Anja T. Umbach

Rapamycin-induced phosphaturia

BACKGROUND The mammalian target of rapamycin (mTOR) is known to stimulate a variety of transport mechanisms including the intestinal phosphate transporter NaPi-IIb. The present study was performed to elucidate whether mTOR similarly regulates the major renal tubular phosphate transporter NaPi-IIa. METHODS To this end, NaPi-IIa was expressed in Xenopus oocytes with or without mTOR and phosphate transport estimated from phosphate-induced (1 mM) current (I(pi)). RESULTS As a result, I(pi) was observed in NaPi-IIa-expressing but not in H(2)O-injected Xenopus oocytes. Co-expression of mTOR significantly enhanced I(pi) in NaPi-IIa-expressing Xenopus oocytes, an effect abrogated by treatment with rapamycin (50 nM for the last 24 h of incubation). In a second series of experiments, the effect of rapamycin was analysed in mice. The in vivo administration of rapamycin (3 microg/g body weight/day) for 3 days resulted in phosphaturia in mice despite a tendency of plasma phosphate concentration to decrease. CONCLUSIONS mTOR contributes to the regulation of renal phosphate transport, and rapamycin thus influences phosphate balance.

Pivotal Role of Serum- and Glucocorticoid-Inducible Kinase 1 in Vascular Inflammation and Atherogenesis

Objective— Atherosclerosis, an inflammatory disease of arterial vessel walls, requires migration and matrix metalloproteinase (MMP)-9–dependent invasion of monocytes/macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor nuclear factor-&kgr;B, which is regulated by inhibitor &kgr;B (I&kgr;B) and thus I&kgr;B kinase. Regulators of nuclear factor-&kgr;B include serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored involvement of SGK1 in vascular inflammation and atherogenesis. Approach and Results— Gene-targeted apolipoprotein E (ApoE)–deficient mice without (apoe −/− sgk1 +/+) or with (apoe −/− sgk1 −/−) additional SGK1 knockout received 16-week cholesterol-rich diet. According to immunohistochemistry atherosclerotic lesions in aorta and carotid artery, vascular CD45+ leukocyte infiltration, Mac-3+ macrophage infiltration, vascular smooth muscle cell content, MMP-2, and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe −/− sgk1 −/−mice than in apoe −/− sgk1 +/+mice. As determined by Boyden chamber, thioglycollate-induced peritonitis and air pouch model, migration of SGK1-deficient CD11b+F4/80+ macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1 −/− than in sgk1 +/+macrophages and in control plasmid–transfected or inactive K127NSGK1-transfected than in constitutively active S422DSGK1-transfected THP-1 cells. Confocal microscopy revealed reduced macrophage number and macrophage MMP-9 content in plaques of apoe −/− sgk1 −/− mice. In THP-1 cells, MMP-inhibitor GM6001 (25 &mgr;mol/L) abrogated S422DSGK1-induced MMP-9 production and invasion. According to reverse transcription polymerase chain reaction, MMP-9 transcript levels were significantly reduced in sgk1 −/−macrophages and strongly upregulated in S422DSGK1-transfected THP-1 cells compared with control plasmid–transfected or K127NSGK1-transfected THP-1 cells. According to immunoblotting and confocal microscopy, phosphorylation of I&kgr;B kinase and inhibitor &kgr;B and nuclear translocation of p50 were significantly lower in sgk1 −/−macrophages than in sgk1 +/+macrophages and significantly higher in S422DSGK1-transfected THP-1 cells than in control plasmid–transfected or K127NSGK1-transfected THP-1 cells. Treatment of S422DSGK1-transfected THP-1 cells with I&kgr;B kinase-inhibitor BMS-345541 (10 &mgr;mol/L) abolished S422DSGK1-induced increase of MMP-9 transcription and gelatinase activity. Conclusions— SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of monocyte/macrophage migration and MMP-9 transcription via regulation of nuclear factor-&kgr;B.

Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2.

Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.

NFκB-sensitive Orai1 expression in the regulation of FGF23 release.

Fibroblast growth factor (FGF23) plasma levels are elevated in cardiac and renal failure and correlate with poor clinical prognosis of those disorders. Both disorders are associated with inflammation and activation of the inflammatory transcription factor NFκB. An excessive FGF23 level is further observed in Klotho-deficient mice. The present study explored a putative sensitivity of FGF23 expression to transcription factor NFκB, which is known to upregulate Orai1, the Ca2+ channel accomplishing store-operated Ca2+ entry (SOCE). In osteoblastic cells (UMR106) and immortalized primary periosteal (IPO) cells, protein abundance was determined by Western blotting, and in UMR106 cells, transcript levels were quantified by RT-PCR, cytosolic Ca2+ activity utilizing Fura-2-fluorescence, and SOCE from Ca2+ entry following store depletion by thapsigargin. As a result, UMR106 and IPO cells expressed Ca2+ channel Orai1. SOCE was lowered by NFκB inhibitor wogonin as well as by Orai1 inhibitors 2-APB and YM58483. UMR106 cell Fgf23 transcripts were increased by stimulation of SOCE and Ca2+ ionophore ionomycin and decreased by Orai inhibitors 2-APB, YM58483 and SK&F96365, by Orai1 silencing, as well as by NFκB inhibitors wogonin, withaferin A, and CAS 545380-34-5. In conclusion, Fgf23 expression is upregulated by stimulation of NFκB-sensitive, store-operated Ca2+ entry.Key messagesOsteoblast UMR106 and IPO cells express Ca2+ channel Orai1.Osteoblast store-operated Ca2+ entry is accomplished by NFκB-sensitive Orai1.Osteoblast Fgf23 transcription is upregulated by increase in the cytosolic Ca2+ activity.Fgf23 transcription is decreased by Orai inhibitors and Orai1 silencing.Fgf23 transcription is lowered by NFκB inhibitors.

Effects of gum arabic (Acacia senegal) on renal function in diabetic mice.

Background/Aims: Gum arabic (GA) is a Ca2+-, Mg2+- and K+-rich dietary fiber used for the treatment of patients with chronic kidney disease in Middle Eastern countries. In healthy mice, GA treatment increases creatinine clearance, renal ADH excretion, as well as intestinal and renal excretion of Mg2+ and Ca2+. GA decreases plasma Pi concentration, urinary Pi and Na+ excretion. The present study explored the effects of GA on renal function in diabetic mice. Methods: Metabolic cage experiments were performed on Akita mice (akita+/–), which spontaneously develop insulin deficiency and thus hyperglycemia. Plasma and urinary concentrations of Na+, K+ and Ca2+ were measured by flame photometry (AFM 5051, Eppendorf, Germany), creatinine by the Jaffé method, phosphate photometrically, urea by an enzymatic method, glucose utilizing a glucometer and an enzymatic kit, aldosterone using an RIA, urinary albumin fluorometrically, and blood pressure by the tail-cuff method. Results: GA (10% in drinking water) significantly increased urinary excretion of Ca2+ and significantly decreased plasma phosphate and urea concentrations, urinary flow rate, urinary Na+, phosphate and glucose excretion, blood pressure and proteinuria. Conclusions: GA treatment decreases blood pressure and proteinuria in diabetic mice and may thus prove beneficial in diabetic nephropathy.

Enhanced FGF23 production in mice expressing PI3K-insensitive GSK3 is normalized by β-blocker treatment

Glycogen synthase kinase (GSK)‐3 is a ubiquitously expressed kinase inhibited by insulin‐dependent Akt/PKB/SGK. Mice expressing Akt/PKB/SGK‐resistant GSK3α/GSK3β (gsk3KI) exhibit enhanced sympathetic nervous activity and phosphaturia with decreased bone density. Hormones participating in phosphate homeostasis include fibroblast growth factor (FGF)‐23, a bone‐derived hormone that inhibits 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3; calcitriol) formation and phosphate reabsorption in the kidney and counteracts vascular calcification and aging. FGF23 secretion is stimulated by the sympathetic nervous system. We studied the role of GSK3‐controlled sympathetic activity in FGF23 production and phosphate metabolism. Serum FGF23, 1,25(OH)2D3, and urinary vanillylmandelic acid (VMA) were measured by ELISA, and serum and urinary phosphate and calcium were measured by photometry in gsk3KI and gsk3WT mice, before and after 1 wk of oral treatment with the β‐blocker propranolol. Urinary VMA excretion, serum FGF23, and renal phosphate and calcium excretion were significantly higher, and serum 1,25(OH)2D3 and phosphate concentrations were lower in gsk3KI mice than in gsk3WT mice. Propranolol treatment decreased serum FGF23 and loss of renal calcium and phosphate and increased serum phosphate concentration in gsk3KI mice. We conclude that Akt/PKB/SGK‐sensitive GSK3 inhibition participates in the regulation of FGF23 release, 1,25(OH)2D3 formation, and thus mineral metabolism, by controlling the activity of the sympathetic nervous system.—Fajol, A., Chen, H., Umbach, A. T., Quarles, L. D., Lang, F., Föller, M. Enhanced FGF23 production in mice expressing PI3K‐insensitive GSK3 is normalized by β‐blocker treatment. FASEB J. 30, 994–1001 (2016). www.fasebj.org

Akt2/PKBβ-sensitive regulation of renal phosphate transport

Aim:  The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport.

SPAK sensitive regulation of the epithelial Na+ channel ENaC

Background/Aims: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK participates in the regulation of NaCl and Na⁺,K⁺,2Cl^- cotransport and thus renal salt excretion. The present study explored whether SPAK has similarly the potential to regulate the epithelial Na⁺ channel (ENaC). Methods: ENaC was expressed in Xenopus oocytes with or without additional expression of wild type SPAK, constitutively active ^T233ESPAK, WNK insensitive ^T233ASPAK or catalytically inactive ^D212ASPAK, and ENaC activity estimated from amiloride (50 µM) sensitive current (I_amil) in dual electrode voltage clamp experiments. Moreover, Ussing chamber was employed to determine I_amil in colonic tissue from wild type mice (spak^wt/wt) and from gene targeted mice carrying WNK insensitive SPAK (spak^tg/tg). Results: I_amil was observed in ENaC-expressing oocytes, but not in water-injected oocytes. In ENaC expressing oocytes I_amil was significantly increased following coexpression of wild-type SPAK and ^T233ESPAK, but not following coexpression of ^T233ASPAK or ^D212ASPAK. Colonic I_amil was significantly higher in spak^wt/wt than in spak^tg/tg mice. Conclusion: SPAK has the potential to up-regulate ENaC.

Intestinal Na+ Loss and Volume Depletion in JAK3-Deficient Mice

Background/Aims: The Janus kinase 3 JAK3 participates in the signaling of immune cells. Lack of JAK3 triggers inflammatory bowel disease, which in turn has been shown to affect intestinal activity of the epithelial Na+ channel ENaC and thus colonic sodium absorption. At least in theory, inflammatory bowel disease in JAK3-deficient mice could lead to intestinal salt loss compromizing extracellular volume maintenance and blood pressure regulation. The present study thus explored whether JAK3 deficiency impacts on colonic ENaC activity, fecal Na+ exretion, blood pressure and extracellular fluid volume regulation. Methods: Experiments were performed in gene-targeted mice lacking functional JAK3 (jak3-/-) and in wild type mice (jak3+/+). Colonic ENaC activity was estimated from amiloride-sensitive current in Ussing chamber experiments, fecal, serum and urinary Na+ concentration by flame photometry, blood pressure by the tail cuff method and serum aldosterone levels by immunoassay. Results: The amiloride (50 µM)-induced deflection of the transepithelial potential difference was significantly lower and fecal Na+ excretion significantly higher in jak3-/- mice than in jak3+/+ mice. Moreover, systolic arterial blood pressure was significantly lower and serum aldosterone concentration significantly higher in jak3-/- mice than in jak3+/+ mice. Both, absolute and fractional renal Na+ excretion were significantly lower in jak3-/- mice than in jak3+/+ mice. Conclusions: JAK3 deficiency leads to impairment of colonic ENaC activity with intestinal Na+ loss, decrease of blood pressure, increased aldosterone release and subsequent stimulation of renal tubular Na+ reabsorption.

Sgk1 Sensitive Pendrin Expression in Murine Platelets

Background: The anion exchanger pendrin (SLC26A4) is required for proper development of the inner ear, and contributes to iodide organification in thyroid glands as well as anion transport in various epithelia, such as airways and renal tubules. SLC26A4 deficiency leads to Pendred syndrome, which is characterized by hearing loss with enlarged vestibular aqueducts and variable hypothyroidism and goiter. Pendrin expression in kidney, heart, lung and thyroid is up-regulated by the mineralocorticoid deoxycorticosterone (DOCA). Platelets express anion exchangers but virtually nothing is known about the molecular identity and regulation of those carriers. Other carriers such as the Na+/H+ exchanger are regulated by the mineralocorticoid-sensitive serum and glucocorticoid inducible kinase SGK1. Methods: The present study utilized i) quantitative reverse transcription polymerase chain reaction (RT-qPCR) to quantify the transcript levels of Slc26a4 as compared to Gapdh and ii) western blotting to assess Slc26a4 protein abundance in murine platelets from gene-targeted mice lacking Sgk1 (sgk1-/-) and respective wild type animals (sgk1+/+) treated without or with a subcutaneous injection of 2.5 mg DOCA for 3 h, or in sgk1+/+ platelets with or without in vitro treatment for 1 h with 10 µg/ml DOCA. Results: Slc26a4 was expressed in platelets, and in vitro DOCA treatment increased Slc26a4 mRNA levels in platelets isolated from sgk1+/+ mice. Moreover, in vivo DOCA treatment significantly up-regulated Slc26a4 mRNA levels in platelets isolated from sgk1+/+ but not sgk1-/- mice. An increase in Sgk1 mRNA levels paralleled that of Slc26a4 mRNA levels in platelets of sgk1+/+ mice. In addition, DOCA treatment further increased Slc26a4 protein abundance in platelets isolated from sgk1+/+ mice. Conclusions: Pendrin is expressed in platelets and is presumably regulated by SGK1 and mineralocorticoids.