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      Tubular and Interstitial Cell Apoptosis in the Streptozotocin-Diabetic Rat Kidney

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          Background/Aims: Angiotensin II (Ang II) mediates progressive nephron loss in diabetes and stimulates apoptotic cell death in several tissues. We studied the extent of apoptosis in streptozotocin (STZ) induced diabetic nephropathy in the rat and the effects of insulin and type 1 (AT<sub>1</sub>) or type 2 (AT<sub>2</sub>) Ang II receptor blockade with losartan or PD123319, respectively. Methods: Three groups of rats were studied after 2 and 12 weeks: (1) controls ; (2) STZ-diabetic rats (STZ rats), and (3) STZ-diabetic rats with insulin implants. Additional rats were treated with losartan (25 mg/kg/day) and/or PD123319 (10 mg/kg/day) for 2 weeks. Kidneys were examined for apoptosis, using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, DNA laddering, and electron microscopy. Immunoblotting determined expression of the proapoptotic protein Bax and of the antiapoptotic protein Bcl-2 in proximal tubules. Results: Diabetes caused a significant increase in apoptosis, involving tubular and interstitial cells of cortex and medulla, but not glomerular cells (2 weeks: controls 264 ± 94 vs. STZ rats 1,501 ± 471 apoptotic nuclei/kidney section; p < 0.02; n = 6–8), an effect reversed by insulin. In STZ rats, ultrastructural examination revealed chromatin condensation and nuclear fragmentation in tubular and interstitial cells. At 2 and 12 weeks, a significant decrease in the expression of the antiapoptotic protein Bcl-2 occurred in STZ rat proximal tubules, with restoration by insulin. In STZ rats, treatment for 2 weeks with losartan or PD123319 inhibited apoptosis in the kidneys, with no additive effect of the combination therapy. Conclusions: Apoptosis occurs in diabetic nephropathy, involving tubular and interstitial cells, an effect reversed by insulin therapy. Furthermore, the effects of AT<sub>1</sub> or AT<sub>2</sub> receptor blockade suggest that Ang II is involved in mediating apoptosis in the diabetic kidney.

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          Most cited references 13

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          Angiotensin II type 2 receptor mediates programmed cell death.

          The function of the recently discovered angiotensin II type 2 (AT2) receptor remains elusive. This receptor is expressed abundantly in fetus, but scantily in adult tissues except brain, adrenal medulla, and atretic ovary. In this study, we demonstrated that this receptor mediates programmed cell death (apoptosis). We observed this effect in PC12W cells (rat pheochromocytoma cell line) and R3T3 cells (mouse fibroblast cell line), which express abundant AT2 receptor but not AT1 receptor. The cellular mechanism appears to involve the dephosphorylation of mitogen-activated protein kinase (MAP kinase). Vanadate, a protein-tyrosine-phosphatase inhibitor, attenuated the dephosphorylation of MAP kinases by the AT2 receptor and restored the apoptotic changes. Antisense oligonucleotide to MAP kinase phosphatase 1 inhibited the AT2 receptor-mediated MAP kinase dephosphorylation and blocked the AT2 receptor-mediated apoptosis. These results suggest that protein-tyrosine-phosphatase, including MAP kinase phosphatase 1 activated by the AT2 receptor, is involved in apoptosis. We hypothesize that this apoptotic function of the AT2 receptor may play an important role in developmental biology and pathophysiology.
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            Angiotensin II induces apoptosis of adult ventricular myocytes in vitro.

            To determine whether angiotensin II (Ang II) activates the suicide program of myocytes, primary cultures of adult rat ventricular myocytes were exposed to 10(-9) M of Ang II, for 24 h. Ang II resulted in a five-fold increase in programmed myocyte cell death (PMCD) documented by the terminal deoxynucleotidyl transferase assay and confirmed by DNA agarose gel electrophoresis. Ang II stimulation was associated with translocation of the epsilon and delta isoforms of protein kinase C (PKC) which was coupled with an increase in cytosolic Ca2+ in the cells. The PKC inhibitor chelerythrine abolished Ang II-mediated increases in cytosolic Ca2+ and PMCD. Similarly, pretreatment of cells with the intracellular Ca2+ chelator BAPTA/AM inhibited the formation of DNA strand breaks. Conversely, the Ca2+ ionophore A23187 markedly increased PMCD. Finally, the AT1 receptor antagonist, losartan, completely blocked Ang II-induced PMCD, whereas the AT2 receptor antagonist, PD123319, did not attenuate this phenomenon. In conclusion, ligand binding of AT1 receptors on myocytes triggers PMCD by a mechanism involving PKC-mediated increases in cytosolic calcium, which result in internucleosomal DNA fragmentation.
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              Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure.

              While tubular cell death is a characteristic of acute renal failure (ARF), the molecular mechanisms that modulate this cell death are unclear. Cell fate in acute renal failure hinges on a balance of survival and mortality factors in a changing environment. We further explored this issue by studying selected cell death-related proteins in experimental renal failure. The expression of genes that promote (c-myc, Bax, BclxS) or protect (Bcl2, BclxL) from cell death was studied by Northern blot, Western blot, and immunohistochemistry in murine kidneys following ARF induced by folic acid or in renal tubular epithelial cells (MCT) stressed in culture. Renal mRNA levels encoding for c-myc and BclxL were elevated in ARF while the Bcl2/Bax ratio was decreased (Bcl2 decreased and Bax increased; P < 0.05). Protein levels of BclxL increased and Bcl2 protein decreased. Expression of tumor necrosis factor (TNF-alpha), a mediator of ARF, was also increased. Immunohistochemistry further demonstrated that BclxL was increased in some tubuli and absent in others, while Bcl2 expression decreased diffusely. Bax staining was also patchy among tubuli and individual cells in the tubular wall and lumen. As a relative deficit of survival factors is present in ARF, MCT epithelium were deprived of serum survival factors. This resulted in apoptosis, decreased Bcl2/Bax and BclxL/Bax ratios (P < 0.05) and sensitization to TNF-alpha-induced apoptosis (P < 0.05). The latter was prevented by enforced overexpression of BclxL (P < 0.01). TNF-alpha increased the mRNA levels encoding for c-myc and decreased BclxL expression. Neither MCT cells nor the kidney expressed BclxS. A relative deficit of survival factors likely contributes to changes in levels of BclxL and Bax in ARF. These deficits predispose to cell death induced by persistent lethal factors such as TNF-alpha that is increased in ARF and a potential source of increased c-myc, a downstream facilitator of cell death. These findings implicate members of the Bcl2 family of proteins as regulators of tubular cell death in ARF and single them out as potential therapeutic targets.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                March 2004
                17 November 2004
                : 96
                : 3
                : e77-e88
                aDivision of Nephrology, Department of Medicine, and bDepartment of Laboratory Medicine, The Ottawa Hospital, Ottawa, Ont.; cKidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ont., Canada
                76749 Nephron Exp Nephrol 2004;96:e77–e88
                © 2004 S. Karger AG, Basel

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                Figures: 8, Tables: 1, References: 52, Pages: 1
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