Renal Epithelial NRK-52E Cells Research Articles

Renoprotective effect of Nesfatin-1 in Adenine-Induced Chronic kidney Disease: An in vitro and in vivo study

Chronic Kidney Disease (CKD) presents a significant global health challenge with limited treatment options. Nesfatin-1, an anorexigenic peptide, has demonstrated antioxidant, anti-inflammatory, and anti-apoptotic properties in various diseases. However, the role of nesfatin-1 in CKD remains unclear. This study investigates the potential renoprotective effects of nesfatin-1 in adenine-induced CKD mice and in NRK-52E renal epithelial cells.Male C57BL/6J mice and NRK-52E renal epithelial cells were administered adenine to induce CKD. Various aspects of renal function, histopathology, oxidative stress, inflammation, apoptosis, and renal interstitial fibrosis were assessed and downstream pathways were investigated.Adenine-fed mice exhibited reduced nesfatin-1 expression and increased markers of kidney damage, including elevated blood urea nitrogen (BUN), serum creatinine, and histological abnormalities, reactive oxygen species (ROS), inflammation, apoptosis, and fibrosis. Treatment with nesfatin-1 in adenine induced mice significantly reversed these changes. Nesfatin-1 also lowered calcium levels and the expression of inflammatory markers, including IL-1β, IL-6, TNF-α, and Nf-kB. Furthermore, nesfatin-1 reduced the expression of apoptotic markers (Caspase-3, Caspase-1, Bax/Bcl2 ratio) and restored the balance of Bcl2 and MMP. Lastly, nesfatin-1 attenuated fibrotic markers (Tgf-β, Smad2/3,4, type IV collagen, α-SMA) in both adenine-induced CKD mice and NRK-52E cells.In conclusion, our results suggest that nesfatin-1 may enhance kidney function in adenine-induced CKD mice and NRK-52E cells. The renoprotective effects of nesfatin-1 are likely associated with its antioxidant, anti-inflammatory, anti-apoptotic, and anti-fibrotic properties.

Activation of Mas and pGCA receptor pathways protects renal epithelial cell damage against oxidative-stress-induced injury

Over-activation of the renin-angiotensin-aldosterone system (RAAS) is a leading cause of cardio-renal complications. Oxidative stress is one of the major contributing factors in the over-activation of RAAS. Angiotensin-converting enzyme2/Angiotensin1–7/MasR and natriuretic peptide/particulate guanylyl cyclase receptor-A pathways play a key role in cardiorenal disease protection. Even though individual activation of these pathways possesses cardiorenal protective effects. However, the dual activation of these pathways under stress conditions and the underlying mechanism has not been explored. The study aimed to investigate whether activation of these pathways by dual-acting peptide (DAP) shows a protective effect in-vitro in oxidative stress-induced renal epithelial cells. Oxidative stress was induced in renal epithelial NRK-52E cells with H2O2. Co-treatment with Ang 1–7, BNP, and DAP was given for 30 min. AT1, MasR, and pGCA expression were measured by RT-PCR. The markers of oxidative stress and apoptosis were measured by confocal microscopy and FACS analysis. A significant increase in AT1, renin, α-SMA, and NFk-β expression and a significant decrease in MasR and pGCA expression was observed in H2O2-induced cells. DAP improved H2O2-induced pathological changes in NRK-52E cells. The effect of DAP was superior to that of Ang1–7 and BNP alone. Interestingly, MasR and pGCA inhibitors could block the effect of DAP in H2O2-induced cells. DAP shows superior anti-RAAS activity, and it is effective against H2O2-induced oxidative stress, apoptosis, fibrosis, and inflammation compared to Ang1–7 and BNP alone. The protective effect is mediated by the dual activation of MasR and pGCA.

Downregulation of nesfatin-1 expression in acute kidney injury in vivo in wistar rats and in vitro in cultured cells

AimsAcute kidney injury (AKI) is a debilitating condition followed by sudden kidney damage or failure within hours or days of its occurrence. AKI is characterized by rapid increase in serum creatinine/BUN and decrease in urine output. Nesfatin-1 is an endogenous peptide reported to possess anorexic, antioxidant and anti-apoptotic properties. Although few clinical studies have shown altered nesfatin-1 levels in hemodialysis patients, however, there are no reports investigating the distribution and expression pattern of nesfatin-1 in AKI. Materials and methodsNesfatin-1 expression was determined in different disease induced models of AKI by immunoblotting, immunofluorescence and RT-PCR. Gene markers of oxidative stress and inflammation were determined by RT-PCR. The expression of different markers of AKI was measured by assay kits and RT-PCR analysis. Key findingsThere was a significant increase in serum levels of creatinine and BUN in AKI rats followed by significant increase in KIM-1 in the kidneys. Significant decrease in nesfatin-1 expression along with increased expression of IL-1β, TNF-α and decreased expression of SOD and catalase was observed in doxorubicin and cisplatin induced AKI rats. However, SOD and catalase expression were upregulated in glycerol induced AKI rats. Moreover, in vitro treatment of renal NRK-52E epithelial cells with nesfatin-1 reversed the changes induced by doxorubicin. SignificanceOur study reports for the first time, nesfatin-1 expression is decreased in kidneys of different models of AKI induced rats as well as cultured NRK-52E renal epithelial cells. Further studies are required to understand the possible molecular mechanism and therapeutic potential of nesfatin-1 in acute kidney injury.

Downregulation of inflammatory mediators by ethanolic extract of Bergenia ligulata (Wall.) in oxalate injured renal epithelial cells

Ethnopharmacological relevanceIn the Indian traditional system of medicine, Bergenia ligulata (Wall.) Engl. has been used for treatment of urolithiasis. Its efficacious nature has led to its incorporation in various commercial herbal formulations such as Cystone and Neeri which are prescribed for kidney related ailments. Aim of the studyTo assess whether ethanolic extract of B. ligulata can mitigate the cascade of inflammatory responses that cause oxidative stress and ultimately cell death in renal epithelial cells exposed to hyperoxaluric conditions. Material and methodsBioactivity guided fractionation using solvents of varying polarities was employed to evaluate the potential of the extracts of B. ligulata to inhibit the crystallization process. Modulation of crystal morphology was visualized through Scanning electron microscopy (SEM) analysis. Cell death was assessed using flow cytometry based assays. Alteration in the inflammatory mediators was evaluated using real time PCR and immunocytochemistry. Phytochemical characterization of the ethanolic extract was carried out using FTIR, LC-MS and GC-MS. ResultsBioactivity guided fractionation for the assessment of antilithiatic activity revealed dose dependent inhibition of nucleation and aggregation process of calcium oxalate crystals in the presence of various extracts, however ethanolic extract showed maximum inhibition and was chosen for further experiments. Studies on renal epithelial NRK-52E cells showed, cytoprotective efficacy of B. ligulata extract against oxalate injury. SEM anaysis further revealed the potential of the extract to modulate the crystal structure and adhesion to renal cell surface. Exposure of the renal cells to the extract led to conversion of the calcium oxalate monohydrate (COM) crystals to the less injurious calcium oxalate dihydrate (COD) form. Expression analysis for oxidative stress and inflammatory biomarkers in NRK-52E cells revealed up-regulation of Mitogen activated protein kinase (MAPK), Osteopontin (OPN) and Nuclear factor- ĸB (NF-ĸB), in response to calcium oxalate insult; which was drastically reduced in the presence of B. ligulata extract. Flow cytometric evaluation pointed to caspase 3 mediated apoptotic cell death in oxalate injured cells, which was attenuated by B. ligulata extract. ConclusionConsidering the complex multifactorial etiology of urolithiasis, ethanolic extract from B. ligulata can be a promising option for the management of kidney stones, as it has the potential to limit inflammation and the subsequent cell death.

PPARα/β Activation Alleviates Age-Associated Renal Fibrosis in Sprague Dawley Rats.

Age-associated renal fibrosis is commonly observed, with a decline in renal function during aging. Although peroxisome proliferator-activated receptors α/β (PPARα/β) activation has been shown to exert beneficial effects on age-associated renal changes, its effects on age-associated renal fibrosis have not been investigated yet. Here, we show that the PPARα/β activator, MHY2013, can significantly alter lipid metabolism in renal tubule epithelial cells and attenuate renal fibrosis in aged Sprague Dawley (SD) rats. We found that MHY2013 significantly increased nuclear translocation and activity of PPARα/β in NRK52E renal epithelial cells. Moreover, the enhanced PPARα/β activity increased the expression of fatty acid oxidation-associated PPARα/β target genes. In addition, transforming growth factor-β (TGF-β)- and oleic acid-induced lipid accumulation and fibrosis-associated gene expression were decreased in NRK52E cells by MHY2013 pretreatment. To evaluate the effects of MHY2013 on age-associated renal fibrosis, aged SD rates were orally administered MHY2013 (1 and 5 mg/kg) daily for 1 month. MHY2013 efficiently increased PPARα/β activation and reduced renal lipid accumulation in aged SD rat kidneys. Furthermore, renal fibrosis was significantly decreased by MHY2013, indicating the importance of renal lipid metabolism in age-associated renal fibrosis. Taken together, our results suggest that activation of PPARα/β signaling during aging prevents age-associated renal fibrosis.

Lipocalin-2 abrogates epithelial cell cycle arrest by PPARγ inhibition

Macrophage-epithelial cross-talk regulates cell cycle progression and represents an important factor in rescuing epithelial cells from cell cycle arrest in order to maintain a healthy epithelial phenotype. However, the underlying mechanisms are still not well defined. We provide evidence that macrophage-secreted lipocalin-2 (Lcn-2) plays a key role during this process. In a co-culture setup using cell cycle arrested NRK52e renal epithelial cells and primary bone marrow-derived macrophages, Lcn-2 restores proliferation through inhibition of peroxisome proliferator-activated receptor (PPAR)-γ. Lcn-2 overexpression in macrophages overcomes epithelial cell cycle arrest and enhances epithelial markers via megalin and the downstream activation of PI3K/Akt signalling pathway, whereas a knockdown of Lcn-2 in macrophages prevented this effect. Our results show that macrophage-secreting Lcn-2 is crucial in rescuing epithelial cells from cell cycle arrest and in promoting epithelial proliferation.Macrophage-epithelial crosstalk regulates cell cycle progression and is essential for the maintenance of a healthy epithelial phenotype. In the present work, the authors show that macrophage-secreted lipocalin-2 is crucial in rescuing epithelial cells from cell cycle arrest and in promoting epithelial proliferation through inhibition of peroxisome proliferator-activated receptor (PPAR)-γ. Lipocalin-2 maintains the epithelial cytoskeleton structure via megalin and downstream activation of thePI3K/AKT signaling pathway.

In vitro inhibition of calcium oxalate crystallization and crystal adherence to renal tubular epithelial cells by Terminalia arjuna.

Urolithiasis is a multifactorial disease and remains a public health problem around the world. Of all types of renal stones, calcium oxalate (CaOx) is the most common composition formed in the urinary system of the patients with urolithiasis. The present study is aimed at evaluating the antiurolithiatic properties of the Tris-Cl extract (TE) of Terminalia arjuna (T. arjuna). The antilithiatic activity of TE of T. arjuna was investigated on nucleation, aggregation, and growth of the CaOx crystals, as well as its protective potency was tested on oxalate-induced cell injury of NRK-52E renal epithelial cells. Also, in vitro antioxidant activity of TE T. arjuna bark was also determined. The TE of T. arjuna exhibited a concentration-dependent inhibition of nucleation and growth of CaOx crystals. Inhibition of aggregation of CaOx crystals remains constant. When NRK-52E cells were injured by exposure to oxalate for 48 h, the TE prevented the cells from injury and CaOx crystal adherence resulting in increased cell viability in a dose-dependent manner. The TE also scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals with an IC50 at 51.72 µg/mL. The results indicated that T. arjuna is a potential candidate for phytotherapy against urolithiasis as it attains the ability to inhibit CaOx crystallization and scavenge DPPH free radicals in vitro along with a cytoprotective role.

Angiotensin-(1–7) abolishes AGE-induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2

Angiotensin-(1–7) (Ang-(1–7))/AT7-Mas receptor axis is an alternative pathway within the renin–angiotensin system (RAS) that generally opposes the actions of Ang II/AT1 receptor pathway. Advanced glycated end product (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may contribute to the development and progression of diabetic nephropathy in part through activation of the Ang II/AT1 receptor system; however, the influence of AGE on the Ang-(1–7) arm of the RAS within the kidney is unclear. The present study assessed the impact of AGE on the Ang-(1–7) axis in NRK-52E renal epithelial cells. MGA exposure for 48h significantly reduced the intracellular levels of Ang-(1–7) approximately 50%; however, Ang I or Ang II expression was not altered. The reduced cellular content of Ang-(1–7) was associated with increased metabolism of the peptide to the inactive metabolite Ang-(1–4) [MGA: 175±9 vs. Control: 115±11fmol/min/mg protein, p<0.05, n=3] but no change in the processing of Ang I to Ang-(1–7). Treatment with Ang-(1–7) reversed MGA-induced cellular hypertrophy and myofibroblast transition evidenced by reduced immunostaining and protein expression of α-smooth muscle actin (α-SMA) [0.4±0.1 vs. 1.0±0.1, respectively, n=3, p<0.05]. Ang-(1–7) abolished AGE-induced activation of the MAP kinase ERK1/2 to a similar extent as the TGF-β receptor kinase inhibitor SB58059; however, Ang-(1–7) did not attenuate the MGA-stimulated release of TGF-β. The AT7-Mas receptor antagonist D-Ala7-Ang-(1–7) abolished the inhibitory actions of Ang-(1–7). In contrast, AT1 receptor antagonist losartan did not attenuate the MGA-induced effects. We conclude that Ang-(1–7) may provide an additional therapeutic approach to the conventional RAS blockade regimen to attenuate AGE-dependent renal injury.

Abstract 234: AGEs Enhance the Metabolism of Angiotensin-(1-7) and Lower Intracellular Peptide Expression

Advancedglycated end products (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may be important mediators of diabetic pathologies. An accompanying abstract described AGE-dependent cellular hypertrophy and myofibroblast transition (MT) in NRK-52E renal epithelial cells that was abolished by treatment with the alternative angiotensin (Ang) product Ang-(1-7); the inhibitory effect of Ang-(1-7) was likely mediated by attenuation of ERK and TGF-β stimulated pathways. AGEs evoke multiple signaling events, but the impact on the Ang-(1-7) system is unknown. The present study addressed whether AGEs suppress the Ang-(1-7) axis, and thus facilitate their deleterious actions. MGA exposure for 48 hrs significantly reduced the cellular expression of Ang-(1-7) [396 ± 59 vs. 219 ± 54 fmol/mg protein; p&lt;0.05] in the NRK-52E cells; however, the cellular content of Ang II and Ang I was not changed. We then assessed the processing pathways of Ang I to Ang-(1-7) in the 100,000 xg soluble fraction, as well as the activity secreted extracellularly in the media. Conversion of Ang I to Ang-(1-7) was also not changed following MGA exposure in the cellular supernatant [56 ± 7 vs. 66 ± 8 fmol/min/mg protein]. In contrast, the metabolism of Ang-(1-7) to Ang-(1-4) was significantly enhanced in the soluble fraction of the MGA exposed cells [175 ± 9 vs. 115 ± 11 fmol/min/mg protein, p&lt;0.05]. The Ang-(1-7) endopeptidase activity was abolished by the thiol inhibitor PCMB, but was insensitive to the metallopeptidase inhibitor JMV390. AGE slightly but significantly induced the metabolism of Ang-(1-7) to Ang-(1-4) in the cell media [1.36 ± 0.01 vs. 1.42 ± 0.01 fmol/min/ml, p&lt;0.05]. In this case, the secreted endopeptidase exhibited marked sensitivity to JMV390 [IC50 = 7.6 nM]. Finally, chronic treatment with the ERK1/2 inhibitor PD98059 abrogated the increase in Ang-(1-4), and suggests that downregulation of the peptidase is linked to AGE-induced MT. We conclude that the cellular actions of AGE may reflect a loss of Ang-(1-7) tone through enhanced metabolism of the peptide. Moreover, we identify two distinct endopeptidase activities that metabolize Ang-(1-7) to Ang-(1-4) and both are increased following AGE exposure.

Nuclear expression of renin-angiotensin system components in NRK-52E renal epithelial cells.

Isolated nuclei of sheep proximal tubules express angiotensin (Ang) receptors as well as angiotensinogen (AGT) and renin. The present study characterized the NRK-52E tubular epithelial cell line for the intracellular expression of renin-angiotensin system (RAS) components. RAS components were visualized by immunofluorescent staining in intact cells and protein expression in isolated nuclei. An antibody to the angiotensin I (Ang I) sequence of AGT (AI-AGT) revealed only cytosolic staining, while an antibody to an internal sequence of AGT (Int-AGT) revealed primarily nuclear staining. Immunoblots of nuclear and cytosolic fractions confirmed the differential cell staining of AGT. Immunostaining for renin was present on nuclei of intact cells. Nuclear renin activity averaged 0.77±0.05 nmol/mg protein/h that was reduced by aliskiren (0.13±0.01 nmol/mg/h, n=3, p<0.01); trypsin activation increased activity three-fold. Peptide staining localized angiotensin II (Ang II) and Ang-(1-7) to the nucleus and peptide content averaged 59±2 and 57±22 fmol/mg (n=4), respectively. Peptide metabolism in isolated nuclei revealed the processing of Ang I to Ang-(1-7) by thimet oligopeptidase. We conclude that the NRK-52E cells express an intracellular RAS localized to the nucleus and may be an appropriate cell model to elucidate the functional relevance of this system.

Targeting JNK by a New Curcumin Analog to Inhibit NF-kB-Mediated Expression of Cell Adhesion Molecules Attenuates Renal Macrophage Infiltration and Injury in Diabetic Mice

Macrophage infiltration contributes to the pathogenesis of diabetic renal injury. However, the regulatory mechanisms between macrophage infiltration and epithelial cell activation are still unclear. Our previous study found that C66, a novel curcumin analog, was able to inhibit inflammatory cytokine expression in vitro and in vivo. This study further elucidated whether C66 can prevent glucose-induced renal epithelial activation and inflammatory macrophage infiltration by a MAPK/NF-κB medicated mechanism. Our data show that pretreatment with C66 not only significantly reduced high glucose (HG)-induced over-expressions of VCAM-1, ICAM-1 and MCP-1, but also remarkably inhibited NF-κB activation, MAPKs phosphorylation, and subsequently macrophage adhesion in renal epithelial NRK-52E cells. Furthermore, we find that MAPKs, especially JNK, play important roles in HG-induced NF-κB activation, which regulates the over-expression of adhesion molecules in HG-stimulated NRK-52E cells. A molecular docking predicted that C66 may target JNK2, which leads to its anti-inflammatory actions. In vivo, administration of C66 or JNK special inhibitor SP600125 at 5 mg/kg markedly decreased diabetes-induced renal adhesion molecule expression, NF-κB activation, inflammatory cell infiltration, and pathological indexes in the kidneys of diabetic mice. These findings provide a perspective on the renoprotective effects of C66 in diabetes, and outline a novel therapeutic strategy of JNK inhibition for the treatment of diabetic nephropathy.

Magnesium deficiency suppresses cell cycle progression mediated by increase in transcriptional activity of p21Cip1 and p27Kip1 in renal epithelial NRK-52E cells

Lack of magnesium suppresses cell growth, but the molecular mechanism is not examined in detail. We examined the effect of extracellular magnesium deficiency on cell cycle progression and the expression of cell cycle regulators in renal epithelial NRK-52E cells. In synchronized cells caused by serum-starved method, over 80% cells were distributed in G1 phase. Cell proliferation and percentage of the cells in S phase in the presence of MgCl(2) were higher than those in the absence of MgCl(2) , suggesting that magnesium is involved in the cell cycle progression from G1 to S phase. After serum addition, the expression levels of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The exogenous expression of p21(Cip1) or p27(Kip1) increased the percentage in G1 phase, whereas it decreased that in S phase. The mRNA levels and promoter activities of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The phosphorylated p53 (p-p53) level was decreased by MgCl(2) addition. Pifithrin-α, a p53 inhibitor, decreased the p-p53, p21(Cip1) and p27(Kip1) levels, and the percentage in G1 phase in the absence of MgCl(2) . Rotenone, a mitochondrial respiratory inhibitor, decreased ATP content and increased the p-p53 level in the presence of MgCl(2) . Together, lack of magnesium may increase p21(Cip1) and p27(Kip1) levels mediated by the decrease in ATP content and the activation of p53, resulting in the suppression of cell cycle progression from G1 to S phase in NRK-52E cells.

Modulation of Antioxidant Enzyme Expression and Activity by Paraquat in Renal Epithelial NRK-52E Cells

Renal toxicity produced by paraquat involves the generation of reactive oxygen species (ROS) which can overwhelm antioxidant defences, leading to oxidant injury. However, there are conflicting reports regarding the activity and/or expression of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) during oxidative stress injury. This study investigated the activity and expression of these enzymes in a renal epithelial cell line following exposure to paraquat. Confluent NRK-52E cells were incubated with increasing concentrations of paraquat (1-100mM) for up to 24 hours. Renal cell death was determined by measurement of lactate dehydrogenase release. Oxidant damage was determined via measurement of malondialdehyde formation and DNA strand breaks. The effects of paraquat on DNA and de novo protein synthesis were determined using radio-labelled thymidine and leucine respectively. ROS generation (superoxide anion and hydroxyl radical formation) was measured using nitro-bluetetrazolium and deoxyribose assays. Antioxidant enzyme activities and expression were measured using established biochemical assays and Western blot analysis. Exposure of confluent NRK-52E cells to paraquat resulted in significant cell death involving increased lipid peroxidation, DNA damage and inhibition of DNA and de novo protein synthesis. Renal cell injury and death were secondary to increased ROS generation. Incubation with paraquat reduced SOD and CAT activities; in contrast, GSH-Px activity increased significantly. Although SOD expression was significantly reduced, catalase expression was unaffected. These results indicate that paraquat mediates renal toxicity via oxidative stress involving both an increase in ROS generation and reductions in SOD and CAT activities with a concomitant reduction in SOD expression.

Diminution of oxalate induced renal tubular epithelial cell injury and inhibition of calcium oxalate crystallization in vitro by aqueous extract of Tribulus terrestris

Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate solution, using phytotherapy is being sought. The present study attempted to evaluate the antilithiatic properties of Tribulus terrestris commonly called as "gokhru" which is often used in ayurveda to treat various urinary diseases including urolithiasis. The activity of Tribulus terrestris was investigated on nucleation and the growth of the calcium oxalate (CaOx) crystals as well as on oxalate induced cell injury of NRK 52E renal epithelial cells. Tribulus terrestris extract exhibited a concentration dependent inhibition of nucleation and the growth of CaOx crystals. When NRK-52E cells were injured by exposure to oxalate for 72 h, Tribulus terrestris extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner. The current data suggests that Tribulus terrestris extract not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role. Our results indicate that it could be a potential candidate for phytotherapy against urolithiasis.

Reduction of oxalate-induced renal tubular epithelial (NRK-52E) cell injury and inhibition of calcium oxalate crystallisation in vitro by aqueous extract of Achyranthes aspera

Despite considerable progress in medical therapy, there is no satisfactory drug to treat kidney stones. Therefore, this study is aimed to look for an alternative treatment by using Achyranthes aspera. Here, the inhibitory potency of A. aspera was investigated on nucleation and the growth of the calcium oxalate (CaOx) crystals as well as on oxalate-induced cell injury of NRK 52E renal epithelial cells in vitro. Data are expressed as mean values of three independent experiments (each in triplicate) and analysed by the analysis of variance (P <0.05) to estimate the differences between values of extracts tested. A. aspera extract exhibited a concentration-dependent inhibition of the growth of CaOx crystals but a similar pattern of inhibition was not observed with increase in the plant extract concentration for the nucleation assay. When NRK 52E cells were injured by exposure to oxalate for 72 hours, A. aspera extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant, the cell viability increased and the lactate dehydrogenase (LDH) release decreased in a concentration-dependent manner. These studies indicate that A. aspera extract besides having a cytoprotective role also has a potential to inhibit both nucleation and the growth of the CaOx crystals and can prove to be a potent candidate for phytotherapy against urolithiasis. Key words: Achyranthes aspera, calcium oxalate, NRK 52E, phytotherapy, urolithiasis

TRPM6 expression and cell proliferation are up-regulated by phosphorylation of ERK1/2 in renal epithelial cells

Transient receptor potential melastatin 6 (TRPM6) is a magnesium channel and expressed in the intestine and renal distal tubules. Little is known about the regulatory mechanism of TRPM6 expression and the role of magnesium influx. EGF increased the phosphorylation of ERK1/2 and TRPM6 expression that were inhibited by U0126 in renal epithelial NRK-52E cells. Furthermore, EGF enhanced the influx of magnesium, whereas U0126 and TRPM6 siRNA inhibited it. EGF increased the proportion of cells in S phase, whereas U0126 and TRPM6 siRNA increased the proportion in G1 phase. The phosphorylation of ERK1/2 may up-regulate TRPM6 expression and magnesium influx, resulting in an increase in cell proliferation with a shift from G1 to S phase.

Search for genes expressed during progression and recovery in the diseased kidney

Kidneys can recovery from some type of injury. After an acute glomerulonephritis or a short-term ischemia, damaged glomerulus or tubules recover their normal structures with proliferation of the survived cells and infiltration of extrarenal cells. On the other hand, a prolonged or continuous injury leads to a progressive renal disease. We have investigated molecular events in reversible and irreversible renal diseases and have made efforts to identify genes pivotal in promoting regeneration or progression of the renal diseases. To identify genes responsible for kidney regeneration, we surveyed the expression pattern of the development genes in ischemia-reperfusion model of rat kidney. The regeneration, molding, and maturation of the restored epithelium after renal ischemia injury have many parallels to the growth and maturation that takes place during kidney organogenesis. We found that leukemia inhibitory factor (LIF) and its receptor (LIFR) expression were up-regulated during the recovery phase after ischemia insult. The increased expression of LIF and LIFR was most marked in the outer medulla, especially in the S3 segment of the proximal tubules. In cultured rat renal epithelial NRK52E cells, LIF expression was enhanced during recovery after adenosine triphosphate (ATP) depletion. Blockade of endogenous LIF with a neutralizing antibody significantly reduced the cell number and DNA synthesis during the recovery period. These results suggest that LIF participates in the regeneration process after tubular injury [Yoshino J, Monkawa T, Tsuji M, et al : Leukemia inhibitory factor is involved in tubular regeneration after experimental acute renal failure. J Am Soc Nephrol 14:3090-3101, 2003]. To search for genes playing important roles in progression of renal diseases, we compared gene expression profiles between an irreversible model and a reversible model of anti-Thy-1 glomerulonephritis using a microarray technology. A single intravenous injection of anti-Thy-1 monoclonal antibody 1-22-3 is known to cause a reversible mesangial proliferative glomerulonephritis. However, monoclonal antibody 1-22-3 injection followed by unilateral nephrectomy leads to progressive glomerulosclerosis with an irreversible course. Among 4854 rat genes on the microarray slide, 189 genes were differentially expressed. The differentially expressed genes were classified into five clusters based on their expression patterns. One of the clusters included genes the expression of which was markedly up-regulated in the irreversible model. The expression levels of the genes belonging to the cluster were low in normal kidney and increased along with disease peaked at day 14, and the up-regulation was more pronounced in the irreversible model than the reversible model. This cluster included collagen type1, laminin, osteopontin, kidney injury molecule-1 (KIM-1), and thymosin β10. These molecules can be new therapeutic targets for slowing the progression of renal diseases or accelerating repair of the injured kidney.

Arachidonic acid-activated Na +-dependent Mg 2+ efflux in rat renal epithelial cells

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg 2+ deficiency. We examined whether the extracellular Mg 2+ concentration affects AA production and whether AA regulates a putative Na +-dependent Mg 2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg 2+-containing medium for 2 days because they enable us to detect Na +-stimulated Mg 2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg 2+ increased AA release both in the absence and presence of extracellular Na +. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 μM), an inhibitor of cytosolic phospholipase A 2 (cPLA 2) and Ca 2+-independent phospholipase A 2 (iPLA 2), and bromoenol lactone (BEL, 10 μM), an inhibitor of iPLA 2. However, LY-311727 (10 μM), a secretory phospholipase A 2 (sPLA 2) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA 2 and iPLA 2 mRNAs, but not sPLA 2. In the mag-fura 2 fluorescence measurements, extracellular Mg 2+ removal caused slight decrease in the intracellular free Mg 2+ concentration ([Mg 2+] i) in the Na +-free condition. The addition of Na + caused a rapid decrease in [Mg 2+] i, indicating the presence of a Na +-dependent Mg 2+ efflux pathway. The Na +-dependent [Mg 2+] i decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 μM), indomethacin (10 μM) and 17-octadecynoic acid (ODYA) (10 μM), enhanced the Na +-dependent [Mg 2+] i decrease. Furthermore, the addition of exogenous AA (30 μM) enhanced the Na +-dependent [Mg 2+] i decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na +/Mg 2+-exchanger inhibitor. These results suggest that extracellular Mg 2+ removal elevates AA release mediated mainly by iPLA 2 and that AA upregulates the Na +-dependent Mg 2+ efflux in NRK-52E cells.

Up-regulation of Na +-dependent Mg 2+ transport by nitric oxide and cyclic GMP pathway in renal epithelial cells

A putative, Na +-dependent Mg 2+ transport pathway controls the intracellular free Mg 2+ concentration ([Mg 2+] i) in various mammalian cells. The characteristics of this Mg 2+ transport pathway have not been clarified. Herein, we examined the regulatory mechanism of Na +-dependent Mg 2+ efflux in renal epithelial NRK-52E cells. Mg 2+ removal from the extracellular bathing solution induced an Na +-dependent [Mg 2+] i decrease in Mg 2+ (5 mM)-loaded cells but not in control cells. Amiloride inhibited the [Mg 2+] i decrease in a dose-dependent manner (IC 50=3 μM). Similarly, atomic absorption spectrophotometry showed that Mg 2+ removal decreased intracellular Mg 2+ content, while it increased Na + content. Calphostin C (1 μM), a protein kinase C inhibitor, and genistein, a tyrosine kinase inhibitor (10 μM), blocked the [Mg 2+] i decrease. The [Mg 2+] i decrease was accompanied by an increase in intracellular nitric oxide (NO) and cyclic GMP contents. ( E)-4-methyl-2-[( E)-hydoxyimino]-5-nitro-6-methoxy-3-hexenamide (0.1 mM), an NO donor, and 8-bromo-cyclic GMP (0.1 mM), a membrane-permeable cyclic GMP analogue, accelerated the [Mg 2+] i decrease. In contrast, N G-monomethyl- l-arginine ( l-NMMA, 0.1 mM), an NO competitive inhibitor, and 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxaline-1-one (ODQ, 10 μM), an NO-sensitive guanylate cyclase inhibitor, significantly blocked the [Mg 2+] i decrease. These results indicate that a decrease in extracellular Mg 2+ concentration induces the production of NO and cyclic GMP, which leads to the up-regulation of Na +-dependent Mg 2+ efflux.

Phosphatidylinositol 3-Kinase Activity Is Required for Epidermal Growth Factor to Suppress Proteolysis

Suppression of protein breakdown occurs commonly in cell growth, but the pathways responsible for controlling proteolysis are poorly understood. Protein breakdown in NRK-52E renal epithelial cells treated with epidermal growth factor (EGF) and intracellular signaling inhibitors or dominant negative signaling molecules contained in an adenoviral vector were measured. The tyrosine kinase inhibitor, herbimycin A, eliminated the suppression of proteolysis induced by EGF. In contrast, the Src inhibitor, PP1, had no effect. Expression of dominant negative H-RasY57 blocked the ability of EGF to stimulate downstream targets of Ras and also reduced the ability of EGF to suppress proteolysis. Inhibiting MEK did not influence the ability of EGF to suppress proteolysis, but the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY249002, stimulated basal proteolysis and completely eliminated the proteolytic response to EGF. Use of an adenovirus that expresses a dominant negative p85 subunit of class 1 PI 3-kinase completely blocked the ability of EGF to suppress proteolysis, whereas use of an adenovirus expressing a K227E constitutively active p110 subunit reproduced the reduction in protein breakdown. It was concluded that EGF suppresses proteolysis by a mechanism that involves Ras and class 1 PI 3-kinase.