benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Fibrosis

Although heat shock protein 72 kDa (HSP72) protects tubular epithelium from a variety of acute insults, its role in chronic renal injury and fibrosis is poorly characterized. In this study, we tested the hypothesis that HSP72 reduces apoptosis and epithelial-to-mesenchymal transition (EMT), important contributors to tubular cell injury in vitro and in vivo. In rats, orally administered geranylgeranylacetone (GGA), an agent that selectively induces HSP72, markedly reduced both apoptosis and cell proliferation in tubular epithelium and decreased both interstitial fibroblast accumulation and collagen I deposition after unilateral ureteric obstruction, a model of chronic renal tubulointerstitial fibrosis and dysfunction. In cultured renal NRK52E cells, exposure to TGF-beta1 induced EMT and apoptosis, major causes of renal fibrosis and tubular atrophy, respectively. Exposure to a pan-caspase inhibitor (ZVAD-FMK) prevented TGF-beta1-induced apoptosis but did not reduce EMT. In contrast, selective HSP72 expression in vitro inhibited EMT caused by TGF-beta1 as indicated by preserving the E-cadherin expression level and alpha-smooth muscle actin induction. Small interfering RNA directed against HSP72 blocked the cytoprotective effects of HSP72 overexpression on EMT in TGF-beta1-exposed cells. Taken together, our data indicate that HSP72 ameliorates renal tubulointerstitial fibrosis in obstructive nephropathy by inhibiting both renal tubular epithelial cell apoptosis and EMT.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cell Line; Cell Proliferation; Cell Transdifferentiation; Diterpenes; Epithelial Cells; Fibrosis; HSP72 Heat-Shock Proteins; Kidney Failure, Chronic; Kidney Tubules, Proximal; Male; Mesenchymal Stem Cells; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1

Recent works from this laboratory demonstrated potent inhibition of Fas-induced apoptosis in alveolar epithelial cells (AECs) by the angiotensin-converting enzyme (ACE) inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998] and induction of dose-dependent apoptosis in AECs by purified angiotensin (ANG) II [R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos and B. D. Uhal. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L885-L889, 1999]. These findings led us to hypothesize that the synthesis and binding of ANG II to its receptor might be involved in the induction of AEC apoptosis by Fas. Apoptosis was induced in the AEC-derived human lung carcinoma cell line A549 or in primary AECs isolated from adult rats with receptor-activating anti-Fas antibodies or purified recombinant Fas ligand, respectively. Apoptosis in response to either Fas activator was inhibited in a dose-dependent manner by the nonthiol ACE inhibitor lisinopril or the nonselective ANG II receptor antagonist saralasin, with maximal inhibitions of 82 and 93% at doses of 0.5 and 5 microg/ml, respectively. In both cell types, activation of Fas caused a significant increase in the abundance of mRNA for angiotensinogen (ANGEN) that was unaffected by saralasin. Transfection with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of Fas-stimulated apoptosis by 70% in A549 cells and 87% in primary AECs (both P < 0.01). Activation of Fas increased the concentration of ANG II in the serum-free extracellular medium 3-fold in primary AECs and 10-fold in A549 cells. Apoptosis in response to either Fas activator was completely abrogated by neutralizing antibodies specific for ANG II (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by Fas requires a functional renin-angiotensin system in the target cell. They also suggest that therapeutic control of AEC apoptosis is feasible through pharmacological manipulation of the local renin-angiotensin system.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Angiotensinogen; Animals; Antibodies; Antisense Elements (Genetics); Apoptosis; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; fas Receptor; Fibrosis; Gene Expression; Humans; Lisinopril; Lung Neoplasms; Male; Membrane Glycoproteins; Neutralization Tests; Peptidyl-Dipeptidase A; Pulmonary Alveoli; Rats; Rats, Wistar; Receptors, Angiotensin; Renin-Angiotensin System; RNA, Messenger; Signal Transduction; Transfection; Tumor Cells, Cultured