Glomerular Mesangial Cell pH Homeostasis Mediates Mineralocorticoid Receptor-Induced Cell Proliferation

Mineralocorticoids (e.g., aldosterone) support chronic inflammatory tissue damage, including glomerular mesangial injury leading to glomerulosclerosis. Furthermore, aldosterone leads to activation of the extracellular signal-regulated kinases (ERK1/2) in rat glomerular mesangial cells (GMC). Because ERK1/2 can affect cellular pH homeostasis via activation of Na+/H+-exchange (NHE) and the resulting cellular alkalinization may support proliferation, we tested the hypothesis that aldosterone affects pH homeostasis and thereby cell proliferation as well as collagen secretion also in primary rat GMC. Cytoplasmic pH and calcium were assessed by single-cell fluorescence ratio imaging, using the dyes BCECF or FURA2, respectively. Proliferation was determined by cell counting, thymidine incorporation and collagen secretion by collagenase-sensitive proline incorporation and ERK1/2-phosphorylation by Western blot. Nanomolar aldosterone induces a rapid cytosolic alkalinization which is prevented by NHE inhibition (10 µmol/L EIPA) and by blockade of the mineralocorticoid receptor (100 nmol/L spironolactone). pH changes were not affected by inhibition of HCO3− transporters and were not dependent on HCO3−. Aldosterone enhanced ERK1/2 phosphorylation and inhibition of ERK1/2-phosphorylation (10 µmol/L U0126) prevented aldosterone-induced alkalinization. Furthermore, aldosterone induced proliferation of GMC and collagen secretion, both of which were prevented by U0126 and EIPA. Cytosolic calcium was not involved in this aldosterone action. In conclusion, our data show that aldosterone can induce GMC proliferation via a MR and ERK1/2-mediated activation of NHE with subsequent cytosolic alkalinization. GMC proliferation leads to glomerular hypercellularity and dysfunction. This effect presents a possible mechanism contributing to mineralocorticoid receptor-induced pathogenesis of glomerular mesangial injury during chronic kidney disease.

Abstract 487: Dihyrosphingosine 1 Phosphate Mediates Collagen Synthesis in Cardiac Fibroblasts Throught Pi3k/akt- Mtor Signalling Pathway

Background: Cardiac fibrosis is one of the hallmarks of cardiac remodelling in cardiomyopathies such as heart Failure (HF). Dyslipidemia plays a role in the progression of HF. The sphingolipid, dihydrosphingosine 1 phosphate (dhS1P) has been shown to bind to high density lipids in plasma. Unlike its analog, spingosine 1 phosphate (S1P), the role of dhS1P in cardiac fibrosis is not known. The aim of this study is to determine the role dhS1P plays in cardiac fibrosis through the PI3K/Akt- mTOR pathway. Method: Neonatal rat cardiac fibroblasts (NCF) were isolated from 1-2 day old pups with enzymic digestion. After pre-treating with the PI3K inhibitor, Wortmannin (W, 0.1 - 10.0μM), cells were stimulated with dhS1P for 48 hours. NCF collagen synthesis was determined by 3H-proline incorporation. NCF were also treated for protein and gene expression analysis. Results: Exogenous addition of 3 μM dhS1P stimulated significant increase in collagen synthesis (p<0.005) which was dose dependently inhibited by W (p < 0.0001, Fig. 1A). Western blot analysis showed that W reduced Akt, mTOR, and S6 activation in the presence of dhS1P. dhS1P also increased protein levels of TGFβ, Coll 1 and TIMP1. W reduced dhS1P elevated TIMP1, and SK1, but not TGFβ1 gene expression (Fig. 1B). Conclusion: Our study demonstrates for the first time that dhS1P can cause cardiac cellular fibrosis via PI3K/Akt- mTOR pathway. Its inhibition may represent a novel therapeutic strategy for cardiac fibrosis.

Dysfunction of Collagen Synthesis and Secretion in Chondrocytes Induced byWisp3Mutation

Wisp3gene mutation was shown to cause spondyloepiphyseal dysplasia tarda with progressive arthropathy (SRDT-PA), but the underlying mechanism is not clear. To clarify this mechanism, we constructed the wild and mutatedWisp3expression vectors and transfected into human chondrocytes lines C-20/A4;Wisp3proteins subcellular localization, cell proliferation, cell apoptosis, andWisp3-mediated gene expression were determined, and dynamic secretion of collagen in transfected chondrocytes was analyzed by14C-proline incorporation experiment. MutatedWisp3protein increased proliferation activity, decreased apoptosis of C-20/A4 cells, and aggregated abnormally in cytoplasm. Expression of collagen II was also downregulated in C-20/A4 cells transfected with mutatedWisp3. Wild typeWisp3transfection increased intracellular collagen content and extracellular collagen secretion, but the mutatedWisp3lost this function, and the peak phase of collagen secretion was delayed in mutatedWisp3transfected cells. Thus abnormal protein distribution, cell proliferation, collagen synthesis, and secretion inWisp3mutated chondrocytes might contribute to the pathogenesis of SEDT-PA.

Downregulation of NOX4 Expression by Roflumilast N-Oxide Reduces Markers of Fibrosis in Lung Fibroblasts

The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin- (BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulatein vitrothe oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F2-isoprostanes that haveper sefibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F2-isoprostane 8-epi-PGF2αwas investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF2α. Proliferation and collagen synthesis were assessed as [3H]-thymidine and [3H]-proline incorporation. Reactive oxygen species (ROS) and F2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF2α per sestimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observedin vivomay be related to its ability to mitigate ROS generation via downregulation of NOX4.

Abstract P348: High Glucose Induces Smad Activation via the Transcriptional Coregulator P300 and Contributes to Cardiac Fibrosis and Hypertrophy

Background: Despite advances in the treatment of heart failure (HF), the mortality remains high, particularly in those individuals with diabetes mellitus. Activated transforming growth factor beta (TGF-ß) contributes to the pathogenesis of diabetic cardiomyopathy. We hypothesized that the transcriptional co-activator p300 regulates glucose induced activation of TGF-ß via acetylation of a specific Lysine residue (Lys19) in the Mad homology 1 domain of Smad 2, and that by inhibiting p300, TGF-ß activity will be reduced and heart failure ameliorated/prevented. Methods: p300 activity and Smad acetylation in normal glucose (5.6 mmol/L - NG) and high glucose (25 mmol/L - HG) media were assessed in H9c2 rat cardiomyoblasts. [H]3 proline incorporation was assessed in cardiac fibroblasts as a marker of collagen synthesis. The role of increased p300 activity was assessed in vitro by using a known p300 inhibitor, curcumin or siRNA directed at p300 and in vivo in a hemodynamically validated model of diabetic cardiomyopathy, the (mRen)2-27 transgenic rat. Results: H9c2 cells exposed to HG demonstrated increased p300 activity c/w NG media, that was reduced by p300 inhibition using curcumin or p300 siRNA (all p<0.01). Increased p300 activity in HG media increased [H]3 proline incorporation (p<0.05). This effect was attenuated by treatment with curcumin/p300 siRNA (p<0.01). Finally, H9c2 cells were stimulated, extracted protein was immunoprecipitated with Smad2, and lys19 acetylation assessed. Acetylation of the Lys19 was reduced in cells pre-incubated with the p300 inhibitor (p<0.05). To determine the functional significance of p300 inhibition, diabetic Ren-2 rats were randomised to receive either curcumin/vehicle for 6 weeks. Curcumin treated diabetic rats had reduced cardiac hypertrophy and improved chamber compliance when c/w untreated diabetic counterparts (all p<0.01). Conclusions: These findings demonstrate that high glucose increases activity of the transcriptional coregulator p300, acetylating Smad2 and promoting cardiac fibrosis and hypertrophy. Inhibition of p300 reduces cardiac hypertrophy and results in improved diastolic function. Modulation of the p300 may be a novel strategy to treat diabetes induced heart failure.

Effects of cardiotonic steroids on dermal collagen synthesis and wound healing

We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na-K-ATPase pathway (Elkareh et al. Hypertension 49: 215–224, 2007). In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid marinobufagenin in a dose- and time-dependent fashion. An eightfold increase in collagen synthesis was noted when cells were exposed to 10 nM marinobufagenin for 24 h ( P < 0.01). Similar increases in proline incorporation were seen following treatment with digoxin, ouabain, and marinobufagenin (10 nM × 24 h, all results P < 0.01 vs. control). The coadministration of the Src inhibitor PP2 or N-acetylcysteine completely prevented collagen stimulation by marinobufagenin. Next, we examined the effect of digoxin, ouabain, and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model. Olive oil was chosen as the digoxin carrier because of a favorable partition coefficient observed for labeled digoxin with saline. This application significantly accelerated in vivo wound healing in rats wounded with an 8-mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both P < 0.01). The data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, as could potentially be exploited to accelerate wound healing.

Short-term peaks in glucose promote renal fibrogenesis independently of total glucose exposure

Postprandial hyperglycemia is implicated as a risk factor predisposing to vascular complications. This study was designed to assess recurrent short-term increases in glucose on markers of renal fibrogenesis. Human renal cortical fibroblasts were exposed to fluctuating short-term (2 h) increases to 15 mM d-glucose, three times a day over 72 h, on a background of 5 mM d-glucose. To determine whether observed changes were due to fluctuating osmolality, identical experiments were undertaken with cells exposed to l-glucose. Parallel experiments were performed in cells exposed to 5 mM d-glucose and constant exposure to either 15 or 7.5 mM d-glucose. Fluctuating d-glucose increased extracellular matrix, as measured by proline incorporation ( P < 0.05), collagen IV ( P < 0.005), and fibronectin production ( P < 0.001), in association with increased tissue inhibitor of matrix metalloproteinase (MMP) ( P < 0.05). Sustained exposure to 15 mM d-glucose increased fibronectin ( P < 0.001), in association with increased MMP-2 ( P = 0.01) and MMP-9 activity ( P < 0.05), suggestive of a protective effect on collagen matrix accumulation. Transforming growth factor-β1 (TGF-β1) mRNA was increased after short-term (90 min) exposure to 15 mM glucose ( P < 0.05) and after 24-h exposure to 7.5 mM ? ( P < 0.05). Normalization of TGF-β1 secretion occurred within 48 h of constant exposure to an elevated glucose. Fluctuating l-glucose also induced TGF-β1 mRNA and a profibrotic profile, however, to a lesser extent than observed with exposure to fluctuating d-glucose. The results suggest that exposure to fluctuating glucose concentrations increases renal interstitial fibrosis compared with stable elevations in d-glucose. The effects are, in part, due to the inherent osmotic changes.