p21对缺血-再灌注损伤后肾小管上皮细胞演变的影响

目的 探讨p21对缺血-再灌注损伤（IRI）后肾小管上皮细胞演变的影响。方法 选择低龄（2个月龄）和高龄（12个月龄）p21（＋／＋）和p21（-／-）鼠，建立左肾IRI模型。于IRI后0、1、3、7d及1、3、6个月光镜下观察肾小管组织学变化，采用免疫组化法检测肾小管上皮细胞增殖细胞核抗原（PCNA）表达，组织化学染色观察肾小管上皮细胞衰老相关β-半乳糖苷酶（SA-β-gal）活力，末端脱氧核糖转移酶介导的生物素化脱氧尿嘧啶缺刻标记技术（TUNEL）检测肾小管上皮细胞凋亡。结果 IRI后0d，肾小管以坏死为主，高龄鼠比低龄鼠严重、p21（-／-）鼠比p21（＋／＋）鼠严重（P均〈0．05）。肾小管上皮细胞凋亡在IRI 1d后出现，7d达高峰，且高龄鼠比低龄鼠明显、p21（-／-）鼠比p21（＋／＋）鼠明显（P均d0．05）。低龄鼠IRI后1个月出现SA—β-gal染色阳性的肾小管上皮细胞，而对侧肾此时未见衰老细胞，3和6个月时衰老的肾小管上皮细胞显著增多，且p21（＋／＋）鼠比p21（-／-）鼠明显（P〈0．05）；p21（＋／＋）高龄鼠IRI后0d双肾即可见大量的SA-β-gal染色阳性肾小管上皮细胞，且较p21（-／-）鼠显著增多（P〈O．05），但1d后，p21（＋／＋）和p21（-／-）鼠IRI肾衰老细胞均明显减少（P均〈0．05），1个月后又呈进行性增加，且p21（＋／＋）鼠始终比p21（-／-）鼠严重。高龄和低龄p21（＋／＋）鼠PCNA阳性染色细胞出现的几率差异无显著性（P〉0．05），但低龄鼠细胞增殖能力要强于高龄鼠；而p21（-／-）鼠的细胞增殖能力明显强于p21（＋／＋）鼠，低龄鼠更为显著（P均〈0．05）。对高龄鼠IRI后1d细胞衰老和凋亡进行相关分析显示，二者呈显著负相关Cp21（＋／＋）鼠：r=-0．82，P〈0．001,p21（-／-）鼠：r=-0．76，P〈0．0013。结论 ①IRI可促进正常肾小管上皮细胞衰老的进程；②已经进入衰老状态的肾小管上皮细胞在遭受IRI刺激后，更易走向死亡[坏死和（或）凋亡]；③p21在IRI所致肾小管上皮细胞演变过程中发挥重要的调控作用。     

A novel free radical scavenger,edarabone, protects against cisplatin-induced acute renal damage in vitro and in vivo

Accumulating evidence suggests that enhanced peroxidative damage caused by reactive oxygen species (ROS) may contribute to the pathogenesis of cisplatin-induced acute renal failure. Nevertheless, little is known about the involvement of oxygen radicals in cisplatin nephropathy. In this study, we investigated the effects of a novel free radical scavenger, 3-methyl-1-phenyl-pyrazolin-5-one (MCI-186; edarabone), on murine proximal tubular cell (PTC) damage induced by exposure to cisplatin in vitro and on renal function in an in vivo model of cisplatin-induced acute renal failure. Edarabone inhibited cisplatin-induced (40 microM, 24 h) cytotoxicity in a concentration-dependent manner (10-5 to 10-3 M). Edarabone also attenuated cisplatin-induced mitochondrial transmembrane potential loss and ROS production of PTCs. In the in vivo study, male Wistar rats were cotreated with cisplatin (5 mg/kg, i.p.) and edarabone (1 or 5 mg/kg, i.v.). Effects of edarabone on the kidney were examined 5 days after treatment. Cisplatin resulted in renal dysfunction, renal tubular damage, mitochondrial damage (assayed by histochemical staining for respiratory chain complex IV), renal protein oxidation (examined by Western blot analysis using a specific antibody for carbonyl group-containing proteins), and tubular apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining). The above changes were attenuated by edarabone treatment. Thus, edarabone exhibited cytoprotective effects in PTCs and renoprotective effects against cisplatin. Our findings suggest that ROS, in particular hydroxyl radicals, are involved in cisplatin nephropathy and that edarabone may be potentially useful in protecting the kidneys and prevention of acute renal failure.     

低氧诱导因子1α基因修饰脂肪干细胞修复小鼠急性肾损伤

背景：干细胞移植为肾损伤的治疗提供了一个新的途径,治疗基因转染干细胞可增强对疾病的治疗效果。目的：探讨低氧诱导因子1α基因修饰的脂肪源性干细胞移植对急性肾损伤小鼠肾脏结构和功能的影响。方法：连续2d向BALB/C裸鼠腹腔注射10mg/kg顺铂诱导急性肾损伤小鼠模型。造模24h后经小鼠尾静脉注射含1×105个脂肪源性干细胞或转染低氧诱导因子1α的脂肪源性干细胞的细胞悬液,3d后留取小鼠血液及肾组织标本进行实验。以注射200μL生理盐水的急性肾损伤小鼠作为模型对照,以正常小鼠作为正常对照。结果与结论：脂肪源性干细胞干预后急性肾损伤小鼠血清肌酐、尿素氮水平降低,肾组织病理改变及肾小管上皮细胞的凋亡病变减轻,肾组织炎症因子RANTES、肿瘤坏死因子α表达降低,白细胞介素10表达升高;其中低氧诱导因子1α基因修饰的脂肪源性干细胞对肾组织细胞凋亡及炎症因子表达作用更明显。免疫荧光染色可见移植的脂肪源性干细胞的存活,但未见其向肾小管上皮细胞转化。结果表明脂肪源性干细胞移植可改善急性肾损伤小鼠的肾脏结构和功能,经低氧诱导因子1α基因修饰后的脂肪源性干细胞作用更显著。     

Attenuation of ischemia/reperfusion-induced renal injury in mice deficient in Na+/Ca2+ exchanger

Using Na+/Ca2+ exchanger (NCX1)-deficient mice, the pathophysiological role of Ca2+ overload via the reverse mode of NCX1 in ischemia/reperfusion-induced renal injury was investigated. Because NCX1(-/-) homozygous mice die of heart failure before birth, we used NCX1(+/-) heterozygous mice. NCX1 protein in the kidney of heterozygous mice decreased to about half of that of wild-type mice. Expression of NCX1 protein in the tubular epithelial cells and Ca2+ influx via NCX1 in renal tubules were markedly attenuated in the heterozygous mice. Ischemia/reperfusion-induced renal dysfunction in heterozygous mice was significantly attenuated compared with cases in wild-type mice. Histological renal damage such as tubular necrosis and proteinaceous casts in tubuli in heterozygous mice were much less than that in wild-type mice. Ca2+ deposition in necrotic tubular epithelium was observed more markedly in wild-type than in heterozygous mice. Increases in renal endothelin-1 content were greater in wild-type than in heterozygous mice, and this reflected the difference in immunohistochemical endothelin-1 localization in necrotic tubular epithelium. When the preischemic treatment with KB-R7943 was performed, the renal functional parameters of both NCX1(+/+) and NCX1(+/-) acute renal failure mice were improved to the same level. These findings strongly support the view that Ca2+ overload via the reverse mode of Na+/Ca2+ exchange, followed by renal endothelin-1 overproduction, plays an important role in the pathogenesis of ischemia/reperfusion-induced renal injury.     

The Duffy antigen receptor for chemokines in acute renal failure: A facilitator of renal chemokine presentation

OBJECTIVE: Acute renal failure remains a major challenge in critical care medicine. Both neutrophils and chemokines have been proposed as key components in the development of acute renal failure. Although the Duffy antigen receptor for chemokines (DARC) is present in several tissues and a highly specific ligand for various chemokines, its exact role in vivo remains unclear. DESIGN: Prospective, controlled experimental study. SETTING: University-based research laboratory. SUBJECTS: C57BL/6 wild-type and DARC gene-deficient mice (DARC-/-). INTERVENTIONS: To unravel the functional relevance of DARC in vivo, we compared wild-type and DARC-/- using neutrophil-dependent models of acute renal failure, induced by either local (renal ischemia-reperfusion) or systemic (endotoxemia, lipopolysaccharide) injury. MEASUREMENTS AND MAIN RESULTS: Plasma creatinine and blood urea nitrogen concentrations served as indicators of renal function or dysfunction. Enzyme-linked immunosorbent assays were used to measure tissue and plasma chemokine concentrations. We also performed immunostaining to localize chemokine expression and flow cytometry to evaluate neutrophil recruitment into the kidney. Following renal injury, wild-type mice developed moderate renal ischemia-reperfusion(lipopolysaccharide, 300% increase in plasma creatinine concentrations) to severe acute renal failure (renal ischemia-reperfusion, 40% mortality) as well as extensive renal neutrophil recruitment. DARC-/- mice exhibited no renal dysfunction (renal ischemia-reperfusion) or only very mild renal dysfunction (lipopolysaccharide, 20% increase in serum creatinine concentrations). DARC-/- mice showed no postischemic neutrophil infiltration. Although DARC-/- and wild-type mice exhibited similar global renal neutrophil-recruitment during endotoxemia, DARC-/- mice showed significantly impaired neutrophil extravasation. Total renal concentrations of the chemokine macrophage inflammatory protein 2, which has been shown to bind to DARC and to be crucial in postischemic acute renal failure, were either identical (lipopolysaccharide) or only moderately different (renal ischemia-reperfusion) between wild-type and DARC-/- mice. Immunostaining revealed an absence of macrophage inflammatory protein-2 in renal endothelial cells of DARC-/- mice. CONCLUSIONS: We suggest that DARC predominantly exerts its effects by controlling spatial chemokine distribution, which in turn regulates neutrophil recruitment and subsequent acute renal failure.     

Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia

Thrombospondin 1 (TSP-1) is a matricellular protein that inhibits angiogenesis and causes apoptosis in vivo and in vitro in several cancerous cells and tissues. Here we identify TSP-1 as the molecule with the highest induction level at 3 hours of IR injury in rat and mouse kidneys subjected to ischemia/reperfusion (IR) injury using the DNA microarray approach. Northern hybridizations demonstrated that TSP-1 expression was undetectable at baseline, induced at 3 and 12 hours, and returned to baseline levels at 48 hours of reperfusion. Immunocytochemical staining identified the injured proximal tubules as the predominant sites of expression of TSP-1 in IR injury and showed colocalization of TSP-1 with activated caspase-3. Addition of purified TSP-1 to normal kidney proximal tubule cells or cells subjected to ATP depletion in vitro induced injury as demonstrated by cytochrome c immunocytochemical staining and caspase-3 activity. The deleterious role of TSP-1 in ischemic kidney injury was demonstrated directly in TSP-1 null mice, which showed significant protection against IR injury-induced renal failure and tubular damage. We propose that TSP-1 is a novel regulator of ischemic damage in the kidney and may play an important role in the pathophysiology of ischemic kidney failure.     

Attenuation of cisplatin-induced acute renal failure is associated with less apoptotic cell death

To clarify the pathophysiologic role of apoptosis in acute renal failure (ARF), we examined whether the attenuation of cisplatin-induced ARF is associated with the change in the degree of apoptotic cell death. The administration of cisplatin (CDDP) (6 mg/kg body weight) in rats induced ARF at day 5, as manifested by a significant increase in serum creatinine (Scr) and tubular damage. CDDP-induced apoptotic cell death was confirmed by electron microscopic examination, agarose gel electrophoresis, and increased cells positive for TaT-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) in the outer medulla of the kidney. Treatment with dimethylthiourea (DMTU)--a scavenger of hydroxyl radicals--or glycine abrogated CDDP-induced increases in Scr, the tubular damage score, and the number of TUNEL-positive cells. Pretreatment with uranyl acetate (UA) induced a significant expression of Bcl-2 in the kidney and ameliorated CDDP-induced increases in Scr, the tubular damage score, and TUNEL-positive cells in the outer stripe of the outer medulla. Our findings indicate (1) that the attenuation of CDDP-induced ARF was associated with less apoptotic cell death and (2) that the induction of the anti-apoptotic protein Bcl-2 attenuated apoptosis and tubular damage. Our results suggest that apoptotic cell death may play an important role in the development of cisplatin-induced ARF.     

骨髓间充质干细胞修复损伤肾小管上皮细胞：可能与可行？

背景：研究表明骨髓间充质干细胞在急性肾损伤后能够通过直接分化为肾小管上皮细胞而促进肾功能的恢复,其修复肾脏的作用机制尚不清楚,能否直接分化为肾小管上皮细胞,目前仍有争议。目的：观察骨髓间充质干细胞输注后急性肾衰竭小鼠肾功能改变,外源性骨髓间充质干细胞在肾组织的分布以及是否向肾小管上皮细胞分化。方法：骨髓间充质干细胞来源于绿色荧光蛋白转基因小鼠。8～10周龄的健康雌性昆白小鼠90只随机随机分为3组。急性肾衰竭组和骨髓间充质干细胞组注射顺铂建立急性肾衰竭模型,骨髓间充质干细胞组在建模后24h经尾静脉输注绿色荧光蛋白转基因小鼠的骨髓间充质干细胞悬液。正常对照组不进行任何干预。建模后第1,4,7,14,28天测定血尿素氮和血肌酐,观察肾组织病理变化,荧光显微镜下观察绿色荧光蛋白阳性的骨髓间充质干细胞在肾组织的分布,共聚焦显微镜下观察骨髓间充质干细胞向肾小管上皮细胞的分化情况。结果与结论：骨髓间充质干细胞组顺铂注射4～14d后,尿素氮、肌酐值比急性肾衰竭组明显降低（P〈0.01或P〈0.05）。骨髓间充质干细胞组第4天肾组织中可见绿色荧光的绿色荧光蛋白细胞,分布在外髓质区肾小管,第7天仍可见少量荧光细胞,同时表达肾小管上皮特异性的功能蛋白megalin。结果提示骨髓间充质干细胞在损伤肾脏可直接分化为肾小管上皮细胞,并改善急性肾衰竭小鼠的肾功能。     

Role of glutathione S-transferase Pi in cisplatin-induced nephrotoxicity

One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyl transpeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have ∼10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum–glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences.     

Endothelial nitric oxide contributes to the renal protective effects of ischemic preconditioning

We determined whether endothelial nitric oxide synthase (eNOS) plays an important role in the renal protective effect of ischemic preconditioning (IP) against the ischemia/reperfusion-induced acute renal failure (ARF) by using eNOS-deficient (eNOS(-/-)) and wild-type (eNOS(+/+)) mice. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. IP, which consists of three cycles of 2-min ischemia followed by 5-min reperfusion, was performed prior to 45-min ischemia. In eNOS(+/+) mice, IP treatment markedly attenuated the ischemia/reperfusion-induced renal dysfunction and significantly improved histological renal damage such as tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. Constitutive nitric oxide synthase activity in the kidney without IP was markedly decreased 6 h after reperfusion, but this decreased response was not observed in eNOS(+/+) mice with IP treatment. The improvement of renal dysfunction in eNOS(+/+) mice with IP treatment was abolished by pretreatment with N(G)-nitro-l-arginine, a nonselective NOS inhibitor, whereas aminoguanidine, an inducible NOS inhibitor, had no effect. Finally, no protective effects of IP on ischemia/reperfusion-induced renal dysfunction and histological damage were observed in eNOS(-/-) mice. These findings strongly support the view that eNOS-mediated NO production plays a pivotal role in the protective effect of IP on ischemia/reperfusion-induced ARF.     

The chemoprotective agent N-acetylcysteine blocks cisplatin-induced apoptosis through caspase signaling pathway

Thiols such as N-acetylcysteine (NAC) are increasingly used in clinical trials of platinum chemotherapy as chemoprotectants. NAC can prevent cisdiamminedichloroplatinum (cisplatin)-induced ototoxicity, nephrotoxicity, and gastrointestinal toxicity; however, the molecular mechanisms of NAC on apoptosis and cisplatin cytotoxicity remain unknown. We investigated cisplatin cytotoxicity and NAC chemoprotection in human tumor cell lines, as assessed by immunoblotting and immunocytochemistry. Cisplatin cytotoxicity was associated with nuclear translocation of apoptosis induction factor, expression of the pro-apoptotic Bax protein, cleavage of caspases 3 and 9, and cleavage of PARP. NAC administration reversed the cytotoxic and apoptotic effects if added concurrent with cisplatin or up to 2 h after cisplatin, but chemoprotection was reduced if NAC administration was delayed more than 2 h and was minimal by 8 h after cisplatin. Expression of tumor suppressor p53 and the cell cycle regulatory protein p21 was stimulated within 5 to 10 min by cisplatin in p53-positive LX-1 small cell lung carcinoma cells, and this effect was blocked by NAC. In p53-negative SKOV3 cells, cisplatin toxicity and NAC chemoprotection remained effective, suggesting that chemoprotection may be mediated through both p53-dependent and -independent pathways. Specific kinase inhibitors demonstrated that cisplatin induced apoptosis through the p38 mitogen-activated protein kinase (MAPK) pathway, not the extracellular signal-regulated kinase MAPK pathway. These results show that NAC blocks both the death receptor and the mitochondrial apoptotic pathways induced by cisplatin. The time course for NAC chemoprotection after cisplatin matches our previous in vivo results and provides an opportunity to manipulate route and timing to maintain cisplatin antitumor efficacy while protecting against chemotherapy side effects.     

Role of p21waf1/cip1 in effects of oxaliplatin in colorectal cancer cells

Clinical studies have shown that oxaliplatin, a novel platinum derivative, is a potent chemotherapeutic agent for colorectal cancer when combined with 5-fluorouracil and leucovorin. Although the toxic activity is based on covalent adducts between platinum and DNA, its actual biological behavior is mostly unknown. In an effort to explore the mechanism of tumor susceptibility to oxaliplatin, we examined the cytotoxic effects of oxaliplatin in colorectal cancer cell lines in reference to p53 gene status. Although p53 gene status did not clearly predict sensitivity to oxaliplatin, p53 wild-type cells including HCT116 were sensitive but HCT116 p53-/- were found to be resistant to oxaliplatin. Oxaliplatin caused strong p21waf1/cip1 induction and G0-G1 arrest in p53 wild-type cells, whereas cisplatin did not induce G0-G1 arrest. Assays using p53 wild but p21waf1/cip1 null HCT116 cells revealed that oxaliplatin did not show G0-G1 arrest and reduced growth-inhibitory effects, suggesting that p21waf1/cip1 may be a key element in oxaliplatin-treated p53 wild-type cells. Although HCT116 is DNA mismatch repair-deficient, a mismatch repair-proficient HCT116+ch3 cell line displayed similar responses with regard to p21waf1/cip1-mediated growth inhibition and G0-G1 arrest. In p53 mutant cells, on the other hand, oxaliplatin caused an abrupt transition from G1 to S phase and eventually resulted in G2-M arrest. This abrupt entry into S phase was associated with loss of the p21waf1/cip1 protein via proteasome-mediated degradation. These findings suggest that p21waf1/cip1 plays a role in oxaliplatin-mediated cell cycle and growth control in p53-dependent and -independent pathways.     

Cisplatin potentiates 1,25-dihydroxyvitamin D3-induced apoptosis in association with increased mitogen-activated protein kinase kinase kinase 1 (MEKK-1) expression

1,25-Dihydroxyvitamin D3 (1,25D3) exhibits potent antitumor activity in the murine squamous cell carcinoma (SCC) SCCVII/SF, and the combination of 1,25D3 with cisplatin (1,25D3/cisplatin) demonstrates even greater activity. Because these agents possess different mechanisms of cytotoxicity, studies were initiated to define the mechanism by which the combination displays enhanced activity. Median dose-effect analysis demonstrates that 1,25D3 and cisplatin act synergistically to inhibit SCC growth. When SCC cells were treated with 1,25D3 (10 nM) and/or cisplatin (0.5 microg/ml), greater caspase-3 activation was observed for the combination than for either agent alone. This suggests that the enhanced cytotoxicity is, at least in part, due to greater induction of apoptosis. No alterations in cellular platinum concentration or platinum-DNA adducts were observed for 1,25D3/cisplatin cotreatment compared with cisplatin treatment alone. Effects of the combination on cisplatin and 1,25D3 signaling pathways in adherent (nonapoptotic) and floating (apoptotic) cells were explored. Cisplatin induced p53 and its downstream targets, p21(Cip1) (p21) and Bax, in both cell populations. In contrast, 1,25D3 reduced p53, p21, and Bax to nearly undetectable levels in adherent cells. In the floating cells, 1,25D3 reduced levels of p53 and p21, but Bax expression was maintained at control levels. Expression of these proteins in cells treated with 1,25D3/cisplatin was similar to treatment with 1,25D3 alone. The two agents also had divergent effects on survival and stress signaling pathways. Phospho-extracellular signal-regulated kinase 1/2 and phospho-Jun levels increased after treatment with cisplatin but decreased after treatment with 1,25D3 and 1,25D3/cisplatin. Moreover, cisplatin decreased levels of mitogen-activated protein kinase kinase kinase (MEKK-1), whereas 1,25D3 up-regulated MEKK-1, and 1,25D3/cisplatin further up-regulated MEKK-1. We propose that the increased cytotoxicity for 1,25D3/cisplatin results from cisplatin enhancement of 1,25D3-induced apoptotic signaling through MEKK-1.     

Impaired IL-18 processing protects caspase-1-deficient mice from ischemic acute renal failure

We sought to determine whether mice deficient in the proinflammatory caspase-1, which cleaves precursors of IL-1 beta and IL-18, were protected against ischemic acute renal failure (ARF). Caspase-1(-/-) mice developed less ischemic ARF as judged by renal function and renal histology. These animals had significantly reduced blood urea nitrogen and serum creatinine levels and a lower morphological tubular necrosis score than did wild-type mice with ischemic ARF. Since caspase-1 activates IL-18, lack of mature IL-18 might protect these caspase-1(-/-) mice from ARF. In wild-type animals, we found that ARF causes kidney IL-18 levels to more than double and induces the conversion of the IL-18 precursor to the mature form. This conversion is not observed in caspase-1(-/-) ARF mice or sham-operated controls. We then injected wild-type mice with IL-18-neutralizing antiserum before the ischemic insult and found a similar degree of protection from ARF as seen in caspase-1(-/-) mice. In addition, we observed a fivefold increase in myeloperoxidase activity in control mice with ARF, but no such increase in caspase-1(-/-) or IL-18 antiserum-treated mice. Finally, we confirmed histologically that caspase-1(-/-) mice show decreased neutrophil infiltration, indicating that the deleterious role of IL-18 in ischemic ARF may be due to increased neutrophil infiltration.     

Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation

Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD(-/-)) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1(-/-) ES cells and Pkd1(+/+) morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1(-/-) and Pkd1(+/+) renal tubular epithelial cells in the early stages of cystogenesis. Pkd1(-/-) cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1(+/+) cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1(-/-) cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1(-/-) mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.     

Sirt1 activation protects the mouse renal medulla from oxidative injury

Sirtuin 1 (Sirt1) is a NAD⁺-dependent deacetylase that exerts many of the pleiotropic effects of oxidative metabolism. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress. Here, we set out to investigate the role of Sirt1 in the kidney. Our initial analysis indicated that it was abundantly expressed in mouse renal medullary interstitial cells in vivo. Knocking down Sirt1 expression in primary mouse renal medullary interstitial cells substantially reduced cellular resistance to oxidative stress, while pharmacologic Sirt1 activation using either resveratrol or SRT2183 improved cell survival in response to oxidative stress. The unilateral ureteral obstruction (UUO) model of kidney injury induced markedly more renal apoptosis and fibrosis in Sirt1^+/– mice than in wild-type controls, while pharmacologic Sirt1 activation substantially attenuated apoptosis and fibrosis in wild-type mice. Moreover, Sirt1 deficiency attenuated oxidative stress–induced COX2 expression in cultured mouse renal medullary interstitial cells, and Sirt1^+/– mice displayed reduced UUO-induced COX2 expression in vivo. Conversely, Sirt1 activation increased renal medullary interstitial cell COX2 expression both in vitro and in vivo. Furthermore, exogenous PGE₂ markedly reduced apoptosis in Sirt1-deficient renal medullary interstitial cells following oxidative stress. Taken together, these results identify Sirt1 as an important protective factor for mouse renal medullary interstitial cells following oxidative stress and suggest that the protective function of Sirt1 is partly attributable to its regulation of COX2 induction. We therefore suggest that Sirt1 provides a potential therapeutic target to minimize renal medullary cell damage following oxidative stress.     

Long-term effects of acetazolamide and sodium chloride loading on cisplatin nephrotoxicity in the rat

The protective effect of acetazolamide or sodium chloride loading on cisplatin nephrotoxicity was investigated in rats. After a single dose of cisplatin (5 mg kg-1 i.p.) kidney function was studied after 5, 28 and 84 days. Acetazolamide (75 mg kg-1 i.p.) was administered as a single dose prior (30 min) to the cisplatin injection. By the time of cisplatin administration, the rats were sodium depleted except the sodium-loaded group. Five days after the cisplatin administration all rats received a regular rat chow for the rest of the experiment. Cisplatin alone caused renal failure that could be observed for up to 12 weeks (ClCr 0.32 +/- 0.13 vs. 0.62 +/- 0.06 ml min-1 x 100 g BW-1) with polyuria (UVol 41.2 +/- 4.5 vs. 18.4 +/- 4.6 ml 24 h-1). Pretreatment with acetazolamide was the most protective manoeuvre tested. Five days after cisplatin, kidney function was significantly better than in rats treated with cisplatin alone (ClCr 0.21 +/- 0.06 vs. 0.03 +/- 0.01 ml min-1 x 100 g BW-1), after 28 days the only sign of nephrotoxicity was polyuria (UVol 28.9 +/- 3.7 vs. 19.0 +/- 2.6 ml 24 h-1) after 84 days no differences could be observed at all. Sodium chloride loading was less protective on cisplatin nephrotoxicity. Impaired renal function could still be observed after 12 weeks (ClCr 0.41 +/- 0.05 vs. 0.62 +/- 0.06 ml min-1 x 100 g BW-1) with no difference in comparison with the rats treated with cisplatin alone. However, since 12 rats died in the group having received cisplatin alone and only one rat in the high-salt group, sodium chloride loading is regarded as being advantageous over sodium depletion on cisplatin nephrotoxicity.     

Ascorbic acid and alpha-tocopherol protect anticancer drug cisplatin induced nephrotoxicity in mice: a comparative study

BACKGROUND: Oxidative stress, resulting from an imbalance between prooxidant and antioxidant systems in favor of the former, largely contributes to immune system deregulation and complications observed in end-stage renal disease (ESRD) and patients treated with hemodialysis. Reactive oxygen species and free radicals are involved in the nephrotoxicity induced by a synthetic anticancer drug cisplatin. METHODS: A comparative study on the nephroprotective effects of antioxidant vitamins (250 and 500 mg/kg, p.o.), vitamin C (ascorbic acid) and vitamin E (alpha-tocopherol), was evaluated using cisplatin (10 mg/kg body wt, i.p.) induced oxidative renal damage in mice. Urea and creatinine in serum were estimated for the renal function. Antioxidant status was estimated in kidney homogenate. RESULTS: We found that both vitamins at 500 mg/kg significantly (P<0.01) protected the nephrotoxicity induced by cisplatin. The cisplatin induced increase of urea and creatinine concentrations were reduced in the vitamins plus cisplatin (250 and 500 mg/kg, p.o.)-treated groups. However the cisplatin induced decline of renal antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities were increased only in the 500 mg/kg vitamins treated groups. Both vitamins at 250 and 500 mg/kg could increase the concentration of reduced glutathione (GSH) and protected the increase of cisplatin induced lipid peroxidation. CONCLUSIONS: Higher doses of vitamins are effective to protect oxidative renal damage and vitamin C is the better nephroprotective agent than vitamin E. The protection is mediated partially by preventing the decline of renal antioxidant status.     

TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity

The purpose of these studies was to examine the role of cytokines in the pathogenesis of cisplatin nephrotoxicity. Injection of mice with cisplatin (20 mg/kg) led to severe renal failure. The expression of cytokines, chemokines, and ICAM-1 in kidney was measured by ribonuclease protection assays and RT-PCR. We found significant upregulation of TNF-alpha, TGF-beta, RANTES, MIP-2, MCP-1, TCA3, IL-1beta, and ICAM-1 in kidneys from cisplatin-treated animals. In addition, serum, kidney, and urine levels of TNF-alpha measured by ELISA were increased by cisplatin. Inhibitors of TNF-alpha production (GM6001, pentoxifylline) and TNF-alpha Ab's reduced serum and kidney TNF-alpha protein levels and also blunted the cisplatin-induced increases in TNF-alpha, TGF-beta, RANTES, MIP-2, MCP-1, and IL-1beta, but not ICAM-1, mRNA. In addition, the TNF-alpha inhibitors also ameliorated cisplatin-induced renal dysfunction and reduced cisplatin-induced structural damage. Likewise, TNF-alpha-deficient mice were resistant to cisplatin nephrotoxicity. These results indicate cisplatin nephrotoxicity is characterized by activation of proinflammatory cytokines and chemokines. TNF-alpha appears to play a central role in the activation of this cytokine response and also in the pathogenesis of cisplatin renal injury.