Sobuj Mia

Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells

Eryptosis, the suicidal erythrocyte death, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to the vascular wall by binding of phosphatidylserine to the CXC chemokine ligand 16 (CXCL16). Stimulators of eryptosis include increased cytosolic Ca(2+) activity, energy depletion, and activation of ceramide-producing sphingomyelinase. The present study explored whether sphingomyelinase triggers erythrocyte adhesion to endothelial cells. To this end, human erythrocytes were exposed for 6 h to bacterial sphingomyelinase (1-10 mU/ml) and phosphatidylserine exposure was estimated from fluorescent annexin-V-binding, cell volume from forward scatter in FACS-analysis, erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber under flow conditions at arterial shear rates, and CXCL16 protein abundance utilizing Western blotting and FACS analysis of fluorescent antibody binding. As a result, sphingomyelinase (≥1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca(2+) removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 μl/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 μg/ml) and following silencing of the CXCL16 gene with small interfering RNA. Pretreatment of HUVEC with sphingomyelinase upregulated CXCL16 protein abundance. Six hours pretreatment of HUVEC with sphingomyelinase (10 mU/ml) or C6-ceramide (50 μM) augmented erythrocyte adhesion following a 30-min treatment with Ca(2+) ionophore ionomycin (1 μM) or following energy depletion by 48-h glucose removal. Thus exposure to sphingomyelinase or C6-ceramide triggers eryptosis followed by phosphatidylserine- and CXCL16-sensitive adhesion of eryptotic erythrocytes to HUVEC.

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.

Downregulation of Kv1.5 K channels by the AMP-activated protein kinase.

Background: The voltage gated K+ channel Kv1.5 participates in the repolarization of a wide variety of cell types. Kv1.5 is downregulated during hypoxia, which is known to stimulate the energy-sensing AMP-activated serine/threonine protein kinase (AMPK). AMPK is a powerful regulator of nutrient transport and metabolism. Moreover, AMPK is known to downregulate several ion channels, an effect at least in part due to stimulation of the ubiquitin ligase Nedd4- 2. The present study explored whether AMPK regulates Kv1.5. Methods: cRNA encoding Kv1.5 was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (α1 β 1γ1), of constitutively active γR70QAMPK (α1 β 1γ1(R70Q)), of inactive mutant αK45RAMPK (α1(K45R)β1γ1), or of Nedd4-2. Kv1.5 activity was determined by two-electrode voltage-clamp. Moreover, Kv1.5 protein abundance in the cell membrane was determined by chemiluminescence and immunostaining with subsequent confocal microscopy. Results: Coexpression of wild-type AMPKWT and constitutively active AMPKγR70Q, but not of inactive AMPKαK45R significantly reduced Kv1.5-mediated currents. Coexpression of constitutively active AMPKγR70Q further reduced Kv1.5 K+ channel protein abundance in the cell membrane. Co-expression of Nedd4-2 similarly downregulated Kv1.5-mediated currents. Conclusion: AMPK is a potent regulator of Kv1.5. AMPK inhibits Kv1.5 presumably in part by activation of Nedd4- 2 with subsequent clearance of channel protein from the cell membrane.

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.

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.

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.

Inhibitory effect of NH4Cl treatment on renal Tgfß1 signaling following unilateral ureteral obstruction.

Background/Aims: Consequences of obstructive nephropathy include tissue fibrosis, a major pathophysiological mechanism contributing to development of end-stage renal disease. Transforming growth factor β 1 (Tgfβ1) is involved in the progression of renal fibrosis. According to recent observations, ammonium chloride (NH4Cl) prevented phosphate-induced vascular remodeling, effects involving decrease of Tgfβ1 expression and inhibition of Tgfβ1-dependent signaling. The present study, thus, explored whether NH4Cl influences renal Tgfβ1-induced pro-fibrotic signaling in obstructive nephropathy induced by unilateral ureteral obstruction (UUO). Methods: UUO was induced for seven days in C57Bl6 mice with or without additional treatment with NH4Cl (0.28 M in drinking water). Transcript levels were determined by RT-PCR as well as protein abundance by Western blotting, blood pH was determined utilizing a blood gas and chemistry analyser. Results: UUO increased renal mRNA expression of Tgfb1, Tgfβ-activated kinase 1 (Tak1) protein abundance and Smad2 phosphorylation in the nuclear fraction of the obstructed kidney tissues, effects blunted in NH4Cl treated mice as compared to control treated mice. The mRNA levels of the transcription factors nuclear factor of activated T cells 5 (Nfat5) and SRY (sex determining region Y)-box 9 (Sox9) as well as of tumor necrosis factor α (Tnfα), interleukin 6 (Il6), plasminogen activator inhibitor 1 (Pai1) and Snai1 were up-regulated in the obstructed kidney tissues following UUO, effects again significantly ameliorated following NH4Cl treatment. Furthermore, the increased protein and mRNA expression of α-smooth muscle actin (α-Sma), fibronectin and collagen type I in the obstructed kidney tissues following UUO were significantly attenuated following NH4Cl treatment. Conclusion: NH4Cl treatment ameliorates Tgfβ1-dependent pro-fibrotic signaling and renal tissue fibrosis markers following obstructive nephropathy.

Down-Regulation of Renal Klotho Expression by Shiga Toxin 2

Background/Aims: Shiga toxin 2 may trigger classical hemolytic uremic syndrome (HUS) eventually leading to renal failure. Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney is involved in the regulation of renal phosphate excretion and also retains renal protective effects. Renal failure is associated with renal depletion of klotho. The present study explored the influence of Shiga toxin 2 on renal klotho expression. Methods: Mice were injected with either solvent or Shiga toxin 2 and urinary flow rate and phosphate excretion were determined in metabolic cages. Renal transcript levels were measured by quantitative RT-PCR and renal protein abundance by Western blotting. Plasma concentrations of 1,25(OH)2D3 and FGF23 were determined by ELISA and plasma phosphate and urea concentrations by photometry. Results: Shiga toxin 2 treatment was followed by increase of plasma urea concentration, urinary flow rate and renal phosphate excretion but not of plasma phosphate concentration. Shiga toxin 2 treatment strongly decreased klotho mRNA expression and klotho protein abundance in renal tissue. Shiga toxin 2 treatment further increased tumor necrosis factor (TnfE) mRNA levels, as well as protein abundance of phosphorylated p38 MAPK in renal tissue. The treatment significantly increased renal Cyp27b1 and decreased renal Cyp24a1 mRNA levels without significantly altering plasma 1,25(OH)2D3 levels. Shiga toxin 2 treatment was further followed by increase of plasma FGF23 concentrations. Conclusion: Shiga toxin 2 treatment stimulated TnfE transcription, down-regulated renal klotho expression and increased FGF23 formation, effects presumably contributing to renal tissue injury. i 2015 S. Karger AG, Basel

Involvement Of Vascular Aldosterone Synthase In Phosphate-Induced Osteogenic Transformation Of Vascular Smooth Muscle Cells

Vascular calcification resulting from hyperphosphatemia is a major determinant of mortality in chronic kidney disease (CKD). Vascular calcification is driven by aldosterone-sensitive osteogenic transformation of vascular smooth muscle cells (VSMCs). We show that even in absence of exogenous aldosterone, silencing and pharmacological inhibition (spironolactone, eplerenone) of the mineralocorticoid receptor (MR) ameliorated phosphate-induced osteo-/chondrogenic transformation of primary human aortic smooth muscle cells (HAoSMCs). High phosphate concentrations up-regulated aldosterone synthase (CYP11B2) expression in HAoSMCs. Silencing and deficiency of CYP11B2 in VSMCs ameliorated phosphate-induced osteogenic reprogramming and calcification. Phosphate treatment was followed by nuclear export of APEX1, a CYP11B2 transcriptional repressor. APEX1 silencing up-regulated CYP11B2 expression and stimulated osteo-/chondrogenic transformation. APEX1 overexpression blunted the phosphate-induced osteo-/chondrogenic transformation and calcification of HAoSMCs. Cyp11b2 expression was higher in aortic tissue of hyperphosphatemic klotho-hypomorphic (kl/kl) mice than in wild-type mice. In adrenalectomized kl/kl mice, spironolactone treatment still significantly ameliorated aortic osteoinductive reprogramming. Our findings suggest that VSMCs express aldosterone synthase, which is up-regulated by phosphate-induced disruption of APEX1-dependent gene suppression. Vascular CYP11B2 may contribute to stimulation of VSMCs osteo-/chondrogenic transformation during hyperphosphatemia.