Effect of glycine on prelethal and postlethal increases in calpain activity in rat renal proximal tubules

Effect of glycine on prelethal and postlethal increases in calpain activity in rat renal proximal tubules. The effect of glycine on hypoxia- and ionomycin-induced increases in calpain activity in rat proximal tubules was determined. Calpain activity was determined both in vitro and in the intact cell using the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7- amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC) and Western blotting for calpain-specific spectrin breakdown products (BDP), respectively. At 7.5 minutes of hypoxia (prelethal injury model) there was a significant (10-fold) increase in in vitro calpain activity that was not inhibited by glycine. At 15 minutes of hypoxia (postlethal injury model) there was a further increase in calpain activity that was inhibited by glycine. Normoxic tubules incubated with the calcium ionophore ionomy-cin (5 µM) for two minutes and 10 minutes had a significant increase in calpain activity that was not inhibited by glycine. After 15 minutes of hypoxia in the presence of glycine, there was an increase in calpain-specific spectrin breakdown products (BDP) in both Triton X-100 soluble and cytosolic extracts from proximal tubules. Glycine in concentrations up to 10mM had no direct effect on the in vitro calpain activity of purified calpains. The present study demonstrates that: (1) prelethal increases in calpain activity stimulated by hypoxia and ionomycin treatment are not affected by glycine; (2) calpain-mediated spectrin breakdown during hypoxia occurs in the presence of glycine; (3) glycine does prevent the additional postlethal increase in calpain activity probably by maintaining membrane integrity to calcium influx

Effect of nitric oxide donors on renal tubular epithelial cell-matrix adhesion

Effect of nitric oxide donors on renal tubular epithelial cell-matrix adhesion.BackgroundNitric oxide (NO) and its metabolite, peroxynitrite (ONOO-), are involved in renal tubular cell injury. We postulated that if NO/ONOO- has an effect to reduce cell adhesion to the basement membrane, this may contribute to tubular obstruction and may be partially responsible for the harmful effect of NO on the tubular epithelium during acute renal failure (ARF).MethodsWe examined the effect of the NO donors (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO), spermine NONOate (SpNO), and the ONOO- donor 3-morpholinosydnonimine (SIN-1) on cell-matrix adhesion to collagen types I and IV and fibronectin using three renal tubular epithelial cell lines: LLC-PK1, BSC-1, and OK.ResultsIn LLC-PK1 cells, DETA/NO (500 μm) had no effect, and SpNO (500 μm) had a modest effect on cell adhesion compared with controls. Exposure to SIN-1 caused a dose-dependent impairment in cell-matrix adhesion. Similar results were obtained in the different cell types and matrix proteins. The effect of SIN-1 (500 μm) on LLC-PK1 cell adhesion was not associated with either cell death or alteration of matrix protein and was attenuated by either the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, the superoxide scavenger superoxide dismutase, or the ONOO- scavenger uric acid in a dose-dependent manner.ConclusionsThese results therefore support the possibility that ONOO- generated in the tubular epithelium during ischemia/reperfusion has the potential to impair the adhesion properties of tubular cells, which then may contribute to the tubular obstruction in ARF

Effect of chemokine receptor CXCR4 on hypoxia-induced pulmonary hypertension and vascular remodeling in rats

<p>Abstract</p> <p>Background</p> <p>CXCR4 is the receptor for chemokine CXCL12 and reportedly plays an important role in systemic vascular repair and remodeling, but the role of CXCR4 in development of pulmonary hypertension and vascular remodeling has not been fully understood.</p> <p>Methods</p> <p>In this study we investigated the role of CXCR4 in the development of pulmonary hypertension and vascular remodeling by using a CXCR4 inhibitor AMD3100 and by electroporation of CXCR4 shRNA into bone marrow cells and then transplantation of the bone marrow cells into rats.</p> <p>Results</p> <p>We found that the CXCR4 inhibitor significantly decreased chronic hypoxia-induced pulmonary hypertension and vascular remodeling in rats and, most importantly, we found that the rats that were transplanted with the bone marrow cells electroporated with CXCR4 shRNA had significantly lower mean pulmonary pressure (mPAP), ratio of right ventricular weight to left ventricular plus septal weight (RV/(LV+S)) and wall thickness of pulmonary artery induced by chronic hypoxia as compared with control rats.</p> <p>Conclusions</p> <p>The hypothesis that CXCR4 is critical in hypoxic pulmonary hypertension in rats has been demonstrated. The present study not only has shown an inhibitory effect caused by systemic inhibition of CXCR4 activity on pulmonary hypertension, but more importantly also has revealed that specific inhibition of the CXCR4 in bone marrow cells can reduce pulmonary hypertension and vascular remodeling via decreasing bone marrow derived cell recruitment to the lung in hypoxia. This study suggests a novel therapeutic approach for pulmonary hypertension by inhibiting bone marrow derived cell recruitment.</p

Upregulation of complement proteins in lung cancer cells mediates tumor progression

IntroductionIn vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture.MethodsStudies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq.ResultsChanges in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner.DiscussionBased on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing

The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury

Vascular smooth muscle cells grown on Matrigel. A model of the contractile phenotype with decreased activation of mitogen-activated protein kinase

Vascular smooth muscle cells have been shown to exist in two phenotypic states which have been designated proliferative and contractile. The properties of rat aortic vascular smooth muscle cells grown on Matrigel were compared with cells grown on untreated plastic culture dishes. Cells grown on Matrigel manifested at least four important properties characteristic of the contractile phenotype as compared with cells grown on plastic. The cells grown on Matrigel had altered morphology similar to in vivo studies of contractile vascular smooth muscle. The cells had a low proliferative index, showed enhanced levels of the smooth muscle isoform of alpha-actin, and had an enhanced contractile response to the vasoconstrictor arginine vasopressin. All of these changes were maintained through at least five passages and could be reversed by replating cells grown on Matrigel back to uncoated plastic dishes. Changes in post-receptor signaling pathways which could account for the altered physiologic responses of the cells were investigated. Cells grown on Matrigel showed no alterations in agonist-induced mobilization of intracellular Ca2+ or agonist-stimulated cAMP levels. However, stimulation of mitogen-activated protein kinase (MAP kinase) by both vasoconstrictors and growth factors was inhibited by 50% in cells grown on Matrigel as compared with plastic. This decrease in agonist-induced MAP kinase was associated with a decrease in the levels of both p42 and p44 MAP kinase protein and a decrease in tyrosine phosphorylation of both isoforms in cells grown on Matrigel. Alterations in MAP kinase activation can account at least in part for the observed physiologic responses of contractile vascular smooth muscle. Growth of vascular smooth muscle cells on Matrigel represents a useful model to examine phenotypic-dependent alterations in post-receptor signaling