and, H3 and H4- acetylation in primary glomerular mesangial cells. Overall, both AT and EZT attenuated diabetic nephropathy; however, AT exhibited greater efficacy despite a similar reduction in circulating cholesterol. HDAC-inhibition may underlie greater efficacy of statins in attenuating kidney injury.

To elucidate the role of the insulin receptor (IR) on kidney nitric oxide generation and blood pressure (BP) control, we generated mice with targeted deletion of renal tubule IR using loxP recombination driven by a Ksp-cadherin promoter. Male knockout (KO) and wild-type (WT) littermates (∼4 mo old) were transitioned through three 1-wk treatments: 1) low-NaCl diet (0.085%); 2) high-NaCl diet (HS; 5%); and 3) HS diet plus 3 mM tempol, a superoxide dismutase mimetic, in the drinking water. Mice were then switched to medium-NaCl (0.5%) diet for 5 days and kidneys harvested under pentobarbital anesthesia. Twenty-four-hour urinary nitrates plus nitrites were significantly higher in the WT mice under HS (2,067 ± 280 vs. 1,550 ± 230 nmol/day in WT and KO, respectively, P < 0.05). Tempol attenuated genotype differences in urinary nitrates plus nitrites. A rise in BP with HS was observed only in KO mice and not affected by tempol (mean arterial pressure, dark period, HS, 106 ± 5 vs. 119 ± 4 mmHg, for WT and KO, respectively, P < 0.05). Renal outer medullary protein levels of nitric oxide synthase (NOS) isoforms by Western blot (NOS1–3 and phosphorylated-S1177-NOS3) revealed significantly lower band density for NOS1 (130-kDa isoform) in the KO mice. A second study, when mice were euthanized under HS conditions, confirmed significantly lower NOS1 (130 kDa) in the KO, with an even more substantial (>50%) reduction of the 160-kDa NOS1 isoform. These studies suggest that the loss of renal IR signaling impairs renal nitric oxide production. This may be important in BP control, especially in insulin-resistant states, such as the metabolic syndrome.

The P2Y2receptor (P2Y2-R) antagonizes sodium reabsorption in the kidney. Apart from its effect in distal nephron, hypothetically, P2Y2-R may modulate activity/abundances of sodium transporters/channel subunits along the nephron via antagonism of aldosterone or vasopressin or interaction with mediators such as nitric oxide (NO), and prostaglandin E2(PGE2) or oxidative stress (OS). To determine the extent of the regulatory role of P2Y2-R in renal sodium reabsorption, in study 1, we fed P2Y2-R knockout (KO; n = 5) and wild-type (WT; n = 5) mice a high (3.15%)-sodium diet (HSD) for 14 days. Western blotting revealed significantly higher protein abundances for cortical and medullary bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), medullary α-1-subunit of Na-K-ATPase, and medullary α-subunit of the epithelial sodium channel (ENaC) in KO vs. WT mice. Molecular analysis of urine showed increased excretion of nitrates plus nitrites (NOx), PGE2, and 8-isoprostane in the KO, relative to WT mice, supporting a putative role for these molecules in determining alterations of proteins involved in sodium transport along the nephron. To determine whether genotype differences in response to aldosterone might have played a role in these differences due to HSD, in study 2 aldosterone levels were clamped (by osmotic minipump infusion). Clamping aldosterone (with HSD) led to significantly impaired natriuresis with elevated Na/H exchanger isoform 3 in the cortex, and NKCC2 in the medulla, and modest but significantly lower levels of NKCC2, and α- and β-ENaC in the cortex of KO vs. WT mice. This was associated with significantly reduced urinary NOx in the KO, although PGE2and 8-isoprostane remained significantly elevated vs. WT mice. Taken together, our results suggest that P2Y2-R is an important regulator of sodium transporters along the nephron. Pre- or postreceptor differences in the response to aldosterone, perhaps mediated via prostaglandins or changes in NOS activity or OS, likely play a role.

Dopamine produced by renal proximal tubules increases sodium excretion via a decrease in renal sodium reabsorption. Dopamine natriuresis is impaired in obese Zucker rats; however, the mechanism is not fully understood. To test the hypothesis that renal expression of one or more of the subtypes are altered in these rats, we measured whole kidney protein levels by immunoblotting of D1-like (D1R and D5R) and D2-like (D2R, D3R, and D4R) dopamine receptors in both male and female obese and lean Zucker rats. In obese males on 1% NaCl diet, D1R, D2R, D4R, and D5R were decreased, while D3R was increased, relative to lean rats. Under a 4% NaCl diet, D2R and D3R levels in obese rats were restored to lean levels. 4% NaCl diet reduced D5R in both body types, relative to 1% NaCl diet. Female rats had higher expression of D1R and D3R than did male; however, the sex difference for D1R was markedly blunted in obese rats. In obese rats, dietary candesartan (angiotensin II type 1 receptor blocker) normalized downregulated D1R and D2R, but either decreased (D3R), did not affect (D4R), or further downregulated (D5R) the other subtypes. Candesartan also decreased D4R in lean rats. In summary, reduced renal protein levels of D1R, D2R, D4R, and D5R in obese Zucker rats could induce salt sensitivity and elevate blood pressure. Increased angiotensin II type 1 receptor activity may be mechanistically involved in the decreased expression of D1R and D2R in obese rats. Finally, reduced D1R and D3R in male rats may contribute to sex differences in blood pressure.

Insulin resistance is associated with hypertension by mechanisms likely involving the kidney. To determine how the major apical sodium transporter of the thick ascending limb, the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is regulated by high-fat feeding, we treated young male, Fischer 344 X Brown Norway (F344BN) rats for 8 wk with diets containing either normal (NF, 4%) or high (HF, 36%) fat, by weight, primarily as lard. HF-fed rats had impaired glucose tolerance, increased urine excretion of 8-isoprostane (a marker of oxidative stress), increased protein levels for NKCC2 (50–125%) and the renal outer medullary potassium channel (106%), as well as increased natriuretic response to furosemide (20–40%). To test the role of oxidative stress in this response, in study 2, rats were fed the NF or HF diet plus plain drinking water, or water containing NG-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor (100 mg/l), or tempol, a superoxide dismutase mimetic (1 mmol/l). The combination of tempol with HF nullified the increase in medullary NKCC2, while l-NAME with HF led to the highest expression of medullary NKCC2 (to 498% of NF mean). However, neither of these drugs dramatically affected the elevated natriuretic response to furosemide with HF. Finally, l-NAME led to a marked increase in blood pressure (measured by radiotelemetry), which was significantly enhanced with HF. Mean arterial blood pressure at 7 wk was as follows (mmHg): NF, 100 ± 2; NF plus l-NAME, 122 ± 3; and HF plus l-NAME, 131 ± 2. Overall, HF feeding increased the abundance of NKCC2. Inappropriately high sodium reabsorption in the thick ascending limb via NKCC2 may contribute to hypertension with insulin resistance.