The role of CD44-hyaluronic acid interaction in exogenous mesenchymal stem cells homing to rat remnant kidney.

BACKGROUND/AIMS: The aim of our study was to reveal the role of CD44-Hyaluronic acid (HA) in the homing and improving renal function of systemically transplanted MSCs in chronic renal failure. METHODS: First, a remnant kidney model was established in rats and the expression of HA was determined using immunohistochemistry (IHC) and western blotting. Next, chemotaxis assay using flow cytometry, and cell migration assay of MSCs were performed in vitro. Then, MSCs were transplanted into rats, thus, sprague-Dawley (SD) rats were randomly divided into sham group, 5/6 nephrectomy (5/6 Nx) group, MSC group and MSC/Anti-CD44 group (n = 8 for all groups). Migration of MSCs to the kidney in these rats was assessed by using cell tracking experiments, and tissue damage was evaluated by morphological analysis using Masson's trichrome staining and periodic acid Schiff staining. RESULTS: HA was significantly observed in 5/6 Nx group, but not in sham group. Meanwhile, HA was discovered induced MSCs migration remarkably (p < 0.05) and anti-CD44 antibody inhibited the migration significantly (p < 0.05) in vitro. In vivo, the GFP-MSCs were observed in MSC group and the cells reduced in MSC/Anti-CD44 groups, especially, in the tubulointerstitium. CONCLUSION: Our findings reveal that CD44-HA has the potential to induce MSCs homing to injured tissue, while its effect on the ability of MSCs, improving tissue function, is not significant.

Involvement of mineralocorticoid receptor in high glucose-induced big mitogen-activated protein kinase 1 activation and mesangial cell proliferation.

We previously demonstrated that high glucose-induced cell proliferation in cultured rat mesangial cells (RMCs) is mediated through activation of big mitogen-activated protein kinase 1 (BMK1). We also found that, in aldosterone-treated rats, mesangial proliferation is associated with BMK1 activation and that these effects were prevented by treatment with a selective mineralocorticoid receptor antagonist, eplerenone. In this study, we investigated the contribution of mineralocorticoid receptors to high glucose-induced BMK1 activation and cell proliferation in RMCs. BMK1 phosphorylation was measured by western blot analysis. Cell proliferation was evaluated by [3H]-thymidine incorporation. High glucose treatment (15.5 mmol/l) increased BMK1 phosphorylation in both the nucleus and cytosol of RMCs. High glucose-induced BMK1 phosphorylation was attenuated by pretreatment with eplerenone (10 micromol/l), mineralocorticoid receptor small interfering RNA or PD98059 (100 micromol/l), a specific inhibitor of extracellular signal-regulated kinase kinase (MEK). Likewise, high glucose-induced increases in [H]-thymidine incorporation were prevented by eplerenone or PD98059 and transfection of dominant-negative MEK5, which is the upstream regulator of BMK1. These results suggest that mineralocorticoid receptors are involved in high glucose-induced BMK1 phosphorylation and cell proliferation. The inhibitory actions of mineralocorticoid receptor antagonists may contribute to their preventive effects on diabetic nephropathy, which have been reported in recent clinical studies.

Gelsolin overexpression alters actin dynamics and tyrosine phosphorylation of lipid raft-associated proteins in Jurkat T cells.

Upon T cell receptor engagement, both the actin cytoskeleton and substrates of tyrosine phosphorylation are remodeled to create a signaling complex at the interface of the antigen-presenting cell and responding T cell. While T cell signaling has been shown to regulate actin reorganization, the mechanisms by which changes in actin dynamics affect early T cell signaling have not been fully explored. Using gelsolin, an actin-binding protein with capping and severing activities, and latrunculin, an actin-depolymerizing agent, we have further investigated the interplay between actin dynamics and the regulation of T cell signaling. Overexpression of gelsolin altered actin dynamics in Jurkat T cells, and alteration of actin dynamics correlated with dysregulation of tyrosine phosphorylation of raft-associated substrates. This perturbation of tyrosine phosphorylation was correlated with inhibition of activation-dependent signaling pathways regulating Erk-1/2 phosphorylation, NF-AT transcriptional activation and IL-2 production. Modification of actin by the depolymerizing agent latrunculin also altered the tyrosine phosphorylation patterns of proteins associated with lipid rafts, and pre-treatment with latrunculin inhibited anti-CD3 mAb-mediated NF-AT activation. Thus, our data indicate that actin cytoskeletal dynamics modulate the tyrosine phosphorylation of raft-associated proteins and subsequent downstream signal transduction.

Augmentation of intrarenal angiotensin II levels in uninephrectomized aldosterone/salt-treated hypertensive rats; renoprotective effects of an ultrahigh dose of olmesartan.

Recent studies have suggested that aldosterone plays a role in the pathogenesis of renal injury. In this study, we investigated whether local angiotensin II (Ang II) activity contributes to the progression of renal injury in aldosterone/salt-induced hypertensive rats. Uninephrectomized rats were treated with 1% NaCl in a drinking solution and one of the following combinations for 6 weeks: vehicle (2% ethanol, s.c.; n=9), aldosterone (0.75 mug/h, s.c.; n=8), aldosterone+Ang II type 1 receptor blocker olmesartan (10 mg/kg/day, p.o.; n=8), or aldosterone+olmesartan (100 mg/kg/day, p.o.; n=9). Aldosterone/salt-treated hypertensive rats exhibited severe proteinuria and renal injury characterized by glomerular sclerosis and tubulointerstitial fibrosis. Aldosterone/salt-induced renal injury was associated with augmented expression of angiotensin converting enzyme and Ang II levels in the renal cortex and medullary tissues. Renal cortical and medullary mRNA expression of transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) as well as the collagen contents were increased in aldosterone/salt-treated hypertensive rats. Treatment with olmesartan (10 or 100 mg/kg/day) had no effect on blood pressure but attenuated proteinuria in a dose-dependent manner. Olmesartan at 10 mg/kg/day tended to decrease renal cortical and medullary Ang II levels, TGF-beta and CTGF expression, and collagen contents; however, these changes were not significant. On the other hand, an ultrahigh dose of olmesartan (100 mg/kg/day) significantly decreased these values and ameliorated renal injury. These data suggest that augmented local Ang II activity contributes, at least partially, to the progression of aldosterone/salt-dependent renal injury.

Involvements of Rho-kinase and TGF-beta pathways in aldosterone-induced renal injury.

Recent studies have suggested a role for aldosterone in the pathogenesis of renal injury. This study investigated the potential contributions of Rho-kinase and TGF-beta pathways to aldosterone-induced renal injury. Rats were uninephrectomized and then treated for 5 wk with 1% NaCl in a drinking solution and one of the following: Vehicle (2% ethanol, subcutaneously; n = 9); aldosterone (0.75 microg/h, subcutaneously; n = 9); or aldosterone + fasudil, a specific Rho-kinase inhibitor (10 mg/kg per d, subcutaneously; n = 8). Phosphorylation of myosin phosphate target subunit-1 (MYPT1) and Smad2/3 in renal cortical tissue was measured by Western blotting with anti-phospho MYPT1 and Smad2/3 antibodies, respectively. Rats that received aldosterone infusion exhibited hypertension and severe renal injury characterized by proteinuria, glomerular sclerosis, and tubulointerstitial fibrosis with increases in alpha-smooth muscle actin staining and numbers of monocytes/macrophages in the interstitium. Renal cortical mRNA levels of types I and III collagen, TGF-beta, connective tissue growth factor, and monocyte chemoattractant protein-1 as well as Smad2/3 phosphorylation were significantly increased in rats that received aldosterone infusion. All of these changes were associated with an increase in renal tissue MYPT1 phosphorylation. Treatment with fasudil did not alter BP but significantly ameliorated proteinuria and renal injury in rats that received aldosterone infusion. Furthermore, fasudil prevented MYPT1 phosphorylation and markedly decreased alpha-smooth muscle actin staining, numbers of monocytes/macrophages, mRNA levels of types I and III collagen, TGF-beta, connective tissue growth factor and monocyte chemoattractant protein-1, and Smad2/3 activity in renal cortical tissues. These results provide evidence, for the first time, that Rho-kinase is substantially involved in aldosterone-induced renal injury through activation of a TGF-beta-dependent pathway.

Synergistic effect of mechanical stretch and angiotensin II on superoxide production via NADPH oxidase in vascular smooth muscle cells.

OBJECTIVE: Mechanical forces and angiotensin II influence the structure and function of vascular cells, and play an important role in reactive oxygen species production. In this study, we examined the effects of mechanical stretch and angiotensin II on the expression of p22-phox and Nox-1, essential membrane components of NADPH oxidase, and superoxide production in rat vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Neither a stretch force nor angiotensin II alone altered p22-phox and Nox-1 expression in VSMCs. Combined stimulation markedly increased p22-phox and Nox-1 mRNA, however, which was associated with increased NADPH oxidase activity, superoxide production and total 8-iso-prostaglandin F2alpha concentration. The increases in p22-phox mRNA levels induced by a stretch force in combination with angiotensin II were prevented by treatment with an angiotensin type I (AT1) receptor antagonist, RNH-6270 (100 nmol/l). Protein expression of the AT1 receptor was upregulated by a stretch force. CONCLUSIONS: These data indicate that mechanical stretch and angiotensin II synergistically increase NADPH oxidase expression in VSMCs, and suggest that part of this mechanism is mediated through an upregulation of the AT1 receptor induced by mechanical stretch. The combined effects of mechanical strain and angiotensin II might promote vascular damage through the production of superoxide in a hypertensive state.

Effects of calcium channel blockade on angiotensin II-induced peritubular ischemia in rats.

Recent studies have indicated that derangement of peritubular capillary (PTC) circulation with consequent tubulointerstitial hypoxia plays a pivotal role in the pathogenesis of renal injury. The present study was performed to determine whether azelnidipine, a new dihydropyridine calcium channel blocker, attenuates angiotensin II (AngII)-induced peritubular ischemia in anesthetized rats. The superficial PTCs were visualized directly using an intravital fluorescence videomicroscope system, and the PTC blood flow was evaluated by analyzing the velocity of fluorescein isothiocyanate-labeled erythrocytes. Intravenous infusion of AngII (50 ng/kg/min, 10 min) significantly increased mean arterial pressure (MAP) and renal vascular resistance (RVR) (by 35 +/- 3% and 110 +/- 32%, respectively), and decreased total renal blood flow (RBF) and PTC erythrocyte velocity (by -34 +/- 4 and -37 +/- 1%, respectively). Treatment with azelnidipine (5 microg/kg/min i.v., 10 min) had no effect on basal MAP, RBF, RVR, or PTC erythrocyte velocity. However, azelnidipine markedly attenuated the AngII-induced increases in MAP (7 +/- 3%) and RVR (40 +/- 4%) and decreases in RBF (-24 +/- 1%) and PTC erythrocyte velocity (-22 +/- 1%). Similar attenuation in the AngII-induced responses of MAP, RBF, RVR, and PTC erythrocyte velocity were observed in rats treated with a higher dose of azelnidipine (20 microg/kg/min i.v., 10 min), which significantly decreased basal MAP and RVR and increased RBF and PTC erythrocyte velocity. These data suggest that calcium channel blockade attenuates AngII-induced peritubular ischemia, which may be involved in its beneficial effects on renal injury.

Aldosterone stimulates reactive oxygen species production through activation of NADPH oxidase in rat mesangial cells.

It has recently been shown that glomerular mesangial injury is associated with increases in renal cortical reactive oxygen species (ROS) levels in rats treated chronically with aldosterone and salt. This study was conducted to determine the mechanisms responsible for aldosterone-induced ROS production in cultured rat mesangial cells (RMC). Oxidative fluorescent dihydroethidium was used to evaluate intracellular production of superoxide anion (O(2)(-)) in intact cells. The lucigenin-derived chemiluminescence assay was used to determine NADPH oxidase activity. The staining of dihydroethidium was increased in a dose-dependent manner by aldosterone (1 to 100 nmol/L) with a peak at 3 h in RMC. Aldosterone (100 nmol/L for 3 h) also significantly increased NADPH oxidase activity from 232 +/- 18 to 346 +/- 30 cpm/5 x 10(4) cells. Immunoblotting data showed that aldosterone (100 nmol/L for 3 h) increased p47phox and p67phox protein levels in the membrane fraction by approximately 2.1- and 2.3-fold, respectively. On the other hand, mRNA expression of NADPH oxidase membrane components, p22phox, Nox-1, and Nox-4, were not altered by aldosterone (for 3 to 12 h) in RMC. Pre-incubation with the selective mineralocorticoid receptor (MR) antagonist, eplerenone (10 micromol/L), significantly attenuated aldosterone-induced O(2)(-) production, NADPH oxidase activation and membranous translocation of p47phox and p67phox. These results suggest that aldosterone-induced ROS generation is associated with NAPDH oxidase activation through MR-mediated membranous translocation of p47phox and p67phox in RMC. These cellular actions of aldosterone may play a role in the pathogenesis of glomerular mesangial injury.

Aldosterone stimulates collagen gene expression and synthesis via activation of ERK1/2 in rat renal fibroblasts.

Recently, we demonstrated that in rats treated chronically with aldosterone and salt, severe tubulointerstitial fibrosis is associated with the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2). Here, we investigated whether aldosterone stimulates collagen synthesis via ERK1/2-dependent pathways in cultured rat renal fibroblasts. Gene expression of mineralocorticoid receptor (MR) and types I, II, III, and IV collagen was measured by real-time polymerase chain reaction (PCR). MR protein expression and ERK1/2 activity were evaluated by Western blotting analysis with anti-MR and anti-phospho-ERK1/2 antibodies, respectively. Collagen synthesis was determined by [3H]-proline incorporation. Significant levels of MR mRNA and protein expression were observed in rat renal fibroblasts. Treatment with aldosterone (0.1 to 10 nmol/L) increased ERK1/2 phosphorylation in a concentration-dependent manner with a peak at 5 minutes. Aldosterone (10 nmol/L) also increased the mRNA levels of types I, III, and IV collagen at 36 hours but had no effect on the type II collagen mRNA level. [3H]-proline incorporation was significantly increased by aldosterone in both the medium and cell layer at 48 hours. Aldosterone-induced ERK1/2 phosphorylation was markedly attenuated by pretreatment with eplerenone (10 micromol/L), a selective MR antagonist, or PD98059 (10 micromol/L), a specific inhibitor of MAPK kinase/ERK kinase, which is the upstream activator of ERK1/2. In addition, both eplerenone and PD98059 prevented the aldosterone-induced increases in types I, III, and IV collagen mRNA and [3H]-proline incorporation. These results suggest that aldosterone stimulates collagen gene expression and synthesis via MR-mediated ERK1/2 activation in renal fibroblasts, which may contribute to the progression of aldosterone-induced tubulointerstitial fibrosis.

Effects of a new calcium channel blocker, azelnidipine, on systemic hemodynamics and renal sympathetic nerve activity in spontaneously hypertensive rats.

Antihypertensive treatment with dihydropyridine calcium channel blockers elicits sympathetic nerve activation, which may contribute to cardiovascular events. However, recent clinical studies showed that treatment with azelnidipine, a new dihydropyridine calcium channel blocker, significantly reduced blood pressure in hypertensive patients while either maintaining or actually decreasing heart rate (HR). In this study, we examined the effects of azelnidipine and amlodipine on systemic hemodynamics and renal sympathetic nerve activity (RSNA) in anesthetized spontaneously hypertensive rats (SHR). We also examined the effects of these agents on baroreflex functions by infusing phenylephrine (30 microg/kg/min, i.v.) and sodium nitroprusside (10 microg/kg/min, i.v.) into azelnidipine- or amlodipine-treated SHR. Fifty min after administration of azelnidipine (10 microg/kg/min for 10 min, i.v.), mean arterial pressure (MAP) significantly decreased from 153+/-5 to 122+/-5 mmHg; however, HR and integrated RSNA did not change significantly (from 352+/-9 to 353+/-10 beats/ min and 115+/-5% of baseline, respectively). Infusion of amlodipine (50 microg/kg/min for 10 min) elicited similar effects on MAP (from 152+/-5 to 120+/-4 mmHg). However, amlodipine significantly increased HR (from 351+/-9 to 375+/-11 beats/min) and integrated RSNA (165+/-5% of baseline). Analyses of baroreflex function curves revealed that azelnidipine-treated rats showed a smaller baroreflex function than amlodipine-treated rats (p<0.05). These data suggest that azelnidipine possesses sympathoinhibitory effects, which may be one reason why it had less pronounced effects on HR in hypertensive patients.

Inhibition of actin polymerization enhances commitment to and execution of apoptosis induced by withdrawal of trophic support.

We have previously shown, using jasplakinolide, that stabilization of the actin cytoskeleton enhanced apoptosis induced upon cytokine withdrawal (Posey and Bierer [1999] J. Biol. Chem. 274:4259-4265). It remained possible, however, that a disruption in the regulation of actin dynamics, and not simply F-actin stabilization, was required to affect the transduction of an apoptotic signal. We have now tested the effects of cytochalasin D, a well-characterized agent that promoted actin depolymerization. Actin depolymerization did not affect CD95 (Fas)-induced death of Jurkat T cells in the time course studied but did enhance the commitment to cytokine withdrawal-induced apoptosis of factor-dependent cell lines. The induction of cell death was not the result of direct cytoskeletal collapse, since treatment of the cells with cytochalasin D in the presence of IL-2 did not promote death. As with jasplakinolide, the enhancement of commitment to apoptosis could be delayed by overexpression of the anti-apoptotic protein Bcl-x(L), but, unlike jasplakinolide, cytochalasin D modestly affected the "execution" stage of apoptosis as well. Taken together, these results suggest that changes in actin dynamics, i.e., the rate of actin polymerization and depolymerization, modulate the transduction of the apoptotic signal committing lymphocytes, withdrawn from required growth factors, to the death pathway.