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HIV disease remains prevalent in the United States and is particularly prevalent in sub-Saharan Africa. Recent investigations revealed that mitochondrial dysfunction in kidney contributes to HIV-associated nephropathy (HIVAN) in Tg26 transgenic mice. We hypothesized that nicotinamide adenine dinucleotide (NAD) deficiency contributes to energetic dysfunction and progressive tubular injury. We investigated metabolomic mechanisms of HIVAN tubulopathy. Tg26 and wild-type (WT) mice were treated with the farnesoid X receptor (FXR) agonist INT-747 or nicotinamide riboside (NR) from 6 to 12 wk of age. Multiomic approaches were used to characterize kidney tissue transcriptomes and metabolomes. Treatment with INT-747 or NR ameliorated kidney tubular injury, as shown by serum creatinine, the tubular injury marker urinary neutrophil-associated lipocalin, and tubular morphometry. Integrated analysis of metabolomic and transcriptomic measurements showed that NAD levels and production were globally downregulated in Tg26 mouse kidneys, especially nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway. Furthermore, NAD-dependent deacetylase sirtuin3 activity and mitochondrial oxidative phosphorylation activity were lower in ex vivo proximal tubules from Tg26 mouse kidneys compared with those of WT mice. Restoration of NAD levels in the kidney improved these abnormalities. These data suggest that NAD deficiency might be a treatable target for HIVAN. NEW & NOTEWORTHY: The study describes a novel investigation that identified nicotinamide adenine dinucleotide (NAD) deficiency in a widely used HIV-associated nephropathy (HIVAN) transgenic mouse model. We show that INT-747, a farnesoid X receptor agonist, and nicotinamide riboside (NR), a precursor of nicotinamide, each ameliorated HIVAN tubulopathy. Multiomic analysis of mouse kidneys revealed that NAD deficiency was an upstream metabolomic mechanism contributing to HIVAN tubulopathy. [ABSTRACT FROM AUTHOR]

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D. NEW & NOTEWORTHY: Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature. [ABSTRACT FROM AUTHOR]

Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis. NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent renal inflammation and chronic kidney disease. [ABSTRACT FROM AUTHOR]

Type 1 Natural Killer T-cells (NKT1 cells) play a critical role in mediating hepatic ischemia-reperfusion injury (IRI). Although hepatic steatosis is a major risk factor for preservation type injury, how NKT cells impact this is understudied. Given NKT1 cell activation by phospholipid ligands recognized presented by CD1d, we hypothesized that NKT1 cells are key modulators of hepatic IRI because of the increased frequency of activating ligands in the setting of hepatic steatosis. We first demonstrate that IRI is exacerbated by a high-fat diet (HFD) in experimental murine models of warm partial ischemia. This is evident in the evaluation of ALT levels and Phasor-Fluorescence Lifetime (Phasor-FLIM) Imaging for glycolytic stress. Polychromatic flow cytometry identified pronounced increases in CD45+CD3+NK1.1+NKT1 cells in HFD fed mice when compared to mice fed a normal diet (ND). This observation is further extended to IRI, measuring ex vivo cytokine expression in the HFD and ND. Much higher interferon-gamma (IFN-g) expression is noted in the HFD mice after IRI. We further tested our hypothesis by performing a lipidomic analysis of hepatic tissue and compared this to Phasor-FLIM imaging using "long lifetime species", a byproduct of lipid oxidation. There are higher levels of triacylglycerols and phospholipids in HFD mice. Since N-acetylcysteine (NAC) is able to limit hepatic steatosis, we tested how oral NAC supplementation in HFD mice impacted IRI. Interestingly, oral NAC supplementation in HFD mice results in improved hepatic enhancement using contrast-enhanced magnetic resonance imaging (MRI) compared to HFD control mice and normalization of glycolysis demonstrated by Phasor-FLIM imaging. This correlated with improved biochemical serum levels and a decrease in IFN-g expression at a tissue level and from CD45+CD3+CD1d+ cells. Lipidomic evaluation of tissue in the HFD+NAC mice demonstrated a drastic decrease in triacylglycerol, suggesting downregulation of the PPAR-γ pathway. [ABSTRACT FROM AUTHOR]

Sodium glucose cotransporter, type 2 inhibitors, such as Empagliflozin, are protective of the kidneys by unclear mechanisms. Our aim was to determine how Empagliflozin affected kidney cortical metabolome and lipidome in mice. Adult male TALLYHO mice (prone to obesity) were treated with a high-milk-fat diet, or this diet containing Empagliflozin (0.01%), for 8 weeks. Targeted and untargeted metabolomics and lipidomics were conducted on kidney cortex by liquid chromatography followed by tandem mass-spectroscopy. Metabolites were statistically analyzed by MetaboAnalyst 5.0, LipidSig (lipid species only) and/or CEU Mass Mediator (untargeted annotation). In general, volcano plotting revealed oppositely skewed patterns for targeted metabolites (primarily hydrophilic) and lipids (hydrophobic) in that polar metabolites showed a larger number of decreased species, while non-polar (lipids) had a greater number of increased species (>20% changed and/or raw p-value < 0.05). The top three pathways regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism, with an amino acid network being highly affected, with 14 of 20 classic amino acids down-regulated. Out of 75 changed polar metabolites, only three were up-regulated, i.e., flavin mononucleotide (FMN), uridine, and ureidosuccinic acid. Both FMN and uridine have been shown to be protective of the kidney. Scrutiny of metabolites of glycolysis/gluconeogenesis/Krebs cycle revealed a 20–45% reduction in several species, including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, although overall lipid quantity was not higher, several lipid species were increased by EMPA, including those of the classes, phosphatidic acids, phosphatidylcholines, and carnitines. Overall, these analyses suggest a protection from extensive metabolic load and the corresponding oxidative stress with EMPA in kidney. This may be in response to reduced energy demands of the proximal tubule as a result of inhibition of transport and/or differences in metabolic pools available for metabolism. [ABSTRACT FROM AUTHOR]

Serum phosphate levels are acutely impacted by the abundance of sodium-phosphate cotransporter IIa (NaPiIIa) in the apical membrane of renal proximal tubule cells. PSD-95/Disks Large/Zonula Occludens (PDZ) domain-containing proteins bind NaPiIIa and likely contribute to the delivery, retention, recovery, and trafficking of NaPiIIa. Shank2 is a distinctive PDZ domain protein that binds NaPiIIa. Its role in regulating NaPiIIa activity, distribution, and abundance is unknown. In the present in vivo study, rats were maintained on a low-phosphate diet, and then plasma phosphate levels were acutely elevated by high-phosphate feeding to induce the recovery, endocytosis, and degradation of NaPiIIa. Western blot analysis of renal cortical tissue from rats given high-phosphate feed showed NaPiIIa and Shank2 underwent degradation. Quantitative immunofluorescence analyses, including microvillar versus intracellular intensity ratios and intensity correlation quotients, showed that Shank2 redistributed with NaPiIIa during the time course of NaPiIIa endocytosis. Furthermore, NaPiIIa and Shank2 trafficked through distinct endosomal compartments (clathrin, early endosomes, lysosomes) with the same temporal pattern. These in vivo findings indicate that Shank2 is positioned to coordinate the regulated endocytic retrieval and downregulation of NaPiIIa in rat renal proximal tubule cells. PDZ domains; proximal tubule cells; microvilli; clathrin; endosomes doi: 10.1152/ajpcell.00183.2010.

Wang XX, Jiang T, Shen Y, Adorini L, Pruzanski M, Gonzalez FJ, Scherzer P, Lewis L, Miyazaki-Anzai S, Levi M. The farnesoid X receptor modulates renal lipid metabolism and diet-induced renal inflammation, fibrosis, and proteinuria. Am J Physiol Renal Physiol 297: F1587-F1596, 2009. First published September 23, 2009; doi:l0.1152/ajprenal.00404.2009.--Diet-induced obesity is associated with proteinuria and glomerular disease in humans and rodents. We have shown that in mice fed a high-fat diet, increased renal expression of the transcriptional factor sterol-regulatory element binding protein-1 (SREBP-1) plays a critical role in renal lipid accumulation and increases the activity of proinflammatory cytokines and profibrotic growth factors. In the current study, we have determined a key role of the farnesoid X receptor (FXR) in modulating renal SREBP-1 activity, glomerular lesions, and proteinuria. We found that feeding a Western-style diet to DBA/2J mice results in proteinuria, podocyte loss, mesangial expansion, renal lipid accumulation, and increased expression of proinflammatory factors, oxidative stress, and profibrotic growth factors. Treatment of these mice with the highly selective and potent FXR-activating ligand 6-[alpha]-ethyl-chenodeoxycholic acid (INT-747) ameliorates triglyceride accumulation by modulating fatty acid synthesis and oxidation, improves proteinuria, prevents podocyte loss, mesangial expansion, accumulation of extracellular matrix proteins, and increased expression of profibrotic growth factors and fibrosis markers, and modulates inflammation and oxidative stress. Our results therefore indicate that FXR activation could represent an effective therapy for treatment of abnormal renal lipid metabolism with associated inflammation, oxidative stress, and kidney pathology in patients affected by obesity. nuclear receptor; diet-induced obesity; obesity-related renal disease doi: 10.1152/ajprenal.00404.2009

Blaine J, Okamura K, Giral H, Breusegem S, Caldas Y, Millard A, Barry N, Levi M. PTH-induced internalization of apical membrane NaPi2a: role of actin and myosin VI. Am J Physiol Cell Physiol 297: C1339-C1346, 2009. First published September 23, 2009; doi: 10.1152/ajpcell.00260.2009--Parathyroid hormone (PTH) plays a critical role in the regulation of renal phosphorous homeostasis by altering the levels of the sodium-phosphate cotransporter NaPi2a in the brush border membrane (BBM) of renal proximal tubular cells. While details of the molecular events of PTH-induced internalization of NaPi2a are emerging, the precise events governing NaPi2a removal from brush border microvilli in response to PTH remain to be fully determined. Here we use a novel application of total internal reflection fluorescence microscopy to examine how PTH induces movement of NaPi2a out of brush border microvilli in living cells in real time. We show that a dynamic actin cytoskeleton is required for NaPi2a removal from the BBM in response to PTH. In addition, we demonstrate that a myosin motor that has previously been shown to be coregulated with NaPi2a, myosin VI, is necessary for PTH-induced removal of NaPi2a from BBM microvilli. apical total internal reflection fluorescence microscopy; parathyroid hormone doi: 10.1152/ajpcell.00260.2009.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate. A m J Physiol Renal Physiol 297: F1466-F1475, 2009. First published August 12, 2009: doi:10.1152/ajprenal.00279.2009.--Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal [P.sub.i] absorption is one of the pharmacological targets that prevents it. The type II Na-[P.sub.i] cotransporter NaPi-2b is the major transporter that mediates [P.sub.i] reabsorption in the intestine. The potential role and regulation of other Na-[P.sub.i] transporters remain unknown. We have identified expression of the type III Na-[P.sub.i] cotransporter PIT-1 in the apical membrane of enterocytes. Na-[P.sub.i] transport activity and NaPi-2b and PIT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-[P.sub.i] diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-[P.sub.i] transport activity and NaPi-2b, but not PiT-l, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-[P.sub.i] diet, there is an increase in BBM Na-[P.sub.i] transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum [P.sub.i] that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-[P.sub.i] transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia. SLC34A2; PiT-1; hyperphosphatemia; chronic kidney disease; dietary [P.sub.i] doi: 10.1152/ajprenal.00279.2009.

Dietary potassium (K) deficiency is accompanied by phosphaturia and decreased renal brush border membrane (BBM) vesicle sodium (Na)-dependent phosphate ([P.sub.i]) transport activity. Our laboratory previously showed that K deficiency in rats leads to increased abundance in the proximal tubule BBM of the apical Na-[P.sub.i] cotransporter NaPi-IIa, but that the activity, diffusion, and clustering of NaPi-IIa could be modulated by the altered lipid composition of the K-deficient BBM (Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, Levi M. Kidney Int 60: 694-704, 2001; Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, Levi M. J Biol Chem 279: 49160-49171, 2004). Here we investigated the role of the renal Na-[P.sub.i] cotransporters NaPi-IIc and PIT-2 in K deficiency. Using Western blotting, immunofluorescence, and quantitative real-time PCR, we found that, in rats and in mice, K deficiency is associated with a dramatic decrease in the NaPi-IIc protein abundance in proximal tubular BBM and in NaPi-IIc mRNA. In addition, we documented the presence of a third Na-coupled [P.sub.i] transporter in the renal BBM, PiT-2, whose abundance is also decreased by dietary K deficiency in rats and in mice. Finally, electron microscopy showed subcellular redistribution of NaPi-IIc in K deficiency: in control rats, NaPi-IIc immunolabel was primarily in BBM microvilli, whereas, in K-deficient rats, NaPiIIc BBM label was reduced, and immunolabel was prevalent in cytoplasmic vesicles. In summary, our results demonstrate that decreases in BBM abundance of the phosphate transporter NaPi-IIc and also PIT-2 might contribute to the phosphaturia of dietary K deficiency, and that the three renal BBM phosphate transporters characterized so far can be differentially regulated by dietary perturbations. hypokalemia; phosphaturia; SLC34A1; SLC34A3; SLC20A2

The principal mediators of renal phosphate ([P.sub.i]) reabsorption are the SLC34 family proteins NaPi-IIa and NaPi-IIc, localized to the proximal tubule (PT) apical membrane. Their abundance is regulated by circulatory factors and dietary [P.sub.i]. Although their physiological importance has been confirmed in knockout animal studies, significant [P.sub.i] reabsorptive capacity remains, which suggests the involvement of other secondary-active [P.sub.i] transporters along the nephron. Here we show that a member of the SLC20 gene family (PIT-2) is localized to the brush-border membrane (BBM) of the PT epithelia and that its abundance, confirmed by Western blot and immunohistochemistry of rat kidney slices, is regulated by dietary [P.sub.i]. In rats treated chronically on a high-[P.sub.i] (1.2%) diet, there was a marked decrease in the apparent abundance of PIT-2 protein in kidney slices compared with those from rats kept on a chronic low-[P.sub.i] (0.1%) diet. In Western blots of BBM from rats that were switched from a chronic low- to high-[P.sub.i] diet, NaPi-IIa showed rapid downregulation after 2 h; PIT-2 was also significantly downregulated at 24 h and NaPi-IIc after 48 h. For the converse dietary regime, NaPi-IIa showed adaptation within 8 h, whereas PIT-2 and NaPi-IIc showed a slower adaptive trend. Our findings suggest that PIT-2, until now considered as a ubiquitously expressed [P.sub.i] housekeeping transporter, is a novel mediator of [P.sub.i] reabsorption in the PT under conditions of acute [P.sub.i] deprivation, but with a different adaptive time course from NaPi-IIa and NaPi-IIc. brush-border membrane; inorganic phosphate; sodium-dependent transport

Heart failure (HF) has been recognized as a common complication of diabetes, with a prevalence of up to 22% in individuals with diabetes and increasing incidence rates. Data also suggest that HF may develop in individuals with diabetes even in the absence of hypertension, coronary heart disease, or valvular heart disease and, as such, represents a major cardiovascular complication in this vulnerable population; HF may also be the first presentation of cardiovascular disease in many individuals with diabetes. Given that during the past decade, the prevalence of diabetes (particularly type 2 diabetes) has risen by 30% globally (with prevalence expected to increase further), the burden of HF on the health care system will continue to rise. The scope of this American Diabetes Association consensus report with designated representation from the American College of Cardiology is to provide clear guidance to practitioners on the best approaches for screening and diagnosing HF in individuals with diabetes or prediabetes, with the goal to ensure access to optimal, evidence-based management for all and to mitigate the risks of serious complications, leveraging prior policy statements by the American College of Cardiology and American Heart Association. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its N[H.sub.2] end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP 17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP 17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAPI7 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa. phosphate transport; PDZ; PDZK1; protein interaction; opossum kidney cells

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa Na[P.sub.i] cotransporter (Na[P.sub.i]-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-Na-[P.sub.i]-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E coimmuno-precipitated with Na[P.sub.i]-IIa from rat renal cortex tissue and HA-Na[P.sub.i]-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound Na[P.sub.i]-IIa and truncation of the COOH-terminal TRL motif from Na[P.sub.i]-IIa abolished this binding, and Far Western blotting showed that the Shank2E- Na[P.sub.i]-IIa interaction occurred directly between the two proteins. Na[P.sub.i]-IIa activity is regulated by moderating its abundance in the apical membrane. High-[P.sub.i] conditions induce Na[P.sub.i]-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-[P.sub.i] conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds Na[P.sub.i]-IIa via PDZ interactions, and undergoes [P.sub.i]-induced internalization. PDZ domains; endocytosis; degradation; epithelia

We previously showed an inverse correlation between membrane cholesterol content and Na-[P.sub.i] cotransport activity during the aging process and adaptation to alterations in dietary [P.sub.i] in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-[P.sub.i] cotransport activity and apical membrane Na-[P.sub.i] protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with [beta]-methyl cyclodextrin ([beta]-MCD) resulted in a significant increase in Na-[P.sub.i] cotransport activity accompanied by a moderate increase in apical membrane Na-[P.sub.i] protein abundance and no alteration of total cellular Na-[P.sub.i] protein abundance. Conversely, acute cholesterol enrichment achieved with [beta]-MCD/cholesterol resulted in a significant decrease in Na-[P.sub.i] cotransport activity with a moderate decrease in apical membrane Na-[P.sub.i] protein abundance and no change of the total cellular Na-[P.sub.i] protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-[P.sub.i] cotransport activity and apical membrane and total cellular Na-[P.sub.i] protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-[P.sub.i] cotransport activity and apical membrane and total cellular Na-[P.sub.i] protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-[P.sub.i] cotransport activity by diverse mechanisms that also include significant interactions of Na-[P.sub.i] protein with lipid microdomains. two-photon fluorescence microscopy; filipin; lipid microdomains; laurdan; opossum kidney cells

Carbohydrate response element-binding protein (ChREBP) is a rat homolog of human Williams-Beuren syndrome region 14 and a member of the basic helix-loop-helix leucine zipper transcription factor family. Its activation was found to be inducible by carbohydrate in the liver nuclear extracts from rats fed a high-sucrose diet. ChREBP is able to bind to the carbohydrate response element on the promoter of L-type pyruvate kinase and initiate the gene transcription. The detailed expression profile and transcriptional regulation of the ChREBP gene in adipocytes have not been characterized. In the present study, we provide evidence showing that 1) the ChREBP gene is expressed in differentiated 3T3-L1 adipocytes and rat adipose tissue; 2) insulin, glucose, and the antidiabetic agent troglitazone can significantly upregulate the gene expression of ChREBP in 3T3-L1 adipocytes, whereas free fatty acids suppress its expression in this cell type; 3) fasting followed by refeeding with a high-carbohydrate diet resulted in a 10-fold increase of ChREBP mRNA level in rat adipose tissue; and 4) ChREBP expression in adipose tissue is not significantly affected by the diabetic state. Taken together, the results we present are consistent with the idea that ChREBP is an important modulator of adipocyte biology and that its expression in adipose tissue is subject to combined regulation by glucose and insulin in vivo. The induction of ChREBP may serve as a novel pharmacological pathway for troglitazone-mediated hypoglycemic effects in vivo. adipogenesis; lipogenesis

Growth hormone (GH) is an important factor in the developmental adaptation to enhance [P.sub.i] reabsorption; however, the nephron sites and mechanisms by which GH regulates renal [P.sub.i] uptake remain unclear and are the focus of the present study. Micropuncture experiments were performed after acute thyroparathyroidectomy in the presence and absence of parathyroid hormone (PTH) in adult (14- to 17-wk old), juvenile (4-wk old), and GH-suppressed juvenile male rats. While the phosphaturic effect of PTH was blunted in the juvenile rat compared with the adult, suppression of GH in the juvenile restored fractional [P.sub.i] excretion to adult levels. In the presence or absence of PTH, GH suppression in the juvenile rat caused a significant increase in the fractional [P.sub.i] delivery to the late proximal convoluted (PCT) and early distal tubule, so that delivery was not different from that in adults. These data were confirmed by [P.sub.i] uptake studies into brush-border membrane (BBM) vesicles. Immunofluorescence studies indicate increased BBM type IIa Na[P.sub.i] cotransporter (NaPi-2) expression in the juvenile compared with adult rat, and GH suppression reduced NaPi-2 expression to levels observed in the adult. GH replacement in the [N-acetyl-[Tyr.sub.1]-D-[Arg.sup.2]]-GRF-(1-29)-[NH.sub.2]treated juveniles restored high NaPi-2 expression and [P.sub.i] uptake. Together, these novel results demonstrate that the presence of GH in the juvenile animal is crucial for the early developmental upregulation of BBM NaPi-2 and, most importantly, describe the enhanced [P.sub.i] reabsorption along the PCT and proximal straight nephron segments in the juvenile rat. development; sodium-phosphate cotransporters; parathyroid hormone; antagonist to growth hormone-releasing factor; brush-border membrane vesicles

Mulroney, Susan E., Craig B. Woda, Nabil Halaihel, Brandon Louie, Kevin McDonnell, Jay Schulkin, Aviad Haramati, and Moshe Levi. Central control of renal sodium-phosphate (NaPi-2) transporters. Am J Physiol Renal Physiol 286: F647-F652, 2004. First published December 23, 2003; 10.1152/ajprenal.00354.2002.--Regulation of phosphate ([P.sub.i]) reabsorption occurs through the up- and downregulation of the renal type-II sodium [P.sub.i] cotransporters (NaPi-2). Recently, renal NaPi2-type expression has been identified in areas of the brain. The present study determined whether brain NaPi-2 is regulated by dietary [P.sub.i] and whether the behavioral and renal adaptations to low-dietary [P.sub.i] are controlled centrally. NaPi-2-like expression in the third ventricle (3V) and amygdala of juvenile Wistar rats was regulated by dietary [P.sub.i], as in the kidneys. When cerebrospinal fluid (CSF) [P.sub.i] concentration was elevated by 3V injections of Pi in rats fed low-[P.sub.i] diet (LPD), the behavioral and renal adaptations to LPD were abolished. Most importantly, NaPi-2 expression was markedly reduced not only in the brain, but also renal proximal tubules, despite the low plasma [P.sub.i] milieu. This was confirmed by the significant reduction in the transport maximum for Pi (from 8.1 [+ or -] 0.2 in LPD + veh 3V to 1.7 [+ or -] 0.1 [micro]mol [P.sub.i]/ml glomerular filtration rate in LPD + 3V [P.sub.i], P < 0.001). These findings indicate that NaPi-2-like transporters in the brain are regulated by both dietary [P.sub.i] and CSF[P.sub.i] concentrations, and most significantly, that the central [P.sub.i] milieu can regulate renal NaPi-2 expression. We hypothesize that central 3V NaPi-2 transporters may act as Pi sensors and help regulate both brain and whole body Pi homeostasis. juvenile rats; behavior; [P.sub.i] sensors

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 33P 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 33P 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. [ABSTRACT FROM AUTHOR]