Fig. 4.

A: serum creatinine kinase (CPK) was not increased by 2,4-dinitrophenol (DNP) and heat exposure. B: periodic acid-Schiff stain with low magnification (×80) showed that tubular injury was evident in the outer medulla (inside the circles). C: TUNEL with low magnification (×80) showed that positive cells were observed mainly in the outer medulla (inside the circle). D: renal ATP after one-time heat exposure. ATP in the kidney harvested soon after a one-time, 30-min heat exposure was decreased. E: renal ATP at euthanization. ATP in the kidney harvested after the 10-day experiment (at 3-h rest period after the second heat exposure) was decreased in the DNP + heat group compared with the nontreated (NT) control group. F: Western blot (WB) analysis of heat shock protein (HSP)70 and mitochondrial protein. HSP70 was increased in the heat and DNP + heat groups. Mitochondrial proteins [cytochrome c oxidase complex IV (COX IV), succinate dehydrogenase subunit A (SDHA), and aconitase-2 (Aco2)] were decreased in the heat and DNP + heat groups. β-Actin was used as an internal control. G–J: quantification of the WB analysis [HSP70 (G), COX IV (H), SDHA (I), and Aco2 (J)]. The intensity and area of bands were analyzed with ImageJ and standardized by β-actin. K: immunohistochemistry with COX IV are presented at low magnification (×80) (left) and high magnification of the outer medulla area (×200) (right). COX IV was less expressed in tubular cells of the outer medulla in the heat and DNP + heat groups. Scale bar = 200 μm. n = 5 mice for A, D, and E and 4 mice for G–J. *P < 0.05; **P < 0.01; ***P < 0.001.