Role of protein kinase C in cisplatin nephrotoxicity

BACKGROUND: Cisplatin is widely used in cancer treatment. The major disadvantage of this antitumor agent is its nephrotoxicity. The mechanism of cisplatin-induced nephrotoxicity has not been clarified. Recent evidence suggests protein kinase C (PKC)-related signal transduction pathways may modulate cisplatin-induced cytotoxicity. METHODS: The effect of cisplatin administration on PKC expression in the kidney and the effect of a PKC inhibitor on cisplatin-induced renal impairment were investigated in rats. RESULTS: A single intraperitoneal injection of 8 mg/kg cisplatin induced remarkable damage in the proximal tubules located in the outer medulla, which was associated with impaired renal function, within 48 h. An immunoblotting study revealed marked expression of alpha-PKC in membrane fractions of medullary tubules prepared from cisplatin-treated rats. In addition, pretreatment with the PKC inhibitor (H-7) protected kidneys from cisplatin-induced damage. CONCLUSIONS: These findings suggest that the nephrotoxic effects of cisplatin may, in part, be related to PKC activation in the renal tubules.     

Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells

Autophagy is a cellular process of bulk degradation of damaged organelles, protein aggregates and other macromolecules in the cytoplasm. It is thought to be a general response to stress contributing to cell death; alternatively it might act as a cytoprotective mechanism. Here we found that administration of cisplatin induced the formation of autophagic vesicles and autophagosomes in mouse kidneys. In cultured proximal tubular cells, the nephrotoxin caused autophagy in a dose- and time-dependent manner prior to apoptosis. Notably, autophagy occurred within hours of cisplatin administration but this was partially suppressed by the p53 inhibitor pifithrin-alpha, suggesting that p53 is involved in autophagic signaling. This cisplatin-induced autophagy was attenuated in renal cells stably transfected with Bcl-2, suggesting an anti-autophagic role for this well-known anti-apoptotic protein. Blockade of autophagy with pharmacological inhibitors (3-methyladenine or bafilomycin) or shRNA knockdown of the autophagic gene Beclin increased tubular cell apoptosis during cisplatin treatment. Our study has found that autophagy occurs in acute kidney injury and this may be an important protective mechanism for cell survival.     

Electrolyte composition of renal tubular cells in gentamicin nephrotoxicity

The effect of long-term gentamicin administration on sodium, potassium, chloride and phosphorus concentrations was studied in individual rat renal tubular cells using electron microprobe analysis. Histological damage was apparent only in proximal tubular cells. The extent of damage was only mild after 7 days of gentamicin administration (60 mg/kg body wt/day) but much more pronounced after 10 days. GFR showed a progressive decline during gentamicin treatment. In non-necrotic proximal tubular cells, sodium was increased from 14.6 +/- 0.3 (mean +/- SEM) in controls to 20.6 +/- 0.4 after 7 and 22.0 +/- 0.8 mmol/kg wet wt after 10 days of gentamicin administration. Chloride concentration was higher only after 10 days (20.6 +/- 0.6 vs. 17.3 +/- 0.2 mmol/kg wet wt). Both cell potassium and phosphorus concentrations were diminished by 6 and 15, and by 8 and 25 mmol/kg wet wt after 7 and 10 days of treatment, respectively. In contrast, no major alterations in distal tubular cell electrolyte concentrations could be observed after either 7 or 10 days of gentamicin administration. As in proximal tubular cells, distal tubular cell phosphorus concentrations were, however, lowered by gentamicin treatment. These results clearly indicate that gentamicin exerts its main effect on proximal tubular cells. Decreased potassium and increased sodium and chloride concentrations were observed in proximal tubular cells exhibiting only mild histological damage prior to the onset of advanced tissue injury. Necrotic cells, on the other hand, showed widely variable intracellular electrolyte concentration patterns.     

Receptor tyrosine kinase and phosphoinositide-3 kinase signaling in malignant mesothelioma

OBJECTIVE: The phosphoinositide-3 kinase signaling pathway is implicated in the development of malignancy and promotes cell-cycle progression and resistance to apoptosis. Malignant mesothelioma tumor specimens demonstrate high levels of the phosphoinositide-3 kinase downstream mediator phosphorylated Akt. Exposure of mesothelioma cell lines to LY294002, a phosphoinositide-3 kinase inhibitor, results in apoptotic cell death and decreased phosphorylated Akt in vitro and tumor burden reduction in vivo. Phosphoinositide-3 kinase is activated by cell-surface receptor tyrosine kinases. We sought to determine which receptors are present in mesothelioma and their role in cellular survival and phosphoinositide-3 kinase signaling. METHODS: Western blot analysis was performed to determine the relative expression of epidermal growth factor receptor, insulin-like growth factor receptor, and platelet-derived growth factor receptor in the mesothelioma cell lines I-45 and REN and the mesothelial line Met5a. After exposure of mesothelioma lines to kinase inhibitors, a cell viability assay was performed, cell-cycle analysis was performed to determine the percentage of apoptosis, and Western blot analysis was performed for phosphorylated Akt. RESULTS: Inhibition of epidermal growth factor receptor resulted in apoptotic cell death and Akt hypophosphorylation in mesothelioma cell lines. Insulin-like growth factor receptor inhibition led to apoptotic cell death without affecting Akt phosphorylation. Platelet-derived growth factor receptor inhibition did not affect cellular survival or phosphoinositide-3 kinase signaling. CONCLUSION: In malignant mesothelioma constitutive activation of phosphoinositide-3 kinase/Akt results in cellular survival and contributes to the malignant phenotype. We have demonstrated that epidermal growth factor receptor inhibition leads to apoptotic cell death through downregulation of phosphoinositide-3 kinase signaling in mesothelioma cell lines, whereas insulin-like growth factor receptor inhibition leads to apoptosis independent of phosphoinositide-3 kinase. Epidermal growth factor receptor, insulin-like growth factor receptor, and phosphoinositide-3 kinase inhibition might be clinically relevant in malignant mesothelioma.     

circSNRK通过上调Akt通路改善肾小管上皮细胞缺血-再灌注损伤

目的  探讨环状RNA SNRK（circSNRK）在缺血-再灌注损伤（IRI）中的作用及机制。方法  构建缺氧-复氧（IRI）细胞模型,检测IRI处理后circSNRK的表达情况及过表达circSNRK对细胞增殖和细胞凋亡的影响。分析circSNRK的作用靶点。将HK2细胞分为空白组（Mock组）、IRI组、对照质粒+IRI组（IRI+NC组）、过表达人circSNRK+IRI组（IRI+circSNRK组）、过表达人circSNRK+IRI+蛋白激酶B（Akt）抑制剂组（IRI+circSNRK+MK2206组）、对照质粒组（NC组）。检测Mock组、IRI组、IRI+NC组、IRI+circSNRK组细胞增殖及凋亡情况。进行circSNRK作用靶点的基因本体（GO）富集分析和京都基因与基因组百科全书（KEGG）聚类分析。检测Mock组、IRI组、IRI+NC组、IRI+circSNRK组细胞CDKN1A、Akt、B细胞淋巴瘤（Bcl）-2、半胱氨酸天冬氨酸蛋白酶（Caspase）-9信使RNA（mRNA）表达水平,p21、Bcl-2、Caspase-9、Akt、p-Akt蛋白表达水平。检测NC组、IRI+NC组、IRI+circSNRK组、IRI+circSNRK+MK2206组细胞增殖及凋亡情况。结果  与Mock组比较,IRI组circSNRK表达水平较低,HK2细胞增殖能力下降,细胞凋亡增多。IRI+circSNRK组细胞增殖能力较IRI+NC组升高,细胞凋亡较IRI+NC组减少。circSNRK可通过51个微小RNA（miRNA）作用于648个靶点。GO富集分析显示,circSNRK作用靶点主要富集于细胞过程和生物调节等生物学过程,细胞部分、细胞和细胞外部分等细胞成分,以及结合、结合蛋白和酶等分子功能。KEGG聚类分析显示,circSNRK作用靶点主要富集在癌症信号通路、磷脂酰肌醇-3-激酶（PI3K）-Akt信号通路和癌症miRNA等相关通路。与Mock组比较,IRI组CDKN1A和Caspase-9 mRNA相对表达量较高,miR-99a-5p RNA表达水平较高,Akt和Bcl-2 mRNA相对表达量较低;与IRI+NC组比较,IRI+circSNRK组CDKN1A和Caspase-9 mRNA相对表达量较低,Akt和Bcl-2 mRNA相对表达量较高,miR-99a-5p RNA表达水平较低,差异均有统计学意义（均为P < 0.05）。IRI组p21和Caspase-9蛋白表达较Mock组增多,p-Akt、Akt和Bcl-2蛋白表达较Mock组减少;IRI+circSNRK组p21和Caspase-9蛋白表达较IRI+NC组减少,p-Akt、Akt和Bcl-2蛋白表达较IRI+NC组增多。circSNRK和Akt上存在miR-99a-5p结合位点。与NC组比较,IRI+NC组细胞增殖能力下降;与IRI+NC组比较,IRI+circSNRK组细胞增殖能力升高;与IRI+circSNRK组比较,IRI+circSNRK+MK2206组细胞增殖能力下降（均为P < 0.05）。IRI+NC组细胞凋亡水平较NC组高;IRI+circSNRK组细胞凋亡水平较IRI+NC组低;IRI+circSNRK+MK2206组细胞凋亡水平较IRI+circSNRK组高。结论  在IRI条件下,circSNRK可影响HK2细胞增殖和凋亡,可能是通过Akt通路发挥作用。     

TLR4 signaling mediates inflammation and tissue injury in nephrotoxicity

The molecular mechanisms of acute kidney injury (AKI) remain unclear. Toll-like receptors (TLRs), widely expressed on leukocytes and kidney epithelial cells, regulate innate and adaptive immune responses. The present study examined the role of TLR signaling in cisplatin-induced AKI. Cisplatin-treated wild-type mice had significantly more renal dysfunction, histologic damage, and leukocytes infiltrating the kidney than similarly treated mice with a targeted deletion of TLR4 [Tlr4(-/-)]. Levels of cytokines in serum, kidney, and urine were increased significantly in cisplatin-treated wild-type mice compared with saline-treated wild-type mice and cisplatin-treated Tlr4(-/-) mice. Activation of JNK and p38, which was associated with cisplatin-induced renal injury in wild-type mice, was significantly blunted in Tlr4(-/-) mice. Using bone marrow chimeric mice, it was determined that renal parenchymal TLR4, rather than myeloid TLR4, mediated the nephrotoxic effects of cisplatin. Therefore, activation of TLR4 on renal parenchymal cells may activate p38 MAPK pathways, leading to increased production of inflammatory cytokines, such as TNF-alpha and subsequent kidney injury. Targeting the TLR4 signaling pathways may be a feasible therapeutic strategy to prevent cisplatin-induced AKI in humans.     

肾损伤分子-1在肾小管损伤中的表达

肾损伤分子-I(Kidneyi.injury molecule-1,KIM-1)是多种急性肾损伤(Acute kidney injury,AKI)早期特异和敏感的标志物,近期研究表明,对肾组织及尿液中KIM-1的检测有助于诊断和鉴别AKI,其应用价值优于血肌酐(Scr)和尿素氮(BUN),此外,KIM-1还参与肾小管上皮细胞的增殖、修复、转分化及肾间质的纤维化过程.本文对近期KIM-1的研究作一综述.     

Deletion of the kinase domain in death-associated protein kinase attenuates tubular cell apoptosis in renal ischemia-reperfusion injury

Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase localized to renal tubular epithelial cells. To elucidate the contribution of DAPK activity to apoptosis in renal ischemia-reperfusion (IR) injury, wild-type (WT) mice and DAPK-mutant mice, which express a DAPK deletion mutant that lacks a portion of the kinase domain, were subjected to renal pedicle clamping and reperfusion. After IR, DAPK activity was elevated in WT kidneys but not in mutant kidneys (1785.7 +/- 54.1 pmol/min/mg versus 160.7 +/- 60.6 pmol/min/mg). Furthermore, there were more TUNEL-positive nuclei and activated caspase 3-positive cells in WT kidneys than in mutant kidneys after IR (24.0 +/- 5.9 nuclei or 9.4 +/- 0.6 cells per high-power field [HPF] versus 6.3 +/- 2.2 nuclei or 4.4 +/- 0.7 cells/HPF at 40 h after ischemia). In addition, the increase in p53-positive tubule cells after IR was greater in WT kidney than in mutant kidneys (9.9 +/- 1.4 cells/HPF versus 0.8 +/- 0.4 cells/HPF), which is consistent with the theory that DAPK activity stabilizes p53 protein. Finally, serum creatinine levels after IR were higher in WT mice than in mutant mice (2.54 +/- 0.34 mg/dl versus 0.87 +/- 0.24 mg/dl at 40 h after ischemia). Thus, these results indicate that deletion of the kinase domain from DAPK molecule can attenuate tubular cell apoptosis and renal dysfunction after IR injury.     

Potassium wasting and other renal tubular defects with rifampin nephrotoxicity

Interstitial nephritis consequent to rifampin was associated with potassium wasting, an acidifying defect, high fractional uric acid excretion, and glucosuria, indicating a multiplicity of renal tubular transport abnormalities. Enlarged kidneys on sonogram and proteinuria were also observed.     

Curcumin counteracts cisplatin-induced nephrotoxicity by preventing renal tubular cell apoptosis

Curcumin has several biological functions particularly antioxidant and anti-inflammatory. The aims of this study are determination of the protective effects of curcumin on cisplatin-induced renal tubular cell apoptosis and related pathways in kidney. Eighteen male Wistar albino rats were randomly divided into three groups (n?=?6): the control, cisplatin (CP), and cisplatin?+?curcumin (CP?+?CUR). Acute renal damage was induced by single dose of cisplatin (7.5?mg/kg) injected by intraperitoneally (i.p). The animals of curcumin-treated group were received daily 200?mg/kg curcumin per os (po), starting from 2 days before the injection of cisplatin to the day of sacrifice. Forty-eight hours after cisplatin injection, samples of cardiac blood and kidneys were harvested from the animals. In this study, the major finding is that curcumin treatment ameliorates the following conditions associated with cisplatin-induced nephrotoxicity: (1) the development of kidney injury (histopathology), (2) inflammatory responses [myeloperoxidase (MPO) and tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, IL-10 levels], (3) the degree of lipid peroxidation [malondialdehyde (MDA) level], (4) renal tubular cell apoptosis (active caspase-3) and expression of related proteins [p53, Fas, and Fas ligand (Fas-L)] by immunohistochemistry, (5) renal dysfunction (serum urea and creatinine). In a conclusion, this study suggests that curcumin has antiapoptotic effect against cisplatin nephrotoxicity, in addition to anti-inflammatory and antioxidant properties.     

The targeting of phosphoinositide-3 kinase attenuates pulmonary metastatic tumor growth following laparotomy

OBJECTIVE: We aimed to characterize a potential role for phosphatidylinositol 3-kinase (PI3k) in leading to accelerated postoperative metastatic tumor growth. BACKGROUND: PI3k enhances tumor cell survival in part by phosphorylating Akt and reducing apoptosis. Postoperatively, apoptosis is reduced within local recurrences and distant metastases. This reduction is associated with the phenomenon of accelerated postoperative tumor growth. METHODS: Balb/c mice underwent a tail vein injection of 1x10 metastatic murine mammary adenocarcinoma 4T1 cells. Animals were divided into the following treatment groups (n=10/group): group A, controls; group B, DMSO intraperitoneally (IP) daily from days 14 to 21; group C, IP LY294002 daily from days 14 to 21; group D, laparotomy only; group E, laparotomy followed by IP DMSO for 7 days; and group F, laparotomy followed by LY294002 IP for 7 days. All laparotomies were performed on day 14. Animals were killed at day 28. Metastatic tumor burden was assessed using the lung/body weight ratio and a histologic metastatic index. Mitotic counts and apoptotic indices were established using a combination of hematoxylin and eosin histology and TUNEL immunohistochemistry. A parallel survival study was performed, and PI3k activity was assessed using western blots for phospho-Akt. RESULTS: Laparotomy was associated with increased systemic tumor burden (P=0.001). Postoperatively, LY294002 significantly attenuated metastatic tumor growth (P<0.001). Effective PI3k inhibition was confirmed by demonstrating a reduced Akt phosphorylation. Moreover, PI3k inhibition led to reduced proliferation, increased apoptosis (P<0.001), and enhanced postoperative survival (P<0.001). CONCLUSIONS: Targeting PI3k with postoperative LY294002 significantly attenuates the acceleration in postoperative metastatic tumor growth seen following laparotomy.     

The pathological role of Bax in cisplatin nephrotoxicity

Nephrotoxicity induced by cisplatin involves tubular cell necrosis and apoptosis; the latter of which may be initiated by multiple mechanisms including activation of the intrinsic mitochondrial pathway. In cultured tubular epithelial cells, cisplatin can activate the proapoptotic protein Bax resulting in cytochrome c release, caspase activation, and apoptosis. Definitive evidence for the involvement of Bax in cisplatin nephrotoxicity in vivo, however, is lacking. We analyzed Bax regulation during cisplatin nephrotoxicity in wild-type mice and determined the pathological role of Bax using mice in which this gene was knocked out. In wild-type mice, cisplatin induced Bax in renal tubular cells which became active, accumulated in the mitochondria, and was accompanied by acute kidney injury. Compared with the wild-type mice renal function, as measured by blood urea nitrogen and serum creatinine, was partially but significantly preserved in Bax knockout mice. The number of apoptotic cells was decreased as was general tissue damage. Additionally, cisplatin-induced cytochrome c release was attenuated in the Bax-deficient mice. This significant decrease in apoptosis and in cytochrome c release was also mirrored in primary cultures of proximal tubular cells prepared from Bax knockout animals. Collectively, our results provide compelling evidence for a role of Bax and its related apoptotic pathway in cisplatin nephrotoxicity.     

Role of CXC chemokine receptor 3 pathway in renal ischemic injury

Chemokines play a major role in the recruitment of leukocytes in inflammation and in the regulation of T helper 1 (Th1)/Th2 immune responses. These mechanisms have been recognized to be important in the pathogenesis of renal ischemia-reperfusion (I/R) injury. The interaction of the CXC chemokine receptor 3 (CXCR3) receptor with its ligands is a key pathogenic pathway in promoting inflammation and in enhancing Th1 immune responses. After the induction of ischemia in the mouse model of renal ischemia, an increase in intrarenal expression of CXCR3 and its ligands was observed. Compared with the wild-type (WT) mice, CXCR3-deficient mice (CXCR3-/-) had significantly lower serum creatinine levels, better survival rate, and significantly less acute tubular necrosis and cellular infiltrates. In the kidney, intracellular staining of infiltrating cells that were recovered from kidneys revealed a lower percentage of CD4+IFN-gamma+ cells in the CXCR3-/- mice compared with the WT mice. Furthermore, adoptive transfer of WT CD3+ cells into CXCR3-/- mice before induction of I/R injury abrogated the protection of CXCR3-/- mice from I/R injury. It is concluded that CXCR3 plays an important role in orchestrating the recruitment of Th1 cells to the ischemic kidney and in mediating I/R injury and therefore may serve as a novel target for the therapy of I/R injury.     

Fluvastatin prevents oxidized low-density lipoprotein-induced injury of renal tubular epithelial cells by inhibiting the phosphatidylinositol 3-kinase/Akt-signaling pathway

BACKGROUND: Lipid abnormalities may play a role in the progression of nephrosis. Recent studies have demonstrated that oxidized low-density lipoprotein (oxLDL) appears to be a detrimental factor for the glomerular mesangium and podocytes. The aim of the present study is to identify the potential effects of oxLDL on tubular epithelial cells (TECs) in vitro and to investigate its associated signal transduction pathway, as well as to examine whether fluvastatin reverses the responses of TECs to oxLDL. METHODS: Hypertrophy of cells was evaluated by measurements of [ 3 H]-leucine incorporation, the total protein contents of cells and cell cycle determined by fluorescence-activated cell sorter (FACS) flow cytometry. TGF-beta beta 1 was quantified by enzyme-linked immunosorbent assay (ELISA). An Akt kinase assay kit was used to determine Akt activity. Akt phosphorylation was determined by Western blot. Wortmannin, the specific inhibitor of phosphatidylinositol-3-kinase (PI3K), was used to reflect the possible involvement of PI3K. RESULTS: The results show that exposure to oxLDL increased Akt activity and phosphorylation in a dose-dependent manner. Stimulating with oxLDL increases the incorporation of [ 3 H]-leucine by 1.3 fold and causes a 1.6-fold increase in total protein content. Compared with that for the control group, the percentage of cells in the G0-G1 phase is 19% higher in the oxLDL group. And the secretion of TGF-beta beta 1 is increased when incubated with oxLDL. Wortmannin treatment significantly inhibits the effects induced by oxLDL, indicating that the effect of oxLDL is PI3K dependent. Fluvastatin significantly decreases Akt phosphorylation and activity in a dose-dependent manner. Incorporation of [ 3 H]-leucine, total protein content, cell cycle distribution and TGF-beta beta 1 expression are all attenuated by fluvastatin, and these effects are prevented with mevalonate. CONCLUSION: These results demonstrate that the PI3K/Akt-signaling transduction system is activated by oxLDL, and may mediate the cell hypertrophy and up-regulation of TGF-beta beta 1 expression from TECs. Fluvastatin may inhibit the activity of PI3K/Akt and prevent injury to TECs from oxLDL.