Martina Feger

Sensitization of erythrocytes to suicidal erythrocyte death following water deprivation.

Background/Aims: Klotho deficiency results in excessive formation of 1,25(OH)2D3, accelerated ageing and early death. Moreover, klotho deficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), glucose depletion, hyperosmotic shock and oxidative stress. Klotho expression is decreased and 1,25(OH)2D3-formation enhanced by dehydration. The present study thus explored whether dehydration influences eryptosis. Methods: Blood was drawn from hydrated or 36h dehydrated mice. Plasma osmolarity was determined by vapour pressure method, plasma 1,25(OH)2D3 and aldosterone concentrations using ELISA, and plasma Ca2+-concentration utilizing photometry. Erythrocytes were exposed to Ca2+-ionophore ionomycin (1 µM, 30 min), energy depletion (12 h glucose removal), hyperosmotic shock (500 mM sucrose added, 2 h) and oxidative stress (100 µM tert-butyl-hydroperoxide, 30 min) and phosphatidylserine exposure at the erythrocyte surface estimated from annexin V binding. Results: Dehydration increased plasma osmolarity and plasma 1,25(OH)2D3 and aldosterone concentrations. Dehydration did not significantly modify phosphatidylserine-exposure of freshly drawn erythrocytes but significantly enhanced the increase of phosphatidylserine-exposure under control conditions and following treatment with ionomycin, glucose-deprivation, hyperosmolarity or tert-butyl-hydroperoxide. Conclusions: Dehydration sensitizes the erythrocytes to spontaneous eryptosis and to the triggering of eryptosis by excessive Ca2+-entry, energy depletion, hyperosmotic shock and oxidative stress.

Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice

Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [1,25(OH)2D3] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyperphosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on 1,25(OH)2D3, FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.

Sgk1 sensitivity of Na+/H+ exchanger activity and cardiac remodeling following pressure overload

Sustained increase of cardiac workload is known to trigger cardiac remodeling with eventual development of cardiac failure. Compelling evidence points to a critical role of enhanced cardiac Na+/H+ exchanger (NHE1) activity in the underlying pathophysiology. The signaling triggering up-regulation of NHE1 remained, however, ill defined. The present study explored the involvement of the serum- and glucocorticoid-inducible kinase Sgk1 in cardiac remodeling due to transverse aortic constriction (TAC). To this end, experiments were performed in gene targeted mice lacking functional Sgk1 (sgk1−/−) and their wild-type controls (sgk1+/+). Transcript levels have been determined by RT-PCR, cytosolic pH (pHi) utilizing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, Na+/H+ exchanger activity by the Na+-dependent realkalinization after an ammonium pulse, ejection fraction (%) utilizing cardiac cine magnetic resonance imaging and cardiac glucose uptake by PET imaging. As a result, TAC increased the mRNA expression of Sgk1 in sgk1+/+ mice, paralleled by an increase in Nhe1 transcript levels as well as Na+/H+ exchanger activity, all effects virtually abrogated in sgk1−/− mice. In sgk1+/+ mice, TAC induced a decrease in Pgc1a mRNA expression, while Spp1 mRNA expression was increased, both effects diminished in the sgk1−/− mice. TAC was followed by a significant increase of heart and lung weight in sgk1+/+ mice, an effect significantly blunted in sgk1−/− mice. TAC increased the transcript levels of Anp and Bnp, effects again significantly blunted in sgk1−/− mice. TAC increased transcript levels of Collagen I and III as well as Ctgf mRNA and CTGF protein abundance, effects significantly blunted in sgk1−/− mice. TAC further decreased the ejection fraction in sgk1+/+ mice, an effect again attenuated in sgk1−/− mice. Also, cardiac FDG-glucose uptake was increased to a larger extent in sgk1+/+ mice than in sgk1−/− mice after TAC. These observations point to an important role for SGK1 in cardiac remodeling and development of heart failure following an excessive work load.

Augmentation of phosphate-induced osteo-/chondrogenic transformation of vascular smooth muscle cells by homoarginine

AIMS Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification. METHODS AND RESULTS Experiments were performed in hyperphosphatemic klotho-hypomorphic mice (kl/kl), in subtotal nephrectomy and vitamin D3-overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 (Pai1) and of osteogenic markers Msx2, Cbfa1, and Alpl. Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1, MSX2, CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine. CONCLUSION Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine.

Effect of Carbon Monoxide Donor CORM-2 on Vitamin D 3 Metabolism

Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by renal 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)2D3 regulates Ca2+ and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)2D3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)2D3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 µM CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)2D3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)2D3 synthesis.

PKB/SGK-Resistant GSK-3 Signaling Following Unilateral Ureteral Obstruction

Background/Aims: Renal tissue fibrosis contributes to the development of end-stage renal disease. Causes for renal tissue fibrosis include obstructive nephropathy. The development of renal fibrosis following unilateral ureteral obstruction (UUO) is blunted in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase SGK1. Similar to Akt isoforms, SGK1 phosphorylates and thus inactivates glycogen synthase kinase GSK-3. The present study explored whether PKB/SGK-dependent phoshorylation of GSK-3α/β impacts on pro-fibrotic signaling following UUO. Methods: UUO was induced in mice carrying a PKB/SGK-resistant GSK-3α/β (gsk-3KI) and corresponding wild-type mice (gsk-3WT). Three days after the obstructive injury, expression of fibrosis markers in kidney tissues was analyzed by quantitative RT-PCR and western blotting. Results: GSK-3α and GSK-3β phosphorylation was absent in both, the non-obstructed and the obstructed kidney tissues from gsk-3KI mice but was increased by UUO in kidney tissues from gsk-3WT mice. Expression of α-smooth muscle actin, type I collagen and type III collagen in the non-obstructed kidney tissues was not significantly different between gsk-3KI mice and gsk-3WT mice but was significantly less increased in the obstructed kidney tissues from gsk-3KI mice than from gsk-3WT mice. After UUO treatment, renal β-catenin protein abundance and renal expression of the β-catenin sensitive genes: c-Myc, Dkk1, Twist and Lef1 were again significantly less increased in kidney tissues from gsk-3KI mice than from gsk-3WT mice. Conclusions: PKB/SGK-dependent phosphorylation of glycogen synthase kinase GSK-3 contributes to the pro-fibrotic signaling leading to renal tissue fibrosis in obstructive nephropathy.

25-Hydroxyvitamin D 3 1-Alpha- Hydroxylase-Dependent Stimulation of Renal Klotho Expression by Spironolactone

Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)2D3 stimulates, by activating the vitamin D3 receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR) or of mineralocorticoid receptor (NR3C2). Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D3 1-alpha-hydroxylase with subsequent activation of the vitamin D3 receptor by 1,25(OH)2D3, an effect possibly independent from the mineralocorticoid receptor.

Inhibition of osteo/chondrogenic transformation of vascular smooth muscle cells by MgCl2 via calcium-sensing receptor.

Objectives: The progression of vascular calcification, an active process promoted by osteo/chondrogenic transformation of vascular smooth muscle cells (VSMCs) is attenuated by activation of the calcium-sensing receptor (CASR). Recent in-vitro studies revealed that vascular calcification could be blunted by Mg2+, but the underlying mechanisms remained elusive. The present study explored whether the effects of MgCl2 on vascular calcification involve the CASR. Methods: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) and in the mouse vascular calcification model of vitamin D3 overload. Results: Phosphate-induced calcium deposition and mRNA expression of the osteogenic markers msh homeobox 2 (MSX2), CBFA1 (core-binding factor &agr; 1), and ALPL (tissue-nonspecific alkaline phosphatase) in HAoSMCs were blunted by additional treatment with MgCl2. MgCl2 upregulated CASR mRNA expression in HAoSMCs in a dose-dependent manner. Furthermore, the inhibitory effects of MgCl2 on phosphate-induced calcium deposition and osteogenic markers mRNA expression were mimicked by the CASR agonist GdCl3 and reversed by additional treatment with the CASR antagonist NPS-2143 or by silencing of the CASR gene in HAoSMCs. MgCl2 also blunted the osteogenic transformation of VSMCs induced by hydroxyapatite particles. High-dosed cholecalciferol treatment induced vascular calcification and upregulated aortic osteogenic markers Msx2, Cbfa1 and Alpl and collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin (Fbn) mRNA expression in mice, effects reduced by additional treatment with MgCl2. These effects were paralleled by increased aortic Casr mRNA expression in cholecalciferol-treated mice, which was further augmented by MgCl2. Conclusion: The protective effects of MgCl2 on osteo/chondrogenic transformation of VSMCs and vascular calcification involve regulation of CASR and CASR-dependent signaling.

Klotho sensitivity of the neuronal excitatory amino acid transporters EAAT3 and EAAT4.

Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)2D3 formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na+-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (Iglu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased Iglu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.

Impact of AMP-Activated Protein Kinase α1 Deficiency on Tissue Injury following Unilateral Ureteral Obstruction

Background AMP-activated protein kinase (Ampk) is a sensor of the cellular energy status and a powerful regulator of metabolism. Activation of Ampk was previously shown to participate in monocyte-to-fibroblast transition and matrix protein production in renal tissue. Thus, the present study explored whether the catalytic Ampkα1 isoform participates in the regulation of the renal fibrotic response following unilateral ureteral obstruction (UUO). Methods UUO was induced in gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and in corresponding wild-type mice (Ampkα1+/+). In the obstructed kidney and, for comparison, in the non-obstructed control kidney, quantitative RT-PCR, Western blotting and immunostaining were employed to determine transcript levels and protein abundance, respectively. Results In Ampkα1+/+ mice, UUO significantly up-regulated the protein abundance of the Ampkα1 isoform, but significantly down-regulated the Ampkα2 isoform in renal tissue. Phosphorylated Ampkα protein levels were significantly increased in obstructed kidney tissue of Ampkα1+/+ mice but not of Ampkα1-/- mice. Renal expression of α-smooth muscle actin was increased following UUO, an effect again less pronounced in Ampkα1-/- mice than in Ampkα1+/+ mice. Histological analysis did not reveal a profound effect of Ampkα1 deficiency on collagen 1 protein deposition. UUO significantly increased phosphorylated and total Tgf-ß-activated kinase 1 (Tak1) protein, as well as transcript levels of Tak1-downstream targets c-Fos, Il6, Pai1 and Snai1 in Ampkα1+/+ mice, effects again significantly ameliorated in Ampkα1-/- mice. Moreover, Ampkα1 deficiency inhibited the UUO-induced mRNA expression of Cd206, a marker of M2 macrophages and of Cxcl16, a pro-fibrotic chemokine associated with myeloid fibroblast formation. The effects of Ampkα1 deficiency during UUO were, however, paralleled by increased tubular injury and apoptosis. Conclusions Renal obstruction induces an isoform shift from Ampkα2 towards Ampkα1, which contributes to the signaling involved in cell survival and fibrosis.