Search

The Na +/H + exchanger isoform 3 (NHE3) facilitates Na + reabsorption and H + secretion by the kidneys. Despite stronger NHE3 abundance in the thick ascending limb (TAL) compared to the S1 and S2 segments of the proximal tubule, the role of NHE3 in the TAL is poorly understood. To investigate the role of NHE3 in the TAL, we generated and phenotyped TAL-specific NHE3 knockout mice (NHE3 TAL-KO). Compared to control mice, NHE3 TAL-KO mice did not show significant differences in body weight, blood pH or plasma Na +, K + or Cl - levels. Fluid intake trended to be higher and urine osmolality was significantly lower in NHE3 TAL-KO mice. Despite a similar GFR, NHE3 TAL-KO mice had a greater urinary K +/creatinine ratio. One proposed role of NHE3 relates to furosemide-induced urinary acidification. Acute bolus treatment with furosemide under anesthesia did not result in differences in the dose dependence of urinary flow rate, Cl - excretion or maximal urinary acidification between genotypes; however, in contrast to control mice, urinary pH returned immediately towards baseline levels in NHE3 TAL-KO mice. Chronic furosemide treatment reduced urine osmolality similarly in both genotypes but metabolic alkalosis, hypokalemia and calciuresis were absent in NHE3 TAL-KO mice. Compared to vehicle, chronic furosemide treatment in control mice resulted in greater NKCC2 and lower Npt2a abundances, effects that were absent in NHE3 TAL-KO mice. In summary, NHE3 in the TAL plays a role for the sustained acidification effect of furosemide. Consistent with this, long-term treatment with furosemide did not result in metabolic alkalosis in NHE3 TAL-KO mice.

Na(+)-glucose cotransporter-2 (SGLT2) inhibitors are the new mainstay of treatment for diabetes mellitus and cardiovascular diseases. Despite the remarkable benefits, the molecular mechanisms mediating the effects of SGLT2 inhibitors on water and electrolyte balance are incompletely understood. The goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function via affecting the renin-angiotensin-aldosterone system (RAAS) and Na(+) channels/transporters along the nephron in Dahl salt-sensitive rats, a model of salt-induced hypertension. Administration of dapagliflozin (Dapa) at 2 mg/kg/day via drinking water for 3 wk blunted the development of salt-induced hypertension as evidenced by lower blood pressure and a left shift of the pressure natriuresis curve. Urinary flow rate, glucose excretion, and Na(+)- and Cl(−)-to-creatinine ratios increased in Dapa-treated compared with vehicle-treated rats. To define the contribution of the RAAS, we measured various hormones. Despite apparent effects on Na(+)- and Cl(−)-to-creatinine ratios, Dapa treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of Dapa on renal Na(+) channels and transporters using RT-PCR, Western blot analysis, and patch clamp. Neither mRNA nor protein expression levels of renal transporters (SGLT2, Na(+)/H(+) exchanger isoform 3, Na(+)-K(+)-2Cl(−) cotransporter 2, Na(+)-Cl(−) cotransporter, and α-, β-, and γ-epithelial Na(+) channel subunits) changed significantly between groups. Furthermore, electrophysiological experiments did not reveal any difference in Dapa treatment on the conductance and activity of epithelial Na(+) channels. Our data suggest that SGLT2 inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the RAAS or the expression or activity of the main Na(+) channels and transporters. NEW & NOTEWORTHY The present study indicates that Na(+)-glucose cotransporter-2 (SGLT2) inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development and magnitude of salt-induced hypertension. Chronic inhibition of SGLT2 increases glucose and Na(+) excretion without secondary effects on the expression and function of other Na(+) transporters and channels along the nephron and hormone levels in the renin-angiotensin-aldosterone system. These data provide novel insights into the effects of SGLT2 inhibitors and their potential use in hypertension.

The sodium-hydrogen exchanger isoform 1 (NHE1) is a plasma membrane transporter with roles in transepithelial Na+/H+ transport, intracellular pH (pHi) and cell volume regulation. Within the kidney, NHE1 is previously documented to be expressed in all nephron segments. However, recent transcriptomic and proteomic analyses, and our staining results in mice, consistently find no expression of NHE1 in the proximal tubule or thick ascending limb, but in the distal nephron, including distal convoluted tubule, connecting tubule, and collecting duct (principal and intercalated cells, PC and IC, respectively). We hypothesized that basolateral NHE1 in IC (co-labelling with H+-ATPase B1 subunit, labeling all 3 types of IC: type A, B, and non-A-non-B) plays an important role in acid-base homeostasis. Due to lack of a suitable animal model, we generated kidney-specific NHE1 knockout (NHE1KS-KO) mice. NHE1KS-KO mice completely lack renal NHE1 protein expression compared to their control littermates (n=6 male mice/genotype). No differences were observed in body weight, food and water intake (determined in their home cages) or concentrations of blood Na+, K+, Ca2+, pH or HCO3-. Urine analysis showed no significant differences in electrolytes, minerals of pH. To mechanistically determine the role of NHE1 for pHi regulation in IC and PC we used confocal microscopy of acutely-split open cortical collecting ducts. This method allows for the analysis of a vast number of PC/IC simultaneously. To study the role of NHE1 we employed a NH4Cl pre-pulse (40 mM) and measured pHi recovery after switching back to a physiological solution. Intracellular pH recovery in PC (n=171) was significantly slower (~50%) in NHE1KS-KO mice compared to controls (n=85). Similar to PC, pHi recovery in type B IC of NHE1KS-KO mice (n=38) was significantly slower (~35%) compared to controls (n=40). No differences in type A IC were observed between NHE1KS-KO mice (n=35) and controls (n=29). Inhibition of ClC-K2 via NPPB (Cl-channel blocker) was used to confirm cell types: PC did not respond with a change in pHi but NPPB increased pHi in type A and decreased pHi in type B ICs. In summary, our results identify for the first time an important role of NHE1 for pHi regulation particularly in PCs and type B ICs. Funding to Dr. Rieg was provided by the NIDDK grant 1R01DK110621, VA Merit Review Award IBX004968A, AHA Transformational Research Award 19TPA34850116, and NIDDK Diabetic Complications Consortium grants DK076169 and DK115255. DK095029 and DK117865 (both to O. Pochynyuk). Dr. Thomas was supported by an AHA postdoctoral grant (828731), V. Tomilin by an AHA Career Development Award 19CDA34660148. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intravenous (IV) iron preparations like ferric carboxymaltose (FCM) and ferric derisomaltose (FDI) are used to treat iron deficiency anemia (IDA) due to heavy uterine bleeding, inflammatory bowel disease, and chronic kidney disease. These preparations greatly differ in terms of their side-effect profiles (e.g, hypophosphatemia). However, studies that directly compare FCM and FDI are limited. The objective of this study was to compare the effects of FCM and FDI in a mouse model of IDA and investigate changes in blood parameters and mineral homeostasis. Female C57Bl/6J mice were fed iron-deficient diet for 5 weeks followed by IV bleeding (0.7% of body weight [b.w.]) for 3 consecutive days. On day 0 (last day of bleeding), baseline blood and urine were analyzed for hematocrit, CBC, Pi and Ca2+. Mice were randomized to vehicle (saline, 2 μl/g b.w.; n=16), FCM (20 mg/kg; n=12), or FDI (20 mg/kg; n=12) treatment via retroorbital injection on day 0 and 7. On day 14, terminal blood, urine, spleen, and heart were collected. At baseline, compared with pre-bleeding, all mice had low hematocrit (22±1 vs 48±0.3%; P9/L; NS) or FDI (7±1 vs 7±1 x109/L; NS); however, WBC were decreased in vehicle-treated mice (4±1 vs 6±1 x109/L; P9/L; P9/L; NS), they were decreased in FCM-treated mice (583±41 vs 771±30 x109/L; Pi compared with vehicle (1.9±0.1 and 2.1±0.1 vs 2.4±0.1 mmol; Pi. Plasma Ca2+ was similar between groups on day 14; but compared with day 0, it was higher in vehicle (2.7±0.1 vs 2.5±0.1 mmol; P2+/creatinine was higher in mice treated with vehicle compared with day 0 (0.4±0.04 vs 0.1±0.01 mmol/mmol; P This work was supported by 1R01DK110621, VA Merit Award IBX004968A, NIDDK Diabetic Complications Consortium RRID:SCR_001415, DK076169 and DK115255 (all to TR). LT was supported by an AHA Postdoctoral Fellowship (828731). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

1,25(OH)2 vitamin D3 (Vit-D3) is a hormone that is formed by the coordinated actions of the skin and kidneys and is involved in the regulation of Ca2+ and phosphate (Pi) homeostasis. Of note, the regulation of these minerals is complex and involves other hormones including parathyroid hormone and fibroblast growth factor 23. Of major importance is the renal Na+/phosphate co-transporter (Npt2a) for long-term Pi homeostasis and Npt2a knockout (Npt2a-/-) mice are hypophosphatemic. However, the impact of Vit-D3 on Pi and Ca2+ in the absence of Npt2a has never been assessed. We hypothesized that exogenous application of Vit-D3 might modulate impaired Pi homeostasis by regulating their Pi transporters in the absence of Npt2a. Here we compared the effects of subcutaneous administration of Vit-D3 (300,000 IU/kg for 4 days, q.d.) or vehicle (5% Ethanol,5% Cremophore EL, and 90% water) in male wild-type (WT) and Npt2a-/- mice (n=6/genotype and treatment). Blood samples were collected before (baseline) and after Vit-D3 treatment. In WT mice, Vit-D3 or vehicle treatment did not change plasma Pi levels compared to baseline. In contrast, Vit-D3 significantly increased (~1.7-fold) plasma Pi levels in Npt2a-/- mice compared to baseline (1.5±0.09 vs. 0.9±0.04 mmol/L, P Funding to Dr. Rieg was provided by the NIDDK grant 1R01DK110621, VA Merit Review Award IBX004968A, AHA Transformational Research Award 19TPA34850116, and NIDDK Diabetic Complications Consortium grants DK076169 and DK115255. Dr. Thomas was supported by an AHA postdoctoral grant (828731). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeting sodium phosphate cotransporter 2a (Npt2a) offers a novel strategy for treating hyperphosphatemia in chronic kidney disease (CKD). Here we review recent studies on the efficacy of Npt2a inhibition, its plasma phosphate (Pi)-lowering effects, as well as potential "off-target" beneficial effects on cardiovascular consequences.Two novel Npt2a-selective inhibitors (PF-06869206 and BAY-767) have been developed. Pharmacological Npt2a inhibition shows a significant phosphaturic effect and consequently lowers plasma Pi and parathyroid hormone (PTH) levels regardless of CKD. However, plasma fibroblast growth factor 23 (FGF23), a master regulator of Pi homeostasis, shows inconsistent responses between these two inhibitors (no effect by PF-06869206 vs. reduction by BAY-767). In addition to the effects on Pi homeostasis, Npt2a inhibition also enhances urinary excretions of Na+, Cl-, and Ca2+, which is recapitulated in animal models with reduced kidney function. The effect of Npt2a inhibition by BAY-767 on vascular calcification has been studied, with positive results showing that oral treatment with BAY-767 (10 mg kg-1) attenuated the increases in plasma Pi and Ca2+ content in the aorta under the setting of vascular calcification induced by a pan-FGF receptor inhibitor. Together, Npt2a inhibition offers a promising therapeutic approach for treating hyperphosphatemia and reducing cardiovascular complications in CKD.Npt2a inhibition significantly increases urinary Pi excretion and lowers plasma Pi and PTH levels; moreover, it exerts pleiotropic "off-target" effects, providing a novel treatment for hyperphosphatemia and exhibiting beneficial potential for cardiovascular complications in CKD.

The present study indicates that Na+-glucose cotransporter-2 (SGLT2) inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development and magnitude of salt-induced hypertension. Chronic inhibition of SGLT2 increases glucose and Na+ excretion without secondary effects on the expression and function of other Na+ transporters and channels along the nephron and hormone levels in the renin-angiotensin-aldosterone system. These data provide novel insights into the effects of SGLT2 inhibitors and their potential use in hypertension.

The Na+/H+ exchanger 3 (NHE3) mediates Na+ (re)absorption in the intestine and the kidney, thereby playing a key role in fluid homeostasis and blood pressure (BP) regulation. NHE3 is expressed in the luminal membrane of the renal proximal tubule, where the majority of renal Na+ reabsorption (60% to 75%) occurs. Whole body NHE3 knockout mice exhibit hypovolemia, hypotension, diarrhea, metabolic acidosis and renal Na+ wasting. Study in kidney-specific (proximal tubule [PT] and thick ascending limb [TAL]) NHE3 knockout mice has reported a rather mild phenotype in terms of Na+ homeostasis and no phenotype for acid-base homeostasis (Kidney International; 2017, 92:397-414). NHE3 is also substantially expressed in the TAL and our data point to a novel role in this nephron segment for urinary concentrating ability. To determine the physiological role of NHE3 in the TAL for salt homeostasis, urinary concentrating ability and BP regulation, we generated a novel tamoxifen (Tam)-inducible TAL-specific NHE3 knockout (NHE3TAL-KO) mice by crossing floxed NHE3 (Control, Con) with UmodCreERT2 mice. Immunofluorescent staining showed that application of Tam (3 days, 67 mg/kg via oral gavage) selectively deleted NHE3 from TAL but not PT. Before Tam, in Con and NHE3TAL-KO mice had comparable food (4.9±0.1 vs 5.3±0.2 g/(day*30g)) and water intake (5.6±0.3 vs 5.9±0.4 g/(day*30g)), urinary osmolality (2435±107.4 vs 2220±111.9 mOsm/kg), urinary pH (6.02±0.06 vs 6.02±0.06), urinary Na+/creatinine (46.3±6.2 vs 52.0±5.2 mmol/mmol) and K+/creatinine (109.0±9.5 vs 97.2±9.2 mmol/mmol). Two weeks after Tam, compared to Con mice, NHE3TAL-KO mice showed lower urinary osmolality (2224±90.1 vs 1826±92.8 mOsm/kg, P

Plasma phosphate (Pi) levels are tightly controlled, and elevated plasma Pilevels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of Pireabsorption: Na+-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70–80% of Pireabsorption. The aim of the present study was to determine the in vitro effects of a novel Npt2a inhibitor (PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a−/−) mice. In OK cells, Npt2a inhibitor caused dose-dependent reductions of Na+-dependent Piuptake (IC50: ~1.4 μmol/L), whereas the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in an ~20% stronger inhibition of Piuptake. The dose-dependent inhibitory effects were present after 24 h of incubation with both low- and high-Pimedia. Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher Kmfor Piin response to Npt2a inhibition with no significant change in apparent Vmax. Higher parathyroid hormone concentrations decreased Piuptake equivalent to the maximal inhibitory effect of Npt2a inhibitor. In vivo, the Npt2a inhibitor induced a dose-dependent increase in urinary Piexcretion in wild-type mice (ED50: ~23 mg/kg), which was completely absent in Npt2a−/−mice, alongside a lack of decrease in plasma Pi. Of note, the Npt2a inhibitor-induced dose-dependent increase in urinary Na+excretion was still present in Npt2a−/−mice, a response possibly mediated by an off-target acute inhibitory effect of the Npt2a inhibitor on open probability of the epithelial Na+channel in the cortical collecting duct.

PURPOSE OF REVIEW: Sodium–glucose cotransporter 2 (SGLT2) inhibitors are antihyperglycemic drugs that act by inhibiting renal sodium–glucose cotransport. Here we present new insights into ‘off target’, or indirect, effects of SGLT2 inhibitors. RECENT FINDINGS: SGLT2 inhibition causes an acute increase in urinary glucose excretion. In addition to lowering blood glucose, there are several other effects that contribute to the overall beneficial renal and cardiovascular effects. Reabsorption of about 66% of sodium is accomplished in the proximal tubule and dependent on the sodium–hydrogen exchanger isoform 3 (NHE3). SGLT2 colocalizes with NHE3, and high glucose levels reduce NHE3 activity. The proximal tubule is also responsible for the majority of phosphate (P(i)) reabsorption. SGLT2 inhibition is associated with increases in plasma P(i), fibroblast growth factor 23 and parathyroid hormone levels in nondiabetics and type 2 diabetes mellitus. Studies in humans identified a urate-lowering effect by SGLT2 inhibition which is possibly mediated by urate transporter 1 (URAT1) and/or glucose transporter member 9 in the proximal tubule. Of note, magnesium levels were also found to increase under SGLT2 inhibition, an effect that was preserved in nondiabetic patients with hypomagnesemia. SUMMARY: Cardiorenal effects of SGLT2 inhibition might involve, in addition to direct effects on glucose homeostasis, effects on NHE3, phosphate, urate, and magnesium homeostasis.

The sodium–hydrogen exchanger isoform 3 (NHE3, SLC9A3) is abundantly expressed in the gastrointestinal tract and is proposed to play essential roles in Na+ and fluid absorption as well as acid–base homeostasis. Mutations in the SLC9A3 gene can cause congenital sodium diarrhea (CSD). However, understanding the precise role of intestinal NHE3 has been severely hampered due to the lack of a suitable animal model. To navigate this problem and better understand the role of intestinal NHE3, we generated a tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout mouse model (NHE3IEC-KO). Before tamoxifen administration, the phenotype and blood parameters of NHE3IEC-KO were unremarkable compared with control mice. After tamoxifen administration, NHE3IEC-KO mice have undetectable levels of NHE3 in the intestine. NHE3IEC-KO mice develop watery, alkaline diarrhea in combination with a swollen small intestine, cecum and colon. The persistent diarrhea results in higher fluid intake. After 3 weeks, NHE3IEC-KO mice show a ∼25% mortality rate. The contribution of intestinal NHE3 to acid–base and Na+ homeostasis under normal conditions becomes evident in NHE3IEC-KO mice that have metabolic acidosis, lower blood bicarbonate levels, hyponatremia and hyperkalemia associated with drastically elevated plasma aldosterone levels. These results demonstrate that intestinal NHE3 has a significant contribution to acid–base, Na+ and volume homeostasis, and lack of intestinal NHE3 has consequences on intestinal structural integrity. This mouse model mimics and explains the phenotype of individuals with CSD carrying SLC9A3 mutations.

AIMS: The kidneys play a major role in maintaining Pi homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal-specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout (NHE3IEC-KO ) mice.METHODS: Mice underwent dietary Pi challenges, and hormones as well as urinary/plasma Pi were determined. Intestinal 33 P uptake studies were conducted in vivo to compare the effects of tenapanor and NHE3IEC-KO . Ex vivo Pi transport was measured in everted gut sacs and brush border membrane vesicles. Intestinal and renal protein expression of Pi transporters were determined.RESULTS: On the control diet, NHE3IEC-KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice. Everted gut sacs and brush border membrane vesicles showed enhanced Pi uptake associated with increased Npt2b expression in NHE3IEC-KO mice. Acute oral Pi loading resulted in higher plasma Pi in NHE3IEC-KO mice. Tenapanor inhibited intestinal 33 P uptake acutely but then led to hyper-absorption at later time points compared to vehicle. In response to high dietary Pi , plasma Pi and FGF23 increased to higher levels in NHE3IEC-KO mice which was associated with greater Npt2b expression. Reduced renal Npt2c and a trend for reduced Npt2a expression were unable to correct for higher plasma Pi .CONCLUSION: Intestinal NHE3 has a significant contribution to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC-KO mice show enhanced, rather than reduced, intestinal Pi uptake.

Hyperphosphatemia results from an imbalance in phosphate (Pi) homeostasis. In patients with and without reduced kidney function, hyperphosphatemia is associated with cardiovascular complications. The current mainstays in the management of hyperphosphatemia are oral Pi binder and dietary Pi restriction. Although these options are employed in patients with chronic kidney disease (CKD), they seem inadequate to correct elevated plasma Pi levels. In addition, a paradoxical increase in expression of intestinal Pi transporter and uptake may occur. Recently, studies in rodents targeting the renal Na+/Pi cotransporter 2a (Npt2a), responsible for ∼70% of Pi reabsorption, have been proposed as a potential treatment option. Two compounds (PF-06869206 and BAY-767) have been developed which are selective for Npt2a. These Npt2a inhibitors significantly increased urinary Pi excretion consequently lowering plasma Pi and PTH levels. Additionally, increases in urinary excretions of Na+, Cl− and Ca2+ have been observed. Some of these results are also seen in models of reduced kidney function. Responses of FGF23, a phosphaturic hormone that has been linked to the development of left ventricular hypertrophy in CKD, are ambiguous. In this review, we discuss the recent advances on the role of Npt2a inhibition on Pi homeostasis as well as other pleiotropic effects observed with Npt2a inhibition.

Epithelial glucose transport is accomplished by Na+ -glucose co-transporters, SGLT1 and SGLT2. In the intestine, uptake of dietary glucose is for its majority mediated by SGLT1, and humans with mutations in the SGLT1 gene show glucose/galactose malabsorption. In the kidney, both transporters, SGLT1 and SGLT2, are expressed and recent studies identified that SGLT2 mediates up to 97% of glucose reabsorption. Humans with mutations in the SGLT2 gene show familial renal glucosuria. In the last three decades, significant progress was made in understanding the physiology of these transporters and their potential as therapeutic targets. Based on the structure of phlorizin, a natural compound acting as a SGLT1/2 inhibitor, initially several SGLT2, and later SGLT1 and dual SGLT1/2 inhibitors have been developed. Interestingly, SGLT2 knockout or treatment with SGLT2 selective inhibitors only causes a fractional glucose excretion in the magnitude of ∼60%, an effect mediated by up-regulation of renal SGLT1. Based on these findings the hypothesis was brought forward that dual SGLT1/2 inhibition might further improve glycaemic control via targeting two distinct organs that express SGLT1: the intestine and the kidney. Of note, SGLT1/2 double knockout mice completely lack renal glucose reabsorption. This review will address the rationale for the development of SGLT1 and dual SGLT1/2 inhibitors and potential benefits compared to sole SGLT2 inhibition.

Lithium is one of the mainstays for the treatment of bipolar disorder despite its side effects on the endocrine, neurological, and renal systems. Experimentally, lithium has been used as a measure to determine proximal tubule reabsorption based on the assumption that lithium and sodium transport go in parallel in the proximal tubule. However, the exact mechanism by which lithium is reabsorbed remains elusive. The majority of proximal tubule sodium reabsorption is directly or indirectly mediated by the sodium-hydrogen exchanger 3 (NHE3). In addition, sodium-phosphate cotransporters have been implicated in renal lithium reabsorption. In order to better understand the role of sodium-phosphate cotransporters involved in lithium (re)absorption, we studied lithium pharmacokinetics in: i) tubule-specific NHE3 knockout mice (NHE3loxloxPax8Cre), and ii) mice challenged with low or high phosphate diets. Intravenous or oral administration of lithium did not result in differences in lithium bioavailability, half-life, maximum plasma concentrations, area under the curve, lithium clearance, or urinary lithium/creatinine ratios between control and NHE3loxloxPax8Cre mice. After one week of dietary phosphate challenges, lithium bioavailability was ~30% lower on low versus high dietary phosphate, possibly the consequence of a smaller area under the curve after oral administration. This was associated with higher apparent lithium clearance after oral administration and lower urinary lithium/creatinine ratios on low versus high dietary phosphate. Collectively, renal NHE3 does not play a role in lithium pharmacokinetics; however, dietary phosphate could have an indirect effect on lithium bioavailability and lithium disposition.

BackgroundCholera toxin (CT)–induced diarrhea is mediated by cyclic adenosine monophosphate (cAMP)–mediated active Cl– secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). Although the constitutive activation of adenylyl cyclase (AC) in response to CT is due to adenosine diphosphate ribosylation of the small G protein α-subunit activating CFTR with consequent secretory diarrhea, the AC isoform(s) involved remain unknown.MethodsWe generated intestinal epithelial cell–specific adenylyl cyclase 6 (AC6) knockout mice to study its role in CT-induced diarrhea.ResultsAC6 messenger RNA levels were the highest of all 9 membrane-bound AC isoforms in mouse intestinal epithelial cells. Intestinal epithelial-specific AC6 knockout mice (AC6loxloxVillinCre) had undetectable AC6 levels in small intestinal and colonic epithelial cells. No significant differences in fluid and food intake, plasma electrolytes, intestinal/colon anatomy and morphology, or fecal water content were observed between genotypes. Nevertheless, CT-induced fluid accumulation in vivo was completely absent in AC6loxloxVillinCre mice, associated with a lack of forskolin- and CT-induced changes in the short-circuit current (ISC) of the intestinal mucosa, impaired cAMP generation in acutely isolated small intestinal epithelial cells, and significantly impaired apical CFTR levels in response to forskolin.ConclusionsAC6 is a novel target for the treatment of CT-induced diarrhea.

BACKGROUND: The kidneys play an important role in phosphate homeostasis. Patients with CKD develop hyperphosphatemia in the later stages of the disease. Currently, treatment options are limited to dietary phosphate restriction and oral phosphate binders. The sodium-phosphate cotransporter Npt2a, which mediates a large proportion of phosphate reabsorption in the kidney, might be a good therapeutic target for new medications for hyperphosphatemia.METHODS: The authors assessed the effects of the first orally bioavailable Npt2a inhibitor (Npt2a-I) PF-06869206 in normal mice and mice that had undergone subtotal nephrectomy (5/6 Nx), a mouse model of CKD. Dose-response relationships of sodium, chloride, potassium, phosphate, and calcium excretion were assessed in response to the Npt2a inhibitor in both groups of mice. Expression and localization of Npt2a/c and levels of plasma phosphate, calcium, parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) were studied up to 24-hours after Npt2a-I treatment.RESULTS: In normal mice, Npt2a inhibition caused a dose-dependent increase in urinary phosphate (ED50 approximately 21 mg/kg), calcium, sodium and chloride excretion. In contrast, urinary potassium excretion, flow rate and urinary pH were not affected dose dependently. Plasma phosphate and PTH significantly decreased after 3 hours, with both returning to near baseline levels after 24 hours. Similar effects were observed in the mouse model of CKD but were reduced in magnitude.CONCLUSIONS: Npt2a inhibition causes a dose-dependent increase in phosphate, sodium and chloride excretion associated with reductions in plasma phosphate and PTH levels in normal mice and in a CKD mouse model.

In general, the mammalian whole body mass-specific metabolic rate correlates positively with maximal urine concentration (Umax) irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass-specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na+-K+-ATPase activity and Na+-K+-ATPase α1-gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na+-K+-ATPase activity (~70%) compared with two rat strains (Sprague-Dawley and Munich-Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na+-K+-ATPase α1-protein (~4- to 6-fold) and mRNA (~13-fold) and higher TAL mitochondrial volume density (~20%) occur in the kangaroo rat compared with both rat strains. Rat TAL Na+-K+-ATPase α1-protein expression is relatively unaffected by body hydration status or, shown previously, by dietary Na+, arguing against confounding effects from two unavoidably dissimilar diets: grain-based diet without water (kangaroo rat) or grain-based diet with water (rat). We conclude that higher TAL Na+-K+-ATPase activity contributes to relationships between whole body mass-specific metabolic rate and high Umax. More vigorous TAL Na+-K+-ATPase activity in kangaroo rat than rat may contribute to its steeper Na+and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

BACKGROUND: The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood.METHODS: Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice.RESULTS: Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo.CONCLUSIONS: Aldosterone acutely activates NCC to modulate renal NaCl excretion.

Introduction: Hyperphosphatemia is common in patients with chronic kidney disease and associated with increased mortality. Oral phosphate binders and dietary phosphate restriction are the current management protocols for patients with hyperphosphatemia; however, their effectiveness is insufficient. In the kidney, the sodium-phosphate cotransporter Npt2a is responsible for bulk uptake of phosphate in the proximal tubule. Recently, an orally bioavailable selective Npt2a inhibitor (Npt2a-I, PF-06869206) has been described to reduce phosphate uptake in HEK cells transfected with mouse or rat Npt2a. So far, its physiological in vivo function has not been tested. Objective: To describe the in vivo effect of renal NPt2a inhibition in C57BL/6J mice Material and Methods: Based on in vitro IC 50 concentrations, we chose to study 30 mg/kg (oral gavage, 1% of body weight) in short-term (3 hours) metabolic cage experiments in C57BL/6J mice. Results: Compared to vehicle (n=14), bolus administration of Npt2a-I (n=12) caused significantly higher (~4-fold) urinary phosphate excretion (104±8 vs 27 ±6 µmol*min -1, P

Objective The human prohormone chromogranin A (CHGA), an index member of the granin family is processed to generate catestatin, a peptide that is hypotensive in action and modulates catecholamine release within the sympathoadrenal system. Hypertensive patients with excess sympathetic activity have diminished catestatin. Often the study of physiological consequences of human genetic variation is confounded by elements such as other variations in obligatory linkage disequilibrium with the variant being studied. Also the phenotype of the variant may be influenced by genetic background that varies amongst individuals. This study addresses the effects of a human catestatin polymorphism (rs9658667) using humanized CHGA mouse models. Methods We created pertinent humanized mouse models wherein the mouse Chga gene locus was replaced by the human ortholog wild-type and the variant versions. This allowed for probing of the effects of catestatin variation in vivo with controls for other variations and global genetic background. Results Both the wild-type and variant human catestatin expressing mouse models were normotensive. The variant catestatin mouse model recapitulated physiological influence of the polymorphism on autonomic traits. These mice had diminished catecholamine, attenuated stress response and increased baroreceptor slopes that would suggest reduced risk of developing hypertension. Elevated plasma glucose, a trait observed in humans was not observed in mice expressing the variant catestatin. Conclusion This functional genomics approach of creating humanized mouse models to study rs9658667 polymorphism recapitulated and validated many of the human trait associations. This approach can also be applied in the study of other human gene polymorphisms.

The small intestine is the major site for nutrient absorption, which is critical in maintenance of euglycemia. Leptin, a key hormone involved in energy homeostasis, directly affects nutrient transport across the intestinal epithelium. Catestatin (CST), a 21-amino acid peptide derived from proprotein chromogranin A, has been shown to modulate leptin signaling. Therefore, we reasoned that leptin and CST could modulate intestinal Na+-glucose transporter 1 (SGLT1) expression in the context of obesity and diabetes. We found that hyperleptinemic db/db mice exhibit increased mucosal mass, associated with an enhanced proliferative response and decreased apoptosis in intestinal crypts, a finding absent in leptin deficient ob/ob mice. Intestinal SGLT1 abundance was significantly decreased in hyperleptinemic, but not leptin-deficient mice, indicating leptin regulation of SGLT1 expression. Phlorizin, a SGLT1/2 inhibitor, was without effect in an oral glucose tolerance test in db/db mice. The alterations in architecture and SGLT1 abundance were not accompanied by changes in the localization of intestinal alkaline phosphatase, indicating intact differentiation. Treatment of db/db mice with CST restored intestinal SGLT1 abundance and intestinal turnover, suggesting a cross-talk between leptin and CST, without affecting plasma leptin levels. Consistent with this hypothesis, we identified structural homology between CST and the AB-loop of leptin and protein-protein docking revealed binding of CST and leptin with the Ig-like binding site III of the leptin receptor. In summary, downregulation of SGLT1 in an obese type 2 diabetic mouse model with hyperleptinemia is presumably mediated via the short form of the leptin receptor and reduces overt hyperglycemia.

Adenylyl cyclase (AC) isoform 6 (AC6) is highly expressed throughout the renal tubule and collecting duct (CD), catalyzes the synthesis of cAMP and contributes to various aspects of renal transport. Several proteins involved in acid–base homeostasis are regulated by cAMP. In the present study, we assess the relative contribution of AC6 to overall acid–base regulation using mice with global deletion of AC6 (AC6(−/−)) or newly generated mice lacking AC6 in the renal tubule and CD (AC6(loxloxPax8Cre)). Higher energy expenditure in AC6(−/−) relative to wild-type (WT) mice, was associated with lower urinary pH, mild alkalosis in conjunction with elevated blood HCO(3)(−) concentrations, and significantly higher renal abundance of the H(+)-ATPase B1 subunit. In contrast with WT mice, AC6(−/−) mice have a less pronounced increase in urinary pH after 8 days of HCO(3)(−) challenge, which is associated with increased blood pH and HCO(3)(−) concentrations. Immunohistochemistry demonstrated that AC6 was expressed in intercalated cells (IC), but subcellular distribution of the H(+)-ATPase B1 subunit, pendrin, and the anion exchangers 1 and 2 in AC6(−/−) mice was normal. In the AC6(−/−) mice, H(+)-ATPase B1 subunit levels after HCO(3)(−) challenge were greater, which correlated with a higher number of type A IC. In contrast with the AC6(−/−) mice, AC6(loxloxPax8Cre) mice had normal urinary pH under baseline conditions but higher blood HCO(3)(−) than controls after HCO(3)(−) challenge. In conclusion, AC6 is required for maintaining normal acid–base homeostasis and energy expenditure. Under baseline conditions, renal AC6 is redundant for acid–base balance but becomes important under alkaline conditions.

Sodium–glucose cotransporters SGLT1 (encoded by SGLT1, also known as SLC5A1) and SGLT2 (encoded by SGLT2, also known as SLC5A2) are important mediators of epithelial glucose transport. While SGLT1 accounts for most of the dietary glucose uptake in the intestine, SGLT2 is responsible for the majority of glucose reuptake in the tubular system of the kidney, with SGLT1 reabsorbing the remainder of the filtered glucose. As a consequence, mutations in the SLC5A1 gene cause glucose/galactose malabsorption, whereas mutations in SLC5A2 are associated with glucosuria. Since the cloning of SGLT1 more than 30 years ago, big strides have been made in our understanding of these transporters and their suitability as drug targets. Phlorizin, a naturally occurring competitive inhibitor of SGLT1 and SGLT2, provided the first insights into potential efficacy, but its use was hampered by intestinal side effects and a short half-life. Nevertheless, it was a starting point for the development of specific inhibitors of SGLT1 and SGLT2, as well as dual SGLT1/2 inhibitors. Since the approval of the first SGLT2 inhibitor in 2013 by the US Food and Drug Administration, SGLT2 inhibitors have become a new mainstay in the treatment of type 2 diabetes mellitus. They also have beneficial effects on the cardiovascular system (including heart failure) and the kidney. This review focuses on the rationale for the development of individual SGLT2 and SGLT1 inhibitors, as well as dual SGLT1/2 inhibition, including, but not limited to, aspects of genetics, genetically modified mouse models, mathematical modelling and general considerations of drug discovery in the field of metabolism.

Caffeine is one of the most widely consumed behavioral substances. We have previously shown that caffeine- and theophylline-induced inhibition of renal reabsorption causes diuresis and natriuresis, an effect that requires functional adenosine A1receptors. In this study, we tested the hypothesis that blocking the Giprotein-coupled adenosine A1receptor via the nonselective adenosine receptor antagonist caffeine changes Na+/H+exchanger isoform 3 (NHE3) localization and phosphorylation, resulting in diuresis and natriuresis. We generated tubulus-specific NHE3 knockout mice (Pax8-Cre), where NHE3 abundance in the S1, S2, and S3 segments of the proximal tubule was completely absent or severely reduced (>85%) in the thick ascending limb. Consumption of fluid and food, as well as glomerular filtration rate, were comparable in control or tubulus-specific NHE3 knockout mice under basal conditions, while urinary pH was significantly more alkaline without evidence for metabolic acidosis. Caffeine self-administration increased total fluid and food intake comparably between genotypes, without significant differences in consumption of caffeinated solution. Acute caffeine application via oral gavage elicited a diuresis and natriuresis that was comparable between control and tubulus-specific NHE3 knockout mice. The diuretic and natriuretic response was independent of changes in total NHE3 expression, phosphorylation of serine-552 and serine-605, or apical plasma membrane NHE3 localization. Although caffeine had no clear effect on localization of the basolateral Na+/bicarbonate cotransporter NBCe1, pretreatment with DIDS inhibited caffeine-induced diuresis and natriuresis. In summary, NHE3 is not required for caffeine-induced diuresis and natriuresis.

Vascular tone can be regulated via nucleotides like ATP and UTP that are derived from erythrocytes or platelets or released from the endothelium (Ralevic & Burnstock 2003). It is known that ATP is an agonist of P2Y2 receptors and UTP is an agonist of P2Y2/4 receptors (Abbracchio et al. 2006). A decrease in blood pressure most likely results from ATP inducing endothelium-dependent relaxation (von Kugelgen et al. 1987, von Kugelgen & Starke 1990, Ralevic & Burnstock 1991a, 2003). Evidence suggests that this vasodilatory response can be mediated by nitric oxide (NO; Ralevic & Burnstock 1991a), endothelial-derived hyperpolarization (EDH; Malmsjo et al. 1999, 2002, Wihlborg et al. 2003) and/or prostacyclin (Hammer et al. 2003, Wihlborg et al. 2003). P2Y2 receptors are found on the endothelium and it was proposed that their activation stimulates the synthesis and release of NO (Ralevic & Burnstock 1991b, Buvinic et al. 2002, Burnstock 2009). Moreover, aortic rings from P2Y2 receptor knockout mice (P2Y2−/−) exhibit impaired vasorelaxation in response to ATP, which suggests that NO release is subsequent to P2Y2 receptor activation (Guns et al. 2005, 2006). Of note, mechanical destruction of the endothelium abolishes the ATP induced vasodilatory effect and produces a direct vasoconstrictory response on vascular smooth muscle cells (Kennedy & Burnstock 1985, Kennedy et al. 1985, Ralevic & Burnstock 1996b). Activation of P2Y4 receptors is associated with vasoconstriction (Dietrich et al. 1996, McMillan et al. 1999, Rubino et al. 1999). In contrast to ATP causing vasodilation, UTP was found to vasoconstrict mouse aortic rings (Boarder & Hourani 1998, Kauffenstein et al. 2010) and rabbit inner ear arteries (von Kugelgen et al. 1987). However, depending on route of administration, species, localization within the vascular tree or vessel type, UTP can cause vasoconstriction, vasodilation or both (Ralevic & Burnstock 1996a,b, Janigro et al. 1997, Horiuchi et al. 2001, Guns et al. 2005, Rayment et al. 2007, Inscho 2009). In recent studies, we demonstrated that P2Y2 receptors play a physiological role in blood pressure regulation and, as a consequence, P2Y2−/− mice were found to have salt-resistant hypertension (Rieg et al. 2007a, Pochynyuk et al. 2010). Direct intra-arterial blood pressure measurements indicated that the blood pressure responses to a P2Y2/4 agonist result in vasodilation via a NO-independent mechanism that possibly involves EDH release subsequently to P2Y2 receptor activation. This was concluded because endothelial NO synthase knockout mice (eNOS−/−) showed an identical blood pressure effect in response to P2Y2/4 receptor activation compared to WT mice. In contrast to WT mice, P2Y2−/− mice responded to P2Y2/4 receptor activation with an increase in blood pressure, possibly a direct effect of P2Y4 receptor activation on vascular smooth muscle cells, which is independent of P2Y2 receptors (Rieg et al. 2011). The vasodilation mediated by EDH requires activation of calcium-activated potassium channels, including KCa2.3 (small-conductance) and KCa3.1 (intermediate-conductance), which are expressed in most endothelial cells (Kohler & Ruth 2010). In contrast, KCa1.1 (big-conductance) is expressed in vascular smooth muscle cells (Feletou 2009, Kohler & Ruth 2010). The activation of calcium-activated potassium channels is speculated to produce hyperpolarization of the endothelium, which is then transmitted (possibly via connexins, see below) to underlying vascular smooth muscle cells causing vasodilation via EDH. Functional studies employing blockers of calcium-activated potassium channels have demonstrated the role of these channels in EDH and subsequent vascular smooth muscle relaxation (Adeagbo & Triggle 1993, Holzmann et al. 1994, Waldron & Garland 1994, Zygmunt & Hogestatt 1996, Eichler et al. 2003). Vascular smooth muscle cells and endothelial cells are functionally linked, and the point of contact between the two cells, the myoendothelial gap junction (MEGJ), plays a key role in the regulation of vascular function (Figueroa & Duling 2009). Initially, it was assumed that a diffusible endothelial factor was the mechanism resulting in hyperpolarization; however, this view was later questioned by experiments demonstrating the involvement of the MEGJ (Griffith et al. 2002, Dora et al. 2003, Chaytor et al. 2005, Mather et al. 2005, Sokoya et al. 2006). It was concluded from these studies that EDH is transferred from the endothelium to the smooth muscle by direct charge transfer through the MEGJ (de Wit & Wolfle 2007, Feletou & Vanhoutte 2009, Grgic et al. 2009, Edwards et al. 2010, de Wit & Griffith 2010, Garland et al. 2011). Gap junction proteins found in the vasculature include as follows: Cx37, Cx40, Cx43 and Cx45 (Figueroa et al. 2004, Lohman et al. 2012); however, the MEGJ is specifically comprised of Cx37 and Cx40 which are speculated to conduct the EDH response as an electrical signal between endothelial and vascular smooth muscle cells (Chaytor et al. 2005, Isakson & Duling 2005, Haddock et al. 2006). To further define the underlying mechanism(s) involved in the P2Y2 receptor-initiated blood pressure responses, possibly via EDH, we studied blood pressure and heart rate in WT, Cx37, Cx40, KCa1.1 and KCa3.1 knockout (−/−) mice in response to systemic application of a P2Y2/4 receptor agonist. We report that P2Y2/4 receptor activation generates a similar biphasic blood pressure response in all mice except for KCa3.1−/− and Cx37−/− mice, which show impaired vascular reactivity. This implies that both KCa3.1 and Cx37 are required for full vascular reactivity in response to P2Y2/4 receptor activation.

The sodium/proton exchanger isoform 3 (NHE3) is expressed in the intestine and the kidney, where it facilitates sodium (re)absorption and proton secretion. The importance of NHE3 in the kidney for sodium chloride homeostasis, relative to the intestine, is unknown. Constitutive tubule-specific NHE3 knockout mice (NHE3(loxloxCre)) did not show significant differences compared to control mice in body weight, blood pH or bicarbonate and plasma sodium, potassium, or aldosterone levels. Fluid intake, urinary flow rate, urinary sodium/creatinine, and pH were significantly elevated in NHE3(loxloxCre) mice, while urine osmolality and GFR were significantly lower. Water deprivation revealed a small urinary concentrating defect in NHE3(loxloxCre) mice on a control diet, exaggerated on low sodium chloride. Ten days of low or high sodium chloride diet did not affect plasma sodium in control mice; however, NHE3(loxloxCre) mice were susceptible to low sodium chloride (about -4 mM) or high sodium chloride intake (about +2 mM) versus baseline, effects without differences in plasma aldosterone between groups. Blood pressure was significantly lower in NHE3(loxloxCre) mice and was sodium chloride sensitive. In control mice, the expression of the sodium/phosphate co-transporter Npt2c was sodium chloride sensitive. However, lack of tubular NHE3 blunted Npt2c expression. Alterations in the abundances of sodium/chloride cotransporter and its phosphorylation at threonine 58 as well as the abundances of the α-subunit of the epithelial sodium channel, and its cleaved form, were also apparent in NHE3(loxloxCre) mice. Thus, renal NHE3 is required to maintain blood pressure and steady-state plasma sodium levels when dietary sodium chloride intake is modified.

Psychiatric patients treated with lithium (Li(+)) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li(+)-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li(+) inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6(-/-)) mice and potentially novel connecting tubule/principal cell-specific AC6 knockout (AC6(loxloxCre)) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li(+) administration increased water intake and reduced urine osmolality in control, AC6(-/-), and AC6(loxloxCre) mice. Consistent with AC6(-/-) mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6(loxloxCre) mice compared with controls under standard conditions, and levels were further reduced after Li(+) administration. AC6(loxloxCre) and control mice had a similar increase in the numbers of proliferating cell nuclear antigen-positive cells in response to Li(+). However, AC6(loxloxCre) mice had a higher number of H(+)-ATPase B1 subunit-positive cells under standard conditions and after Li(+) administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell-to-principal cell ratio.

Adenylyl cyclases (AC) catalyze formation of cAMP, a critical component of G protein-coupled receptor signaling. So far, nine distinct membrane-bound AC isoforms (AC1-9) and one soluble AC (sAC) have been identified and, except for AC8, all of them are expressed in the kidney. While the role of ACs in renal cAMP formation is well established, we are just beginning to understand the function of individual AC isoforms, particularly with regard to hormonal regulation of transporter and channel phosphorylation, membrane abundance, and trafficking. This review focuses on the role of different AC isoforms in regulating renal water and electrolyte transport in health as well as potential pathological implications of disordered AC isoform function. In particular, we focus on modulation of transporter and channel abundance, activity, and phosphorylation, with an emphasis on studies employing genetically modified animals. As will be described, it is now evident that specific AC isoforms can exert unique effects in the kidney that may have important implications in our understanding of normal physiology as well as disease pathogenesis.

In the kidney, the sodium-glucose cotransporters SGLT2 and SGLT1 are thought to account for >90 and ∼3% of fractional glucose reabsorption (FGR), respectively. However, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40–50%, mimicking values in Sglt2 knockout mice. Here, we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1−/−) mice. Treatment in diet with the SGLT2-I for 3 wk maintained 1.5- to 2-fold higher urine glucose/creatinine ratios in Sglt1−/− vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1−/− vs. WT after 24 h (−33 vs. −11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64 ± 2% in WT and 17 ± 2% in Sglt1−/−. Additional intraperitoneal application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ∼10-fold and reduced FGR to 44 ± 3% in WT and to −1 ± 3% in Sglt1−/−. The absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97 and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by SGLT2-I, the increase in SGLT1-mediated glucose reabsorption explains why only 50–60% of filtered glucose is excreted.

Our previous work has shown that gene knockout of the sodium-glucose cotransporter SGLT2 modestly lowered blood glucose in streptozotocin-diabetic mice (BG; from 470 to 300 mg/dl) and prevented glomerular hyperfiltration but did not attenuate albuminuria or renal growth and inflammation. Here we determined effects of the SGLT2 inhibitor empagliflozin (300 mg/kg of diet for 15 wk; corresponding to 60–80 mg·kg−1·day−1) in type 1 diabetic Akita mice that, opposite to streptozotocin-diabetes, upregulate renal SGLT2 expression. Akita diabetes, empagliflozin, and Akita + empagliflozin similarly increased renal membrane SGLT2 expression (by 38–56%) and reduced the expression of SGLT1 (by 33–37%) vs. vehicle-treated wild-type controls (WT). The diabetes-induced changes in SGLT2/SGLT1 protein expression are expected to enhance the BG-lowering potential of SGLT2 inhibition, and empagliflozin strongly lowered BG in Akita (means of 187–237 vs. 517–535 mg/dl in vehicle group; 100–140 mg/dl in WT). Empagliflozin modestly reduced GFR in WT (250 vs. 306 μl/min) and completely prevented the diabetes-induced increase in glomerular filtration rate (GFR) (255 vs. 397 μl/min). Empagliflozin attenuated increases in kidney weight and urinary albumin/creatinine ratio in Akita in proportion to hyperglycemia. Empagliflozin did not increase urinary glucose/creatinine ratios in Akita, indicating the reduction in filtered glucose balanced the inhibition of glucose reabsorption. Empagliflozin attenuated/prevented the increase in systolic blood pressure, glomerular size, and molecular markers of kidney growth, inflammation, and gluconeogenesis in Akita. We propose that SGLT2 inhibition can lower GFR independent of reducing BG (consistent with the tubular hypothesis of diabetic glomerular hyperfiltration), while attenuation of albuminuria, kidney growth, and inflammation in the early diabetic kidney may mostly be secondary to lower BG.