Supplementary MaterialsSupplementary figures and desks. cisplatin-induced DNA damage and exacerbates tubular injury through the upregulation of p53-dependent pro-apoptotic signaling. Acute kidney injury must be cautiously monitored when ATM inhibitors become available in medical practice in the future. resulted in kidney fibrosis via upregulation of the production of profibrotic cytokines like TGF and CTGF13,14. ATM inhibition inhibited cell cycle arrest after aristrochiac acid treatment and ameliorated the profibrotic gene upregulation. In UNC-1999 novel inhibtior addition, p53, a UNC-1999 novel inhibtior major downstream molecule of ATM, plays an essential role in apoptosis induction after injury, and the inhibition of p53 ameliorated kidney injury and and (encoding megalin) in cisplatin-injected mice, which was significantly lower in the mice that received KU55933 and cisplatin. (encoding Kim-1) expression was upregulated in cisplatin-treated mice, and there was no difference between the mice with or without co-administration of KU55933. However, upregulation of another tubular injury marker, (encoding Ngal), was marked in the kidneys of the mice that received KU55933 and cisplatin. Profibrotic cytokines, and and and was significantly reduced in the mice that received KU55933 and cisplatin (Fig.?4b). expression was not affected by KU55933, but the expression of and and expression did not differ between cisplatin with or without KU55933 (Fig.?4b). Regarding cell cycle markers, we evaluated the expression of UNC-1999 novel inhibtior regulatory molecules associated with the G1/S phase, including and and and mRNA expression was increased in the mice that received cisplatin and KU55933, whereas and mRNA manifestation did not differ among all groups, Rabbit polyclonal to PCMTD1 suggesting that most cells arrested in the G1 phase34. Open in a separate window Figure 4 qPCR analysis of isolated proximal tubular epithelia from the mice that received cisplatin by FACS. (a) Isolation of tdTomato+ tubular epithelial cells using FACS as described in the experimental scheme in Fig.?3a. (b) qPCR of RNA from isolated tubular epithelia for the representative markers of mature tubules (and and and and (encoding p21), and (encoding PUMA) in cisplatin-treated mice, which was slightly higher in the mice that also received KU55933 (Fig.?5d). Considering the upregulation of pro-apoptotic genes, we performed TUNEL staining to evaluate apoptotic cells. TUNEL+ cells were found in cisplatin-treated kidneys, and there were significantly more in the kidneys from mice treated with cisplatin and KU55933 (Fig.?5e,f). As previous studies found that cisplatin can induce mitochondrial injury through activation of the p53-PUMA axis8, we evaluated the expression of TOM20, a protein of the mitochondrial membrane. Immunostaining for TOM20 was weak in the kidneys from mice that received cisplatin, but it was even weaker in those of mice that received both KU55933 and cisplatin (Fig.?5g). Open in a separate window Figure 5 Activation of p53 signaling in proximal tubular epithelia in the cisplatin-treated mice with ATM inhibition. (a) Western blot of protein lysate from isolated tubular epithelia for p53, CDK2, and GAPDH. Representative pictures from n?=?2. Western blotting with n?=?4C6?in each group is shown. Optical density of (b) p53 and (c) CDK2 bands were normalized against those of GAPDH. The normalized density of the samples from the control mice was arbitrarily set to 1 1. (d) qPCR of RNA from isolated tubular epithelia for the downstream signaling of ATM and p53. (e) TUNEL staining of kidney sections (4 days after treatment) and (f) quantification of TUNEL?+?cells. UNC-1999 novel inhibtior (g) Immunostaining of kidney sections (4 days after treatment) for Tom20. (h) Kaplan-Meier curve for animal survival. Pifithrin- slightly improved the mortality rate of cisplatin nephropathy after KU55933 administration. Log rank test. n?=?8?in the pifithrin–treated group and n?=?10?in the other group. (i) The BUN increase at 4 days after treatment did not differ between the two groups: n?=?10 per group. (j) PAS staining of kidney sections (4 days after treatment). For all groups, data are means SEM, *p? ?0.05 vs control, #p? ?0.05 vs cisplatin, Bar = 100 m in (e,j) and = 50 m in (h). We further examined whether additional treatment with pifithrin-, a selective p53 inhibitor, can UNC-1999 novel inhibtior prevent the acceleration of cisplatin nephropathy by ATM inhibition. Pifithrin- slightly improved the mortality price of cisplatin nephropathy with ATM inhibition (Fig.?5h), though it didn’t improve renal function or renal histology (Fig.?5i,j). These total results suggested that pifithrin- improved the repair process after serious.