Cisplatin induces primary necrosis through poly(ADP-ribose) polymerase 1 activation in kidney proximal tubular cells / 대한해부학회지

Treatment with cisplatin for cancer therapy has a major side effect such as nephrotoxicity; however, the role of poly (ADP-ribose) polymerase 1 (PARP1) in necrosis in response to cisplatin nephrotoxicity remains to be defined. Here we report that cisplatin induces primary necrosis through PARP1 activation in kidney proximal tubular cells derived from human, pig and mouse. Treatment with high dose of cisplatin for 4 and 8 hours induced primary necrosis, as represented by the percentage of propidium iodide-positive cells and lactate dehydrogenase release. The primary necrosis was correlated with PARP1 activation during cisplatin injury. Treatment with PJ34, a potent PARP1 inhibitor, at 2 hours after injury attenuated primary necrosis after 8 hours of cisplatin injury as well as PARP1 activation. PARP1 inhibition also reduced the release of lactate dehydrogenase and high mobility group box protein 1 from kidney proximal tubular cells at 8 hours after cisplatin injury. Oxidative stress was increased by treatment with cisplatin for 8 hours as shown by 8-hydroxy-2'-deoxyguanosine and lipid hydroperoxide assays, but PARP1 inhibition at 2 hours after injury reduced the oxidative damage. These data demonstrate that cisplatin-induced PARP1 activation contributes to primary necrosis through oxidative stress in kidney proximal tubular cells, resulting in the induction of cisplatin nephrotoxicity and inflammation.

Influence of Fenofibrate on Apoptosis of Human Renal Proximal Tubular Cells Induced by Free Fatty Acids / 中国医科大学学报

Objective To explore the influence of fenofibrate on apoptosis of human renal proximal tubular cells(HK?2)induced by free fatty acid (FFAs). Methods Methyl azo thiazole blue(determined by MTT)was applied for detection of HK?2 cell proliferation capacity;Spectrophotometry was used to determine the expression of MDA and SOD;MCP?1 and IL?8 in culture supernatant of cells were detected by ELISA;Real?time PCR and Western blot were used to evaluate mRNA and protein expression of Bax and Bcl?2 respectively. Results FFAs inhibited HK?2 cell prolifera?tion in a dose and time dependence. mRNA and protein expression of Bax significantly increased compared with control,but mRNA and protein ex?pression of Bcl?2 decreased. After preincubation of fenofibrate,the inhibition of HK?2 cell proliferation by FFAs was alleviated;expression of SOD increased and expression of MDA,MCP?1 and IL?8 were decreased;mRNA and protein expression of Bax was significantly decreased,but mRNA and protein expression of Bcl?2 was increased. Conclusion FFAs can induce apoptosis of renal proximal tubular cell in a dose and time dependent manner. Fenofibrate can inhibite HK?2 cell apoptosis by decreasing oxidative stress,inhibiting inflammation,up?regulating expression of Bcl?2 and down?regulating expression of Bax,which alleviated nephrotoxicity of FFAs.

BOIOGITO INCREASES FATTY ACID - METABOLISM IN PROXIMAL TUBULAR CELLS THROUGH THE PEROXISOME PROLIFERATORS-ACTIVATED RECEPTOR (PPAR) Α AGONISTIC ACTIVITY / Монголын эм зүй, эм судлал

The promotion of fatty acid metabolism, to which PPARα contributes, has been suggested that it would be participate to maintain the proximal tubular cell function in kidney. The loading on the proximal tubular cell of fatty acids could arise the inflammation and cell death in obesity. One of the “Kampo” medicines, Boiogito (BO) is used for the remedy of overweight women exhibiting chronic fatigues as well as edema in the lower extremities or knees. BO would exhibit the prevention of the proximal tubular cell damage and improvement of kidney function by reducing the portion of fatty acids. In this study, BO was orally administered high fatty acid combined with bovine serum albumin for mice to evaluate the mRNA expression of PPARα quantified by PCR. The increase of PPARα mRNA expression was observed BO administration, followed by reduce the volume of fatty acids in kidney.KEY WORDS: Boiogito, Fatty acid metabolism, PPARα, Proximal Tubular CellINTRODUCTIONObesity is a risk factor for incidence of albuminuria and chronic kidney disease 1, 2, and an accumulating visceral fat would be involved in the regulation of primary stage of nephropathy 3, microalbuminuria. Fatty acids are major contributor to these kidney disorders caused by obesity 4. The binding fatty acids with albumin represents in blood generally, taking up by proximal tubular cells after glomerular filtration from albumin. A peroxisome proliferator - activated receptor (PPARα) has been suggested that it would regulate the fatty acid metabolism. Because the glomerular filtration rate and renal blood flow would increase in overweight patients 5, a large quantity of free fatty acids should be loaded into proximal tubular cells. Therefore, the investigation concerning to PPARα stimulator can be regarded as the fatty acid metabolism - regulation. One of the “Kampo” medicines, Boiogito (BO) is used for the remedy of the inflammation and cell death in obesity, is composed of eight crude drugs: Aluminum Silicate Hydrate with Silicon Dioxide, Astragalus Root, Atractylodes Rhizome, Ginger, Glycyrrhiza, Jujube, Sinomenium Stem and Rhizome. In this study, to clarify the therapeutic mechanisms of BO, we focused on the up - regulating for fatty acid metabolism through the PPARα activation.METHODSKampo formulaeBO was prepared according to the prescription for a one-day dose 6: 3.0 g Aluminum Silicate Hydrate with Silicon Dioxide, 5.0 g Astragalus Root, 3.0 g Atractylodes Rhizome, 1.0 g Ginger, 2.0 g Glycyrrhiza, 4.0 g Jujube, 4.0 g Sinomeniumstem and Rhizome.

Bilirubin Activates Transcription of HIF-1alpha in Human Proximal Tubular Cells Cultured in the Physiologic Oxygen Content

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1alpha protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1alpha was increased by 1.69+/-0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1alpha expression by bilirubin. HIF-1alpha expression decreased by 10 microM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1alpha concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1alpha mRNA expression. In coonclusion, bilirubin enhances HIF-1alpha transcription as well as the up-regulation of HIF-1alpha protein translation through the attenuation of ROS and subunits of NADPH oxidase.

TGF-beta and Fas Expression in Human Proximal Tubular Cell (HPTC) in the Presence of Proteinuria / 대한신장학회잡지

BACKGROUND: Glomerular diseases of diverse origins are characterized by heavy proteinuria and tubulointerstitial changes in pathology. Numerous studies have recently demonstrated that interstitial fibrosis and tubular atrophy are better predictors of renal disease progression compared with glomerular pathology. One of the important mechanisms of these tubulointerstitial injury is tubulointerstitial damage due to increased protein trafficking across the proximal tubular epithelial cells. We tested the hypothesis that tubular cells exposed to high concentration of protein express TGF-beta, which can be related to tubulointerstitial fibrosis, and Fas antigen, which can be associated with tubular cell apoptosis. METHODS: Cultured human proximal tubular cells were incubated with varying concentrations of BSA (1, 10 mg/mL) and nephrotic range proteinuria, due to diabetic nephropathy (1, 10 mg/mL), with or without inactivation of complement. After 24 hr-incubation period, the expressions of TGF-beta and Fas mRNA were examined by RT-PCR. RESULTS: The amount of expression of TGF-beta was increased in BSA 10 mg/mL group (0.78+/-0.12, p=0.016) and in diabetic proteinuria 10 mg/mL group (0.7+/-0.08, p=0.012) compared to control group which was incubated in medium alone (0.48+/-0.02), and the amount of expression of Fas was increased in BSA 10 mg/mL group (0.97+/-0.09, p=0.021) and showed increased tendency in diabetic proteinuria 10 mg/mL group (0.94+/-0.14, p=0.067) also. Furthermore, the anti TGF-beta antibody ameliorated the increased albumin-induced expression of Fas. CONCLUSION: Collectively, our results showed that protein overload increased the expression of TGF-beta & Fas, which can play an important role in tubulointerstitial atrophy by inducing apoptosis of renal tubular cells.

TGF-beta and Fas Expression in Human Proximal Tubular Cell (HPTC) in the Presence of Proteinuria / 대한신장학회잡지

BACKGROUND: Glomerular diseases of diverse origins are characterized by heavy proteinuria and tubulointerstitial changes in pathology. Numerous studies have recently demonstrated that interstitial fibrosis and tubular atrophy are better predictors of renal disease progression compared with glomerular pathology. One of the important mechanisms of these tubulointerstitial injury is tubulointerstitial damage due to increased protein trafficking across the proximal tubular epithelial cells. We tested the hypothesis that tubular cells exposed to high concentration of protein express TGF-beta, which can be related to tubulointerstitial fibrosis, and Fas antigen, which can be associated with tubular cell apoptosis. METHODS: Cultured human proximal tubular cells were incubated with varying concentrations of BSA (1, 10 mg/mL) and nephrotic range proteinuria, due to diabetic nephropathy (1, 10 mg/mL), with or without inactivation of complement. After 24 hr-incubation period, the expressions of TGF-beta and Fas mRNA were examined by RT-PCR. RESULTS: The amount of expression of TGF-beta was increased in BSA 10 mg/mL group (0.78+/-0.12, p=0.016) and in diabetic proteinuria 10 mg/mL group (0.7+/-0.08, p=0.012) compared to control group which was incubated in medium alone (0.48+/-0.02), and the amount of expression of Fas was increased in BSA 10 mg/mL group (0.97+/-0.09, p=0.021) and showed increased tendency in diabetic proteinuria 10 mg/mL group (0.94+/-0.14, p=0.067) also. Furthermore, the anti TGF-beta antibody ameliorated the increased albumin-induced expression of Fas. CONCLUSION: Collectively, our results showed that protein overload increased the expression of TGF-beta & Fas, which can play an important role in tubulointerstitial atrophy by inducing apoptosis of renal tubular cells.

Peripheral Blood Mononuclear Cell Culture Supernatant Increase Fas Expression in human Proximal Tubular Cell After the Initiation of Hemodialysis in Chronic Renal Failure Patients / 대한신장학회잡지

Residual renal function rapidly declines after the initiation of hemodialysis and its mechanisms are supposed to be associated with frequent hypotensive episodes during hemodialysis and subsequent ischemic injury to remnant nephron, but blood-membrane interaction might play an important role because of its ability to activate complement system and other various humoral and cellular mechanisms. Blood monocytes are activated by complements, bacterial contaminants and activated monocytes are known to secrete multiple proinflammatory cytokines such as TNF-alpha, IL-1 beta. The expression of TNF-alpha and IL-1 beta in mRNA and protein level were examined by RT-PCR and ELISA respectively in patients with ESRD after the initiation of hemodialysis. Author also investigated the mRNA expression of Fas in human proximal tubular cell culture in the presence of TNF-alpha and PBMC culture supernatant before and after the initiation of hemodialysis. Compared to PBMC separated before the initiation of HD, the amount of cytokine mRNA from PBMC separated after the initiation of HD showed increased tendency from 0.97+/-0.2 to 1.12+/-0.28 for TNF-alpha(p=0.29), from 1.03+/-0.18 to 1.10+/-027 for IL-1 beta (p=0.54). TNF-alpha and IL-l beta protein level in PBMC culture supernatant also showed increased tendency from 2.25+/-0.5 to 4.254+/-3.77 for TNF-alpha (p=0.10), from 3.5+/-2.08 to 4.0+/-4.3 for IL-1 beta(p=0,25). TNF-alpha increased Fas mRNA expression dose-dependently compared to control but it was not statistically significant(p=0,37, 0.22). Compared to the the level of Fas expression in HPTC cultured in the presence of pre HD PBMC supernatant, the level of Fas expression increased significantly in the presence of post HD PBMC supernatant (0.64+/- 057 vs 1.05+/- 0.12, p=0.01). As a conclusion, cytokine gene expression and secretion can increase as a result of blood-membrane interaction and these might have some influence on the loss of residual renal function in CRF patients maintained on hemodialysis.

Effects of Bene Jones protein and TGF-?_1 on the proliferation of rat renal proximal tubular cells in vitro / 中国病理生理杂志

AIM: To study the effect of Bene Jones protein (BJP) from multiple myeloma(MM) patient and TGF-? 1 on cultured renal proximal tubular cell(PTC) proliferation. METHODS:[ 3H] TdR incorporation was used to study the effect of ?BJP and TGF-? 1 on cultured rat NRK.52E PTC proliferation,the expression of TGF-? 1 in the supernatant of PTC cultured with BJP was assessed with ELISA. RESULTS:① [ 3H] TdR incorporation of PTC was inhibited by BJP in a dose-dependent manner,when co-cultured with 100-800 ?mol/L BJP and 2.0 ?g/L TGF-? 1, the [ 3H] TdR incorporation was lower than that of BJP alone, especially when BJP≥400 ?mol/L; ②The expression of TGF-? 1 in the supernatant of PTC cultured with BJP was increased ,especially when BJP≥400 ?mol/L( P

Effect of Calcium Channel Blockers on Endothelin-1 Production by HDL in Cultured Human Proximal Tubular Cells / 대한신장학회잡지

BACKGROUND: Human high density lipoprotein (HDL) is known to stimulate endothelin-1 (ET-1) production through the phospholipase C (PLC)/Ca2+/ protein kinase C (PKC) pathway. Calcium channel blockers may be involved in the decrease of HDL- induced ET-1 production. This study was designed to evaluate whether HDL-induced ET-1 production was affected by Ca2+ channel blockers in cultured human proximal tubular cells (PTC). METHODS: The human PTC were obtained from human nephrectomized tissues, and cultured in six different media, which were bovine serum free (SF) DMEM/F12 medium alone, and five other SF DMEM/F12 media with 200 microgram/ml of HDL, with 200 microgram/ml of HDL and each 10 micrometer of diltiazem, nifedipine, and verapamil solved in 100% ethanol 0.1 volume%, and with 200 microgram/ml of HDL and 0.1 volume% of 100% ethanol as a control. After 24 hours of exposure, ET-1 in the supernatant was measured by radioimmunoassay, and ET-1 level in each well were marked as pg ET-1/mg cell protein/ 24 hr in consideration of cell count. RESULTS: In SF medium, ET-1 production was 1.803+/-0.295pg/mg cell protein/24 hr. In SF medium with 200 microgram/ml of HDL, ET-1 production significantly increased from 1.803+/-0.295 to 10.860+/-0.476 pg/mg cell protein/24 hr (P<0.05). In SF medium with 200 microgram/ml of HDL and 100% ethanol 0.1 volume%, ET-1 production significantly decreased from 10.860+/-0.476 to 6.700+/-1.273pg/mg cell protein/ 24 hr (P<0.05). In SF media with 200 microgram/ml of HDL and each 10 micrometer of diltiazem, nifedipine, and verapamil solved in 100% ethanol 0.1 volume%, ET- 1 production was decreased from 6.700+/-1.273 to 4.043+/-1.550 by diltiazem (P<0.05), to 3.260+/-0.752pg/ mg cell protein/24 hr by verapamil (P<0.05), and to 4.414+/-1.567pg/mg cell protein/24 hr by nifedipine (P=0.067). CONCLUSION: These results suggest that the HDL- induced ET-1 production in cultured human PTC was significantly decreased by diltiazem and verapamil, and it seemed to be decreased by nifedipine.

Effect of nitric oxide on iron-mediated cytotoxicity in primary cultured renal proximal tubules / 中华肾脏病杂志

Objective To explore the possible mechanism of nitric oxide(NO) involved in iron-mediated cytotoxicity on renal tubular cells, meanwhile to estimate the effect of reactive oxygen sepcies scavenger on iron-mediated cytotoxicity and its relation to nitric oxide. Methods in this study, the relationship between NO production and lactate dehydrogenase(LDH) release were observed in primary subconfluent proximal tubular cells coincubated with different doses of NTA-Fe and lipopolysaccharide(LPS) alone or in combination. NO production was monitored by NO2 -- concentration in supernatant based on Griess reaction. Meanwhile, semi-quantitative RT-PCR was applied to detect the inducible nitric oxide synthase (iNOS) mRNA level induced by NTA-Fe and LPS together. In addition, experimental groups were exposed to reactive oxygen species (ROS) scavengers to determine the impact of the interaction between NO and ROS on iron-mediated cytotoxicity. Results After 12-hour coincubation, NTA-Fe could increase both LDH release and NO2 production in a dose-dependent manner (P 0. 05 ) although tubular injury was aggravated (P