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. 2010 Jun;298(6):F1332-40.
doi: 10.1152/ajprenal.00737.2009. Epub 2010 Mar 10.

Omi/HtrA2 protease is associated with tubular cell apoptosis and fibrosis induced by unilateral ureteral obstruction

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Omi/HtrA2 protease is associated with tubular cell apoptosis and fibrosis induced by unilateral ureteral obstruction

Jinu Kim et al. Am J Physiol Renal Physiol. 2010 Jun.

Abstract

Kidney fibrosis, a typical characteristic of chronic renal disease, is associated with tubular epithelial cell apoptosis. The results of our recent studies have shown that Omi/HtrA2 (Omi), a proapoptotic mitochondrial serine protease, performs a crucial function in renal tubular epithelial apoptotic cell death in animal models of acute kidney injury, including cisplatin toxicity and ischemia-reperfusion insult. However, the role of Omi in tubulointerstitial disease-associated fibrosis in the kidney remains to be clearly defined. We evaluated the potential function and molecular mechanism of Omi in ureteral obstruction-induced kidney epithelial cell apoptosis and fibrosis. The mice were subjected to unilateral ureteral obstruction (UUO) via the ligation of the left ureter near the renal pelvis. UUO increased the protein level of Omi in the cytosolic fraction of the kidney, with a concomitant reduction in the mitochondrial fraction. UUO reduced the X-linked inhibitor of apoptosis protein (XIAP), a substrate of Omi, and pro-caspase-3, whereas it increased cleaved poly(ADP-ribose) polymerase (cleaved PARP) and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells. When mice were treated with ucf-101, an inhibitor of the proteolytic activity of Omi (6.19 microg/day ip), on a daily basis beginning 2 days before UUO and continuing until the end of the experiment, the Omi inhibitor protected XIAP cleavage after UUO and reduced the increment of PARP cleavage and the numbers of TUNEL-positive cells. Furthermore, the Omi inhibitor significantly attenuated UUO-induced increases in fibrotic characteristics in the kidney, including the atrophy and dilation of tubules, expansion of the interstitium, and increases in the expression of collagens, alpha-smooth muscle actin, and fibronectin. In conclusion, Omi/HtrA2 is associated with apoptotic signaling pathways in tubular epithelial cells activated by unilateral ureteral obstruction, thereby resulting in kidney fibrosis.

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Figures

Fig. 1.
Fig. 1.
Collagen deposition and apoptosis in mouse kidneys subjected to unilateral ureteral obstruction (UUO). The left kidneys of male BALB/c mice were subjected to UUO as described in materials and methods. Three or five days after UUO, the kidneys were harvested, hemisected (A), and cut into 2-μm thickness for Masson trichrome staining (B). The blue color indicates collagen. C: collagen deposition was quantified by counting the number of blue pixels in a 0.1-mm2 field of the kidney section using LabWorks analysis software (10 fields/kidney). D: apoptosis was evaluated via a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The arrows indicate TUNEL-positive cells. E: TUNEL-positive cells were quantified as the number of positive nuclei in a 0.1-mm2 field of the kidney section using LabWorks analysis software (10 fields/kidney). 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to stain the nuclei blue. The red color emitted at 650–710 nm was used to detect general morphology without any treatment. Day 0 indicates before UUO. Values are means ± SE (n = 4). Scale bars: 1 mm in A and 100 μm in B and D.
Fig. 2.
Fig. 2.
Activation of Omi/HtrA2 (Omi) protease in kidneys subjected to UUO. Kidneys were harvested at 0, 3, or 5 days after the UUO induction. A: Omi, copper-zinc superoxide dismutase (CuZnSOD) in the cytosolic fraction (C), and manganese superoxide dismutase (MnSOD) expression in the mitochondrial fraction (M) were determined by Western blot analysis using antibodies against these proteins. Coomassie blue staining was used to detect proteins on the SDS-PAGE gels. B: cytosolic Omi (cyto-Omi) in the cytosolic fraction and mitochondrial Omi (mito-Omi) in the mitochondrial fraction expressions were evaluated by Western blot analysis using antibodies against those proteins, with β-actin used as a control to ensure equal loading of cytosolic fractions (C and D). The protein bands were quantified using LabWorks analysis software. Values are means ± SE (n = 4).
Fig. 3.
Fig. 3.
X-linked inhibitor of apoptosis protein (XIAP) expression in the kidneys subjected to UUO. Kidneys were harvested 0, 3, or 5 days after UUO. A: XIAP expression in the whole kidney lysate was determined by Western blot analysis using antibodies against this protein, with β-actin utilized as a loading marker. B: protein bands were quantified using LabWorks analysis software. Values are means ± SE (n = 4).
Fig. 4.
Fig. 4.
Pro-caspase-3, cleaved poly(ADP-ribose) polymerase (cleaved PARP) expression, and caspase-3 activity in the kidneys subjected to UUO. Kidneys were harvested 0, 3, or 5 days after UUO. A: pro-caspase-3 and cleaved PARP expressions in the whole kidney lysate were evaluated via Western blot analysis using antibodies against these proteins, with β-actin used as a loading marker. B and C: protein bands were quantified using LabWorks analysis software. D: caspase-3 activity in the whole kidney lysate was measured using a Caspase-3 Fluorescent Assay Kit. Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (Ac-DEVC-AMC) was used as a substrate of the caspase-3 enzyme. Values are means ± SE (n = 4). AMC, 7-amino-4-methylcoumarin.
Fig. 5.
Fig. 5.
Effect of ucf-101 on Omi release into the cytoplasm by UUO. Male BALB/c mice were treated with either vehicle or ucf-101 (Omi inhibitor) for 5 days, beginning 2 days before and continuing for 3 days after UUO or sham-operation (sham). The kidneys were harvested 3 days after the operation. A: cyto-Omi in the cytosolic fraction and mito-Omi in the mitochondrial fraction expressions were evaluated by Western blot analysis using antibodies against these proteins, with β-actin used as an equal loading marker of the cytosolic fraction. B and C: protein bands were quantified using LabWorks analysis software. Values are means ± SE (n = 4). NS, no significant difference. *P < 0.05 vs. respective sham.
Fig. 6.
Fig. 6.
Effect of ucf-101, an inhibitor of Omi, on apoptotic signal activation and apoptosis induced by UUO. Male BALB/c mice were treated with either vehicle or ucf-101 for 5 days, beginning 2 days before UUO or sham. Kidneys were harvested 3 days after the operation. A: XIAP, pro-caspase-3, and cleaved PARP protein levels in the whole kidney lysate were determined via Western blot analysis using antibodies against these proteins, with β-actin used as an equal loading marker. BD: protein bands were quantified using LabWorks analysis software. E: caspase-3 activity in the whole kidney lysate was measured using a Caspase-3 Fluorescent Assay Kit. Ac-DEVC-AMC was used a substrate of the caspase-3 enzyme. F: apoptosis was determined via TUNEL assay. Arrows indicate TUNEL-positive cells. White rectangular areas were separated into TUNEL, DAPI, and merged images. G: TUNEL-positive cells were quantified as the number of positive nuclei in a 0.1-mm2 field of the kidney section (10 fields/kidney). Blue indicates DAPI-stained nuclei. Red emitted at 650–710 nm is used here to view the general morphology of the tissue without any treatment. Values are means ± SE (n = 4). Scale bars = 50 μm. *P < 0.05 vs. respective sham.
Fig. 7.
Fig. 7.
Ucf-101 reduces α-smooth muscle actin (SMA), fibronectin expression, and collagen deposition in kidneys subjected to UUO. Male BALB/c mice were treated with either vehicle or ucf-101 beginning 2 days before UUO or sham and continuing until the kidneys were harvested 3 days after the operation. A: α-SMA and fibronectin expressions were determined via Western blot analysis using antibodies against these proteins, with β-actin used as an equal loading marker. B and C: protein bands were quantified using LabWorks analysis software. D and F: α-SMA expression and collagen deposition were determined via immunofluorescence staining and Masson's trichrome staining, respectively. D: arrows indicate α-SMA-positive cells. F: blue indicates collagen. E and G: positive areas of α-SMA and Masson's trichrome were quantified by counting the number of positive-color pixels in a 0.1-mm2 field of the kidney section using LabWorks analysis software (10 fields/kidney). Values are ± SE (n = 4). Scale bars = 50 μm. *P < 0.05 vs. respective sham.
Fig. 8.
Fig. 8.
Ucf-101 reduces XIAP, α-SMA expression, and caspase-3 activity 10 days after UUO. Male BALB/c mice were treated with either vehicle or ucf-101 beginning 2 days before UUO or sham and continuing until the kidneys were harvested 10 days after the operation. A: XIAP and α-SMA expressions were determined via Western blot analysis using antibodies against these proteins. β-Actin expression was determined to evaluate the equal loading marker of samples. B and C: protein bands were quantified using LabWorks analysis software. D: caspase-3 activity was measured using a Caspase-3 Fluorescent Assay Kit. Ac-DEVC-AMC was used a substrate of the caspase-3 enzyme. E: scheme of Omi pathway in UUO. Values are means ± SE (n = 4). Scale bars = 50 μm. *P < 0.05 vs. respective sham.

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