Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models

Abstract

Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is an 18-kDa drug- and cholesterol-binding protein localized to the outer mitochondrial membrane and implicated in a variety of cell and mitochondrial functions. To determine the role of TSPO in ischemia-reperfusion injury (IRI), we used both in vivo and in vitro porcine models: an in vivo renal ischemia model where different conservation modalities were tested and an in vitro model where TSPO-transfected porcine proximal tubule LLC-PK(1) cells were exposed to hypoxia and oxidative stress. The expression of TSPO and its partners in steroidogenic cells, steroidogenic acute regulatory protein (StAR) and cytochrome P-450 side chain cleavage CYP11A1, as well as the impact of TSPO overexpression and exposure to TSPO ligands in vitro in hypoxia-ischemia conditions were investigated. Hypoxia induced caspase activation, reduction of ATP content, and LLC-PK(1) cell death. Transfection and overexpression of TSPO rescued the cells from the detrimental effects of hypoxia and reoxygenation. Moreover, TSPO overexpression was accompanied by a reduction of H(2)O(2)-induced necrosis. TSPO drug ligands did not affect TSPO-mediated functions. In vivo, TSPO expression was modulated by IRI and during regeneration particularly in proximal tubule cells, which do not express this protein at the basal level. Under the same conditions, StAR and CYP11A1 protein and gene expression was reduced without apparent relation to TSPO changes. Pregnenolone was identified and measured in the pig kidney. Pregnenolone synthesis was not affected by the experimental conditions used. Taken together, these results indicate that changes in TSPO expression in kidney regenerating tissue could be important for renal protection and maintenance of kidney function.

Fig. 1.

Effect of ischemia-reperfusion on blood creatinine concentrations. Blood creatinine concentrations, an index of renal function, were measured 1, 3, and 7 days after different experimental conditions [warm ischemia for 60 min and reperfusion (WI60min)]; [cold ischemia for 24 h (CI24h)]; [warm ischemia for 60 min and cold ischemia for 24 h (WI60CI24h)]. Creatinine concentrations in sham-operated group (Sham) were also measured. Unif, uninephrectomized. Creatinine concentrations in experimental groups were significantly increased over control levels at day 3 and decreased to basal values by day 7 (°P < 0.05, °°P < 0.01 vs. Unif and Sham). Creatinine levels at day 3 were significantly greater in CI24h and WI60minCI24h than in WI60min (n = 3 for all time points; *P < 0.05, **P <0.01 vs. WI60min).

Fig. 2.

Representative Western immunoblotting analysis of kidney homogenate using anti-steroidogenic acute regulatory protein (StAR; A–C) and anti-CYP11A1 (D–F) and quantitative real-time PCR measurement of StAR and CYP11A1 (G and H). Kidneys were removed during reperfusion (3 h, 3 days, 7 days) after 24 h of cold preservation (A and D), 60 min of warm ischemia and cold preservation (B and E), or 60 min of warm ischemia without transplantation (C and F); n = 3/group. For StAR, each lane of renal tissue contained 20 μg of cytosolic proteins and, respectively, 2.8m 1.4, and 0.7 μg from testis tissue. For CYP11A1, each lane of renal tissue contained 35 μg of mitochondrial proteins and, respectively, 1.4, 0.7, and 0.35 μg from testis tissue. F: quantitation of StAR mRNA expression in normal kidney (control), kidneys which underwent warm ischemia at body temperature for 60 min (IC 60), kidneys conserved at 4°C for 24 h in University of Wisconsin solution (UW) after 60-min warm ischemia (IC 60 UW), and kidneys conserved at 4°C for 24 in UW (UW). Measurements were performed at both 3 h and 7 days postreperfusion. Testis is used as a positive control for the expression of StAR. G: expression of CYP11A1 in similar tissues and conditions. Values are means ± SE by t-test to control. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3.

Pregnenolone concentrations in renal (A–C) and in testis (D) tissue homogenate. Kidneys were removed during reperfusion (3 h, 3 days, 7 days) after 24 h of cold preservation (A), 60 min of warm ischemia and cold preservation for 24 h (B), or 60 min of warm ischemia without transplantation (C). Values are means ± SE (n = 6/group). Control is representative of the value determined in fresh kidney tissue.

Fig. 4.

Effect of ischemia-reperfusion on translocator protein 18 kDa (TSPO). TSPO staining is indicated by black arrows. Negative control was performed by omitting the primary antibody, and the positive control was performed using adrenal tissue. The representative staining concerns the cortical part of the adrenal where steroidogenesis is located. TSPO staining appears to be parallel with tissue regeneration in kidneys following ischemia-reperfusion lesions. Original magnification ×200.

Fig. 5.

Localization of TSPO-V5-tagged protein in transfected LLC-PK₁ cells. The intracellular distribution of TSPO-V5 protein was examined using immunostaining and confocal microscopy. A: basal expression in nontransfected cells. B: TSPO-V5 subcellular localization in transfected cells. C: representative immunoblot of 3 independent experiments. GAPDH was used as a loading control.

Fig. 6.

Effect of peripheral-type benzodiazepine receptor (PBR) transfection on cell death after hypoxia (A) and reoxygenation (B). LDH activity was measured spectrophotometrically, by following the rate of conversion of NADH to NAD⁺, at 340 nm. LDH release was calculated as [supernatant LDH/(supernatant LDH + cell lysate LDH) × 100]. Values are means ± SE of 6 independent experiments. **P < 0.05 TSPO-transfected LLC-PK₁ cells vs. LLC-PK₁ with empty virus (EV).

Fig. 7.

Renal protein densitometry (A) and representative immunoblots (B) of the regulation of apoptosis by AIF, Bax, and Bcl-xL in the different experimental groups. *P < 0.05 TSPO-transfected cells vs. nontransfected cells, transfected cells plus ligands vs. nontransfected cells plus ligands, and transfected cells plus clonazepam (Clz) vs. nontransfected cells plus ligands

Fig. 8.

Effect of H₂O₂ in the different experimental groups. Cell injury was measured by LDH (A) and MTT assay (B). Caspase 8 (C) and -9 (D) activities were determined and are presented, respectively. *P < 0.05 transfected cells vs. nontransfected cells.

Fig. 9.

Effect of TSPO transfection on cytochrome c release. Top: immunoblot analysis of cytosolic and mitochondrial fraction in the different experimental groups. β-Actin antibody was used as an internal control for cytosolic fractions, and a cytochrome oxydase (COX) antibody was used as an internal control of the mitochondrial fractions. Bottom: relative amount of normalized cytochrome c protein level. *P < 0.05 transfected cells vs. nontransfected cells.

Fig. 10.

Schematic representation of the putative role of TSPO in ischemia-reperfusion injury (IRI). Left: classic steroidogenesis pathway, by which StAr transports free cholesterol to TSPO to transfer it inside the mitochondria to be processed by metabolic enzymes (among which is CYP11A1) into pregnenolone and further steroid hormones. Right: oxidative stress during IRI modulates the expression and conformation of TSPO, favoring the monomeric form. This specific conformation allows TSPO to have a protective effect against lesional mechanisms like apoptosis and maintain mitochondrial integrity. It may affect the downstream steroidogenic metabolism as well. The role of cholesterol in this conformation is yet to be determined.