Comparison of the medium cutoff dialyzer and postdilution hemodiafiltration on the removal of small and middle molecule uremic toxins.

BACKGROUND: The medium cutoff (MCO) dialyzer increases the removal of several middle molecules more effectively than high-flux hemodialysis (HD). However, comparative data addressing the efficacy and safety of MCO dialyzers vs. postdilution hemodiafiltration (HDF) in Korean patients are lacking. METHODS: Nine patients with chronic HD were included in this pre-post study. Patients underwent HD with an MCO dialyzer for 4 weeks, followed by a 2-week washout period using a high-flux dialyzer to minimize carryover effects, and then turned over to postdilution HDF for 4 weeks. Reduction ratios and differences in the uremic toxins before and after dialysis were calculated from the MCO dialysis, postdilution HDF, and high-flux HD. In the in vitro study, EA.hy926 cells were incubated with dialyzed serum. RESULTS: Compared to postdilution HDF, the MCO dialyzer achieved significantly higher reduction ratios for larger middle molecules (myoglobin, kappa free light chain [κFLC], and lambda FLC [λFLC]). Similarly, the differences in myoglobin, κFLC, and λFLC concentrations before and after the last dialysis session were significantly greater in MCO dialysis than in postdilution HDF. The expression of Bax and nuclear factor κB was decreased in the serum after dialysis with the MCO dialyzer than with HDF. CONCLUSION: Compared with high-volume postdilution HDF, MCO dialysis did not provide greater removal of molecules below 12,000 Da, whereas it was superior in the removal of larger uremic middle molecule toxins in patients with kidney failure. Moreover, these results may be expected to have an anti-apoptotic effect on the human endothelium.

Stanniocalcin­1 suppresses TGF­ß­induced mitochondrial dysfunction and cellular fibrosis in human renal proximal tubular cells.

Stanniocalcin­1 (STC1), a multifunctional glycoprotein with antioxidant and anti­inflammatory properties, serves an important role in kidney protection. STC1 is one of the few hormones targeted to the mitochondria to regulate mitochondrial quality control by suppressing oxidative stress and mitochondrial damage. However, the mechanisms underlying the effect of STC1 remain unclear. The present study aimed to investigate the protective role of recombinant STC1 (rSTC1) in renal fibrosis and to identify the mechanisms underlying cellular fibrosis in HK2 human renal proximal tubular cells. Semi­quantitative PCR, western blot analysis and confocal microscopy were used to detect the mRNA levels, protein levels and mitochondrial membrane potential (MMP). Mitochondrial superoxide production was evaluated using MitoSox staining. rSTC1 attenuated TGF­ß­induced downregulation of AMP­activated protein kinase and uncoupling protein 2 (UCP2). Treatment of HK2 cells with TGF­ß reduced the MMP and increased the production of reactive oxygen species (ROS). In addition, TGF­ß treatment upregulated fibrotic markers, such as α­SMA and fibronectin, in HK2 cells. Treatment with rSTC1 and TGF­ß suppressed mitochondrial ROS production by recovering the MMP and reversed the upregulation of fibrotic markers in HK2 cells. The effects of rSTC1 were reversed when UCP2 expression was silenced. The present study revealed a novel role of STC1 in preventing TGF­ß induced cellular fibrosis in HK2 cells.

Renoprotective Effects of Maslinic Acid on Experimental Renal Fibrosis in Unilateral Ureteral Obstruction Model via Targeting MyD88.

Maslinic acid (MA), also named crategolic acid, is a pentacyclic triterpene extracted from fruits and vegetables. Although various beneficial pharmacological effects of MA have been revealed, its effect on renal fibrosis remains unclear. This study was designed to clarify whether MA could attenuate renal fibrosis and determine the putative underlying molecular mechanisms. We demonstrated that MA-treated mice with unilateral ureteral obstruction (UUO) developed a histological injury of low severity and exhibited downregulated expression of fibrotic markers, including α-smooth muscle actin (α-SMA), vimentin, and fibronectin by 38, 44 and 40%, and upregulated expression of E-cadherin by 70% as compared with untreated UUO mice. Moreover, MA treatment restored the expression levels of α-SMA, connective tissue growth factor, and vimentin to 10, 7.8 and 38% of those induced by transforming growth factor (TGF)-ß in NRK49F cells. MA decreased expression of Smad2/3 phosphorylation and Smad4 in UUO kidneys and TGF-ß treated NRK49F cells (p < 0.05, respectively). Notably, MA specifically interferes with MyD88, an adaptor protein, thereby mitigating Smad4 nuclear expression (p < 0.01 compared to TGF-ß treated group) and ameliorating renal fibrotic changes (p < 0.01 for each fibrotic markers compared to TGF-ß induced cells). In addition, in the UUO model and lipopolysaccharide-induced NRK49F cells, MA treatment decreased the expression of IL-1ß, TGF-α and MCP-1, ICAM-1, associated with the suppression of NF-κB signaling. These findings suggest that MA is a potential agent that can reduce renal interstitial fibrosis, to some extent, via targeting TGF-ß/Smad and MyD88 signaling.

Glycol chitosan-based tacrolimus-loaded nanomicelle therapy ameliorates lupus nephritis.

BACKGROUND: Recently, we developed hydrophobically modified glycol chitosan (HGC) nanomicelles loaded with tacrolimus (TAC) (HGC-TAC) for the targeted renal delivery of TAC. Herein, we determined whether the administration of the HGC-TAC nanomicelles decreases kidney injury in a model of lupus nephritis. Lupus-prone female MRL/lpr mice were randomly assigned into three groups that received intravenous administration of either vehicle control, an equivalent dose of TAC, or HGC-TAC (0.5 mg/kg TAC) weekly for 8 weeks. Age-matched MRL/MpJ mice without Faslpr mutation were also treated with HGC vehicle and used as healthy controls. RESULTS: Weekly intravenous treatment with HGC-TAC significantly reduced genetically attributable lupus activity in lupus nephritis-positive mice. In addition, HGC-TAC treatment mitigated renal dysfunction, proteinuria, and histological injury, including glomerular proliferative lesions and tubulointerstitial infiltration. Furthermore, HGC-TAC treatment reduced renal inflammation and inflammatory gene expression and ameliorated increased apoptosis and glomerular fibrosis. Moreover, HGC-TAC administration regulated renal injury via the TGF-ß1/MAPK/NF-κB signaling pathway. These renoprotective effects of HGC-TAC treatment were more potent in lupus mice compared to those of TAC treatment alone. CONCLUSION: Our study indicates that weekly treatment with the HGC-TAC nanomicelles reduces kidney injury resulting from lupus nephritis by preventing inflammation, fibrosis, and apoptosis. This advantage of a new therapeutic modality using kidney-targeted HGC-TAC nanocarriers may improve drug adherence and provide treatment efficacy in lupus nephritis mice.

Anti-Apoptotic Effect of G-Protein-Coupled Receptor 40 Activation on Tumor Necrosis Factor-α-Induced Injury of Rat Proximal Tubular Cells.

G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic ß-cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-α mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit induced by TNF-α treatment. TNF-α treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-α treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-α and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-κB signaling pathway.

Chronic and acute stress monitoring by electrophysiological signals from adrenal gland.

We present electrophysiological (EP) signals correlated with cellular cell activities in the adrenal cortex and medulla using an adrenal gland implantable flexible EP probe. With such a probe, we could observe the EP signals from the adrenal cortex and medulla in response to various stress stimuli, such as enhanced hormone activity with adrenocorticotropic hormone, a biomarker for chronic stress response, and an actual stress environment, like a forced swimming test. This technique could be useful to continuously monitor the elevation of cortisol level, a useful indicator of chronic stress that potentially causes various diseases.

Histone deacetylase inhibitor, CG200745 attenuates renal fibrosis in obstructive kidney disease.

Tubulointerstitial fibrosis is a common feature of kidney disease. Histone deacetylase (HDAC) inhibitors have been reported to attenuate renal fibrosis progression. Here, we investigated the effect of CG200745, a novel HDAC inhibitor, on renal fibrosis development in a mouse model of unilateral ureteral obstruction (UUO). To examine the effects of CG200745 on renal fibrosis in UUO, C57BL/6 J male mice were divided into three groups: control, UUO, and CG200745 (30 mg/kg/day)-treated UUO groups. CG 200745 was administered through drinking water for 1 week. Human proximal tubular epithelial (HK-2) cells were also treated with CG200745 (10 µM) with or without TGF-ß (2 ng/mL). Seven days after UUO, plasma creatinine did not differ among the groups. However, plasma neutrophil gelatinase-associated lipocalin (NGAL) levels were markedly increased in the UUO group, which were attenuated by CG200745 treatment. UUO kidneys developed marked fibrosis as indicated by collagen deposition and increased α-smooth muscle actin (SMA) and fibronectin expression. CG200745 treatment attenuated these fibrotic responses and suppressed UUO-induced production of transforming growth factor-beta1 (TGF-ß) and phosphorylation of Smad-2/3. CG200745 treatment also attenuated UUO-induced inflammation as indicated by the expression of inflammatory markers. Furthermore, CG200745 attenuated phosphorylation of p38 mitogen-activated protein kinase in UUO kidneys. In HK-2 cells, TGF-ß induced the expression of α-SMA and fibronectin, which were attenuated by CG200745 cotreatment. These results demonstrate that CG200745, a novel HDAC inhibitor, has a renoprotective effect by suppressing renal fibrosis and inflammation in a UUO mouse model.

Resveratrol attenuates 4-hydroxy-2-hexenal-induced oxidative stress in mouse cortical collecting duct cells.

Resveratrol (RSV) may provide numerous protective eff ects against chronic inflammatory diseases. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress, and aldehyde products formed during lipid peroxidation, such as 4-hydroxy-2-hexenal (HHE), might be responsible for tubular injury. This study aimed at investigating the eff ects of RSV on renal and its signaling mechanisms. While HHE treatment resulted in decreased expression of Sirt1, AQP2, and nuclear factor erythroid 2-related factor 2 (Nrf2), mouse cortical collecting duct cells (M1) cells treated with HHE exhibited increased activation of p38 MAPK, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and increased expression of NOX4, p47(phox), Kelch ECH associating protein 1 (Keap1) and COX2. HHE treatment also induced NF-κB activation by promoting IκB-α degradation. Meanwhile, the observed increases in nuclear NF-κB, NOX4, p47(phox), and COX2 expression were attenuated by treatment with Bay 117082, N-acetyl-l-cysteine (NAC), or RSV. Our findings indicate that RSV inhibits the expression of inflammatory proteins and the production of reactive oxygen species in M1 cells by inhibiting NF-κB activation.

Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells.

BACKGROUND: Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine's effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects. METHODS: Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis. RESULTS: The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-κB activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-κB inhibitor, Bay 11-7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression. CONCLUSIONS: Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-κB signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

Activation of G-protein-coupled receptor 40 attenuates the cisplatin-induced apoptosis of human renal proximal tubule epithelial cells.

G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion and inflammation. However, the pathophysiological roles of GPR40 in kidney disease have not yet been identified. In the present study, we investigated the expression of GPR40 during cisplatin-induced kidney injury using male Sprague-Dawley rats that were treated with 8 mg/kg cisplatin. Control rats were treated with saline. Following treatment with cisplatin, the protein expression of GPR40 in the kidneys was decreased in association with an increase in serum creatinine levels and the Bax/Bcl-2 expression ratio. To further investigate the function of GPR40, the human renal proximal tubule epithelial cell line (HK-2) was cultured with cisplatin in the absence or presence of GW9508, a selective GPR40 agonist. Pre-treatment of the HK-2 cells with GW9508 attenuated the decrease in cell viability induced by treatment with cisplatin. Treatment with cisplatin increased the number of cells with condensed nuclei, which was ameliorated by GW9508 pre-treatment. TUNEL assay also revealed that pre-treatment with GW9508 ameliorated cisplatin-induced apoptosis. Treatment with cisplatin increased the Bax/Bcl-2 expression ratio and cleaved caspase-3 expression, and promoted the activation of nuclear factor-κB (NF-κB). These changes were attenuated by pre-treatment with GW9508. The cisplatin-induced generation of reactive oxygen species (ROS) and the activation of the Src/epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) pathway were also counteracted by pre-treatment with GW9508. Thus, the activation of GPR40 attenuates cisplatin-induced apoptosis by inhibiting the generation of ROS, the activation of the Src/EGFR/ERK signaling pathway and the nuclear activation of NF-κB and pro-apoptotic factors.

Farnesoid X receptor ligand prevents cisplatin-induced kidney injury by enhancing small heterodimer partner.

The farnesoid X receptor (FXR) is mainly expressed in liver, intestine and kidney. We investigated whether 6-ethyl chenodeoxycholic acid (6ECDCA), a semisynthetic derivative of chenodeoxycholic aicd (CDCA, an FXR ligand), protects against kidney injury and modulates small heterodimer partner (SHP) in cisplatin-induced kidney injury. Cisplatin inhibited SHP protein expression in the kidney of cisplatin-treated mice and human proximal tubular (HK2) cells; this effect was counteracted by FXR ligand. Hematoxylin and eosin staining revealed the presence of tubular casts, obstructions and dilatations in cisplatin-induced kidney injury, which was attenuated by FXR ligand. FXR ligand also attenuated protein expression of transforming growth factor-ß1 (TGF-ß1), Smad signaling, and the epithelial-to-mesenchymal transition process, inflammatory markers and cytokines, and apoptotic markers in cisplatin-treated mice. Cisplatin induced NF-κB activation in HK2 cell; this effect was attenuated by pretreatment with FXR ligand. In SHP knockdown by small interfering RNA, cisplatin-induced activation of TGF-ß1, p-JNK and Bax/Bcl-2 ratio was not attenuated, while SHP overexpression and FXR ligand inhibited expression of these proteins in cisplatin-pretreated HK2 cells. In conclusion, FXR ligand, 6ECDCA prevents cisplatin-induced kidney injury, the underlying mechanism of which may be associated with anti-fibrotic, anti-inflammatory, and anti-apoptotic effects through SHP induction.

Renoprotective effects of the direct renin inhibitor aliskiren on gentamicin-induced nephrotoxicity in rats.

This study aimed to examine the protective effects of aliskiren on gentamicin-induced nephropathy. Rats were injected with gentamicin (100 mg/kg per day) for 14 days. Aliskiren was infused for two weeks. Human proximal tubular epithelial cell lines (HK-2) were cultured with gentamicin in the absence or presence of aliskiren. Inflammatory profibrotic and apoptotic markers were evaluated in vivo and in vitro. Aliskiren treatment attenuated the decreased creatinine clearance, increased fractional sodium excretion, glomerulosclerosis and tubulointerstitial fibrosis and counteracted the increased ED-1 expression in gentamicin-treated rats. The levels of inflammatory cytokines (TNF-α, IL-1ß and IFN-γ) and adhesion molecules (MCP-1, ICAM-1 and VCAM-1) increased in the gentamicin-treated kidneys. These changes were restored by aliskiren co-treatment. Aliskiren effectively reversed transforming growth factor-ß-induced fibrotic responses such as induction of α-smooth muscle actin in gentamicin-treated rat kidneys. Along with these changes, aliskiren also attenuated the increase in nuclear factor κB and phosphorylated extracellular signal-regulated kinase (pERK 1/2) levels in HK-2 cells cultured with gentamicin. In addition, aliskiren decreased the number of TUNEL-positive nuclei and reduced the expression of proapoptotic markers in gentamicin-treated HK-2 cells. These findings suggest that aliskiren attenuates gentamicin-induced nephropathy by suppression of inflammatory, profibrotic and apoptotic factors through inhibition of the nuclear factor κB, Smads and mitogen-activated protein kinase signaling pathways.

Paricalcitol attenuates 4-hydroxy-2-hexenal-induced inflammation and epithelial-mesenchymal transition in human renal proximal tubular epithelial cells.

4-Hydroxy-2-hexenal (HHE), the aldehyde product of lipid peroxidation, may be responsible for the pathogenesis of progressive renal disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to be renoprotective through its anti-inflammatory and antifibrotic effects in various experimental nephropathy models. In this study, we investigated the effects of paricalcitol on inflammation and epithelial-mesenchymal transition (EMT) after HHE-induced renal tubular epithelial cell injury. To investigate the molecular mechanisms underlying HHE-induced renal tubular cell injury, the human proximal tubular epithelial (HK-2) cells cultured with 10 µM HHE in the presence or absence of paricalcitol. In HK-2 cells, paricalcitol attenuated the HHE-induced expression of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, and prevented nuclear factor-κB (NF-κB) activation. The expression of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 was attenuated by paricalcitol pretreatment. In addition, HHE increased the expression of the transforming growth factor (TGF)-ß/Smad signaling proteins and fibrotic proteins, such as α-smooth muscle actin and connective tissue growth factor; this inducible expression was suppressed by pretreatment with paricalcitol. Treatment with HHE resulted in the activation of the ß-catenin signaling pathway, and paricalcitol pretreatment reduced the expression of ß-catenin in HHE-treated HK-2 cells. Coimmunoprecipitation shows that paricalcitol induced vitamin D receptor (VDR)/ß-catenin complex formation in HK-2 cells. Also immunofluorescence staining revealed that co-localization of VDR and ß-catenin in the nuclei. ICG-001, an inhibitor of ß-catenin, decreased the expression of TGF-ß1 and attenuated HHE-induced tubular EMT. These results show that paricalcitol attenuated HHE-induced renal tubular cell injury by suppressing inflammation and EMT process through inhibition of the NF-κB, TGF-ß/Smad, and ß-catenin signaling pathways.

Increased Phosphorylation of PI3K/Akt/mTOR in the Obstructed Kidney of Rats with Unilateral Ureteral Obstruction.

The present study aimed to investigate changes in the mammalian target of rapamycin (mTOR) signaling pathway in the obstructed kidney of rats with unilateral ureteral obstruction (UUO). Male Sprague-Dawley rats were unilaterally obstructed by ligation of the left proximal ureter for 7 days. Control rats were treated in the same way except that no ligature was made. The expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K), Akt, and mTOR were determined in the kidney by semiquantitative immunoblotting. The protein expression levels of transforming growth factor (TGF)-ß1, Bax, and Bcl-2 were also determined in the kidney. The phosphorylation of PI3K, Akt, and mTOR was increased in the kidney of ureteral obstruction rats compared with the control. In the obstructed kidney, the protein expression of TGF-ß1 and Bax was also increased, whereas Bcl-2 expression was decreased. In conclusion, the phosphorylation of PI3K/Akt/mTOR was increased in the obstructed kidney of rats with UUO.

Renoprotective effects of sildenafil in DOCA-salt hypertensive rats.

BACKGROUND/AIMS: Sildenafil, the first selective phosphodiesterase-5 (PDE5) inhibitor to be widely used for treating erectile dysfunction, has been investigated with regard to its cardioand renoprotective effects in animal models. This study further investigated the renoprotective effects of sildenafil and their molecular mechanisms in deoxycorticosterone acetate (DOCA)-salt hypertensive (DSH) rats. METHODS: DOCA strips (200 mg/kg) were implanted in rats 1 week after unilateral nephrectomy. These rats were fed on a control diet, with or without sildenafil (50 mg·kg(-1)day(-1)), for 2 weeks. Systolic blood pressure (SBP) was measured by the tail cuff method, and the urinary albumin-to-creatinine ratio (ACR) was calculated. The extent of glomerulosclerosis and tubulointerstitial fibrosis was determined by Masson's trichrome stain. Renal expression of ED-1, transforming growth factor-ß1 (TGF-ß1), Bax, and Bcl-2 were determined by semiquantitative immunoblotting, polymerase chain reaction (PCR), and immunohistochemistry. TUNEL staining was used for detecting apoptotic cells. RESULTS: The increased SBP in DSH rats was not attenuated by sildenafil treatment. The decreased creatinine clearance and increased ACR in DSH rats, compared with control animals, were attenuated by sildenafil treatment. Further, sildenafil treatment attenuated glomerulosclerosis and tubulointerstitial fibrosis in DSH rats and counteracted the increased expression of ED-1, TGF-ß1, and Bax and the decreased expression of Bcl-2 in the kidneys of these rats. The increase in the number of apoptotic cells in DSH rats was attenuated by sildenafil treatment. CONCLUSION: Sildenafil effectively prevented the progression of renal injury in DSH rats via its anti-inflammatory, antifibrotic, and antiapoptotic effects.

Paricalcitol prevents cisplatin-induced renal injury by suppressing apoptosis and proliferation.

The present study was performed to examine whether paricalcitol may prevent the cisplatin-induced kidney injury. Furthermore, potential molecular mechanisms underlying the protective effect of paricalcitol were explored. Male Sprague-Dawley rats were treated with vehicle (n=12), cisplatin (n=12, 6 mg/kg/day, i.p.), or cisplatin+paricalcitol (n=12, 0.2 µg/kg/day, s.c.) for 4 days. In another series of experiment, HK-2 cells were treated with cisplatin (50 µM), with or without paricalcitol (0.2 ng/ml). Paricalcitol counteracted the cisplatin-induced decline in renal function. Paricalcitol also suppressed the expression of TGF-ß1, Smad signaling, and the subsequent epithelial-to-mesenchymal process in cisplatin-treated rats. The expression of P-p53 and p21 was increased in cisplatin-induced nephropathy. These changes were completely prevented or significantly attenuated with paricalcitol co-treatment. The expression of p27(kip1) was increased in cisplatin-treated rats, which was, however, further augmented by the paricalcitol co-treatment. In HK-2 cells, cisplatin increased the expression of p-ERK1/2 and P-p38. Cisplatin also increased the expression of fibronectin and CTGF. Cisplatin increased the expression of pro-apoptotic markers. The expression of CDK2 and Cyclin E as well as that of PCNA was increased. These changes were completely prevented or significantly attenuated by the paricalcitol pretreatment. In contrast, cisplatin increased the expression of p27(kip1), which was further augmented by the paricalcitol-pretreatment. These results suggest that paricalcitol may ameliorate cisplatin-induced renal injury by suppressing the fibrotic, apoptotic and proliferative factors. Its underlying mechanisms may include inhibition of TGF-ß1, mitogen-activated protein kinase signaling, p53-induced apoptosis, and augmentation of p27(kip1).

Activation of the Renal PI3K/Akt/mTOR Signaling Pathway in a DOCA-Salt Model of Hypertension.

The present study investigated the changes that occurred in the mammalian target of rapamycin (mTOR) signaling pathway in the kidney as a result of deoxycorticosterone acetate (DOCA)-salt hypertension. Rats were implanted with DOCA strips (200 mg/kg) 1 week after unilateral nephrectomy and were then supplied with 0.9% saline to drink. Four weeks after DOCA implantation, systolic blood pressure (SBP) was measured by use of the tail-cuff method. The expression levels of phosphorylated phosphatidylinositol-3-kinase (PI3K), Akt, and mTOR, as well as the protein expression levels of ED-1 and cyclooxygenase-2 (COX-2), transforming growth factor-ß1 (TGF-ß1), α-smooth muscle actin (SMA), caspase-3, Bax, and Bcl-2, were then examined in the kidney by semiquantitative immunoblotting. DOCA-salt hypertensive rats were found to have significantly increased SBP as well as an increased kidney weight-to-body weight ratio. Moreover, the phosphorylation of PI3K, Akt, and mTOR was increased in the kidney of DOCA-salt hypertensive rats compared with the control, as was the protein expression of ED-1, COX-2, TGF-ß1, and α-SMA. The expression levels of caspase-3 and Bax were increased significantly, whereas Bcl-2 expression was decreased. In conclusion, the phosphorylation of PI3K/Akt/mTOR was increased in the kidney of DOCA-salt hypertensive rats.

4-Hydroxy-2-hexenal-induced apoptosis in human renal proximal tubular epithelial cells.

BACKGROUND: The aldehyde products of lipid peroxidation such as 4-hydroxy-2-hexenal (HHE) might be responsible for the pathogenesis of kidney injury. The present study was aimed to investigate the effects of HHE on renal tubular epithelial cells and its signaling mechanisms. METHODS: Human proximal tubular epithelial (HK-2) cells were treated with 10 µM of HHE. Cell viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The fluorescent probe 2',7'-dichlorofluorescein diacetate was used to measure intracellular levels of reactive oxygen species (ROS). The protein expression of NF-κB, mitogen-activated protein kinase (MAPK), pro-apoptoic Bax and anti-apoptotic protein Bcl-2 was determined by semiquantitative immunoblotting. Apoptosis was assessed by flow cytometry analysis after the cells were stained by fluorescein isothiocyanate-conjugated annexin V protein and propidium iodine. RESULTS: Treatment with various doses of HHE resulted in dose-dependent decreases of cell viability and increases of ROS. HHE increased the expression of p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). HHE induced NF-κB activation and IκB-α degradation. Increased nuclear NF-κB activation was blocked by inhibitors of ERK (PD98059) or JNK (SP600125), but not affected by p38 MAPK inhibitor (SB203580). Flow cytometry analysis revealed HHE-induced apoptosis. HHE decreased the expression of Bcl-2, while it increased that of Bax, which was attenuated by the treatment of NF-κB inhibitor (Bay 11-7082) or N-acetyl-L-cysteine (NAC). An inhibition of NF-κB prevented HHE-induced apoptosis. CONCLUSIONS: HHE-induced tubular cell apoptosis is mediated by modulation of Bax and Bcl-2 via ROS generation. HHE-mediated accumulation of ROS may induce redox-sensitive transcription factor, NF-κB, through activation of ERK and JNK, resulting in cellular apoptosis in HK-2 cells.

Bone-marrow-derived mesenchymal stem cell transplantation enhances closing pressure and leak point pressure in a female urinary incontinence rat model.

PURPOSE: The purpose of this study was to determine whether periurethral injection of allogenic mesenchymal stem cells (MSCs) could increase the leak point pressure (LPP) in a rat model of stress urinary incontinence. MATERIALS AND METHODS: Female Sprague-Dawley rats (230-240 g, n = 30) were divided into 3 groups: sham operation (group C), saline-treated (group S) and MSC-treated (group M). Bilateral pudendal nerve dissection followed by normal saline or MSC injection on both sides of the urethra was done. LPP and closing pressure (CP) testing was performed after the treatment. The specific markers for smooth muscle cells in the transplantation sites of the urethra were determined. RESULTS: Both the LPP and CP were significantly lower in group S than controls. However, these were restored to the control values in group M (p < 0.05). The LPPs of groups C, S and M were 29.1 ± 2.1, 22.0 ± 2.2 and 43.1 ± 3.2 cm H(2)O, respectively. The CPs of groups C, S and M were 27.1 ± 3.1, 21.1 ± 3.2, and 32.1 ± 2.1 cm H(2)O, respectively. The injected MSCs stained positive for muscle-specific markers. CONCLUSION: This study suggests that MSCs might differentiate into muscle lineage cells and may contribute to the repair of damaged muscle tissue.

Altered regulation of renal nitric oxide, atrial natriuretic peptide and cyclooxygenase systems in aldosterone escape in rats.

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO), atrial natriuretic peptide (ANP) and cyclooxygenase (COX) systems in the kidney in association with the aldosterone escape. Male Sprague-Dawley rats were used. Aldosterone (200 microg/day) was infused through entire time course. The control group was kept on a low sodium diet (0.02 mEq/day), and the experimental group was supplied with a higher sodium diet (2. /day). Four days after beginning the regimen, the kidneys were taken. The protein expression of NO synthase (NOS) and COX isoforms was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed aldosterone escape in the experimental group. Accordingly, the renal content and the urinary excretion of NO increased. The expression of nNOS was increased in the inner medulla. Neither the expression of eNOS nor that of iNOS was changed. The expression and the catalytic activity of soluble guanylyl cyclase remained unaltered. The mRNA expression of ANP was increased. Neither the expression of NPR-A or NPR-C nor the activity of particulate guanylyl cyclase was altered in the papilla. The protein expression of COX-2 was increased in the inner medulla, while that of COX-1 remained unchanged. In conclusion, the upregulation of nNOS, ANP, and COX-2 may be causally related with the aldosterone escape.