Cultures Of Renal Tubular Cells Research Articles

Rapamycin Is Not Protective against Ischemic and Cisplatin-Induced Kidney Injury.

Autophagy plays an important role in the pathogenesis of acute kidney injury (AKI). Although autophagy activation was shown to be associated with an increased lifespan and beneficial effects in various pathologies, the impact of autophagy activators, particularly, rapamycin and its analogues on AKI remains obscure. In our study, we explored the effects of rapamycin treatment in in vivo and in vitro models of ischemic and cisplatin-induced AKI. The impact of rapamycin on the kidney function after renal ischemia/reperfusion (I/R) or exposure to the nephrotoxic agent cisplatin was assessed by quantifying blood urea nitrogen and serum creatinine and evaluating the content of neutrophil gelatinase-associated lipocalin, a novel biomarker of AKI. In vitro experiments were performed on the primary culture of renal tubular cells (RTCs) that were subjected to oxygen-glucose deprivation (OGD) or incubated with cisplatin under various rapamycin treatment protocols. Cell viability and proliferation were estimated by the MTT assay and real-time cell analysis using an RTCA iCELLigence system. Although rapamycin inhibited mTOR (mammalian target of rapamycin) signaling, it failed to enhance the autophagy and to ameliorate the severity of AKI caused by ischemia or cisplatin-induced nephrotoxicity. Experiments with RTCs demonstrated that rapamycin exhibited the anti-proliferative effect in primary RTCs cultures but did not protect renal cells exposed to OGD or cisplatin. Our study revealed for the first time that the mTOR inhibitor rapamycin did not prevent AKI caused by renal I/R or cisplatin-induced nephrotoxicity and, therefore, cannot be considered as an ideal mimetic of the autophagy-associated nephroprotective mechanisms (e.g., those induced by caloric restriction), as it had been suggested earlier. The protective action of such approaches like caloric restriction might not be limited to mTOR inhibition and can proceed through more complex mechanisms involving alternative autophagy-related targets. Thus, the use of rapamycin and its analogues for the treatment of various AKI forms requires further studies in order to understand potential protective or adverse effects of these compounds in different contexts.

A Low Tacrolimus Concentration/Dose Ratio Increases the Risk for the Development of Acute Calcineurin Inhibitor-Induced Nephrotoxicity.

Fast tacrolimus metabolism is linked to inferior outcomes such as rejection and lower renal function after kidney transplantation. Renal calcineurin-inhibitor toxicity is a common adverse effect of tacrolimus therapy. The present contribution hypothesized that tacrolimus-induced nephrotoxicity is related to a low concentration/dose (C/D) ratio. We analyzed renal tubular epithelial cell cultures and 55 consecutive kidney transplant biopsy samples with tacrolimus-induced toxicity, the C/D ratio, C0, C2, and C4 Tac levels, pulse wave velocity analyses, and sublingual endothelial glycocalyx dimensions in the selected kidney transplant patients. A low C/D ratio (C/D ratio < 1.05 ng/mL×1/mg) was linked with higher C2 tacrolimus blood concentrations (19.2 ± 8.7 µg/L vs. 12.2 ± 5.2 µg/L respectively; p = 0.001) and higher degrees of nephrotoxicity despite comparable trough levels (6.3 ± 2.4 µg/L vs. 6.6 ± 2.2 µg/L respectively; p = 0.669). However, the tacrolimus metabolism rate did not affect the pulse wave velocity or glycocalyx in patients. In renal tubular epithelial cells exposed to tacrolimus according to a fast metabolism pharmacokinetic profile it led to reduced viability and increased Fn14 expression. We conclude from our data that the C/D ratio may be an appropriate tool for identifying patients at risk of developing calcineurin-inhibitor toxicity.

Nephrotoxicity induced by the venom of Hypnale hypnale from Sri Lanka: Studies on isolated perfused rat kidney and renal tubular cell lines

The hump-nosed pit viper Hypnale hypnale is responsible for a high number of snakebite cases in southwestern India and Sri Lanka. Although most patients only develop local signs and symptoms of envenoming, there is a growing body of evidence indicating that these envenomings may be associated with systemic alterations, including acute kidney injury. In this study we evaluated the renal toxicity of H. hypnale venom by using a perfused isolated rat kidney system and by assessing cytotoxicity in two different renal tubular cell lines in culture. The venom caused alterations in several renal functional parameters, such as reduction on perfusion pressure, renal vascular resistance, and sodium and chloride tubular transport, whereas glomerular filtration rate and urinary flow initially decreased and then increased after venom perfusion. In addition, this venom was cytotoxic to proximal and distal renal tubular cells in culture, with predominance of necrosis over apoptosis. Moreover, the venom affected the mitochondrial membrane potential and induced an increment in reactive oxygen species in these cells. Taken together, our results demonstrate a nephrotoxic activity of H. hypnale venom in these experimental models, in agreement with clinical observations.

Gene Pathways Analysis of the Effects of Suspension Culture on Primary Human Renal Proximal Tubular Cells

Drug-induced acute kidney injury causes massive morbidity and mortality at exorbitant cost, yet there is currently no effective method for preclinical in vitro testing for nephrotoxicity. Proximal tubule cells are a key target for nephrotoxins, but heretofore, it has been a challenge to maintain their differentiation in vitro. One promising approach is to culture them in suspension, under physiological levels of shear, so as to induce and maintain structural and functional differentiation. Advances in materials, additive manufacturing, and injection molding have reduced the complexity and cost of suspension cultures hardware by orders of magnitude, making it a viable alternative for high throughput screening. This study defines the global transcriptome responses of human renal proximal tubular cells to suspension culture. GSEA/Cytoscape/ClueGO analysis showed near perfect concurrence with GPS-SIGORA analysis in the areas of mineral absorption and ribosome assembly, and defined the nucleic acid and protein mechanisms underlying the transcriptome response to suspension culture. Proximal tubular cells in suspension culture showed increased growth and viability assayed as reducing potential compared to cells cultured under static conditions. Suspension culture of human renal proximal tubular cells now allows investigations in basic cell biology, toxicology, drug screening, and tissue engineering, free of artificial matrices, feeder layers, fetal gene expression, or the need for complex engineering technologies.

Knockout of the interleukin-36 receptor protects against renal ischemia-reperfusion injury by reduction of proinflammatory cytokines

IL-36, a newly named member of the IL-1 cytokine family, includes 3 isoforms, IL-36α, IL-36β, and IL-36γ, all of which bind to a heterodimer containing the IL-36 receptor (IL-36R). Little is known about the role of the IL-36 axis in acute kidney injury (AKI) pathogenesis. Therefore, we evaluated IL-36 function in the bilateral renal ischemia-reperfusion injury model of AKI using IL-36R knockout and wild-type mice. IL-36R was found to be expressed in the kidney, mainly in proximal tubules. In IL-36R knockout mice, plasma creatinine, blood urea nitrogen, and IL-6 levels after ischemia-reperfusion injury were significantly lower than those in wild-type mice. Immunohistological analysis revealed mild tubular injury. IL-36α/β/γ levels were increased after ischemia-reperfusion injury, and IL-36α was expressed in lymphocytes and proximal tubular cells, but post-ischemia-reperfusion injury mRNA levels of IL-6 and TNF-α were low in IL-36R knockout mice. In primary cultures of renal tubular epithelial cells, IL-36α treatment upregulated NF-κB activity and Erk phosphorylation. Notably, in patients with AKI, urine IL-36α levels were increased, and IL-36α staining in renal biopsy samples was enhanced. Thus, IL-36α/IL-36R blockage could serve as a potential therapeutic target in AKI.

D-serine, a novel uremic toxin, induces senescence in human renal tubular cells via GCN2 activation

The prevalence of chronic kidney disease (CKD), characterized by progressive renal dysfunction with tubulointerstitial fibrosis, is increasing because of societal aging. Uremic toxins, accumulated during renal dysfunction, cause kidney damage, leading to renal deterioration. A recent metabolomic analysis revealed that plasma D-serine accumulation is associated with faster progression of renal dysfunction in CKD patients. However, the causal relationship and the underlying mechanisms remain unclear. Herein, we demonstrated that D-serine markedly induced cellular senescence and apoptosis in a human proximal tubular cell line, HK-2, and primary culture of human renal tubular cells. The former was accompanied by G2/M cell cycle arrest and senescence-associated secretory phenotype, including pro-fibrotic and pro-inflammatory factors, contributing to tubulointerstitial fibrosis. Integrated stress response mediated by the general control nonderepressible 2 played an important role in D-serine-induced tubular cell toxicity and pro-fibrotic phenotypes, accelerating CKD progression and kidney aging. D-serine upregulated the L-serine synthesis pathway. Furthermore, D-serine-induced suppression of tubular cell proliferation was ameliorated by L-serine administration, indicating that D-serine exposure induced an L-serine-deprived state in tubular cells, compensated by L-serine synthesis. Thus, this study unveils molecular mechanisms underlying D-serine-induced tubular damage and pro-fibrotic phenotypes, suggesting that D-serine is a uremic toxin involved in CKD pathogenesis.

Preconditioning of primary human renal proximal tubular epithelial cells without tryptophan increases survival under hypoxia by inducing autophagy

Hypoxia plays a significant role in the pathogenesis of acute kidney injury (AKI). Autophagy protects from AKI. Amino acid deprivation induces autophagy. The effect of L-tryptophan depletion on survival and autophagy in cultures of renal proximal tubular epithelial cells (RPTECs) under hypoxia was evaluated. RPTECs were preconditioned in a medium containing or not tryptophan, following culture under hypoxia and treatment with or without the autophagy inhibitor chloroquine. Cell survival was assessed by cell imaging, the level of certain proteins by western blotting and cellular ATP fluorometrically. Preconditioning of RPTECs in a medium without tryptophan activated general control nonderepressible 2 kinase and induced changes that favored autophagy and cell survival under hypoxic conditions. Additionally, it increased cellular ATP, while it inhibited apoptosis. Inhibition of autophagy nullified the induced increase in cellular ATP and cell survival by the absence of tryptophan. The absence of tryptophan increased p53, although its effect on p53's transcriptional targets was heterogeneous. In accordance with the decreased apoptosis, expression of p21 increased, while expression of Bax decreased. The expression of BNIP3L, which may be pro-apoptotic or pro-autophagic, increased. Considering the decreased apoptosis, it is likely that tryptophan depletion enhances autophagy through a p53-mediated increase of BNIP3L. Preconditioning of primary human RPTECs in a medium without tryptophan increases their survival under hypoxia by inducing autophagy. Identifying new molecular mechanisms that protect renal tissue from hypoxia could be proved clinically important in the prevention of AKI.

Mechanisms of Inflammatory Injury of Renal Tubular Cells in a Cellular Model of Pyelonephritis.

Previously, we have assembled a cellular model of pyelonephritis which contains a primary culture of renal tubular epithelial cells, mononuclear leukocytes, and bacterial lysate or lipopolysaccharide. After cocultivation of renal cells with leukocytes and bacterial lysate, proinflammatory changes were observed in the renal cells, followed by nitrosative and oxidative stress and cell death. The interaction of bacterial antigens not only with leukocytes, but also with epithelial cells of the renal tubules, was partially mediated by signaling pathways involving Toll-like receptors (TLR2 and TLR4). Activation of these receptors led to increased levels of oxidative stress and synthesis of proinflammatory cytokines (TNF, IL-6, IL-1α) in the renal epithelium, while TLR4 blockade decreased the severity of these processes. Apart from the fact that activation of inflammatory signaling in response to bacterial antigens is observed directly in the renal cells, the presence of leukocytes significantly amplifies the inflammatory response as measured by the level of cytokines generated in the ensemble. In the presence of activated leukocytes, higher expression of TLR2 on the surface of renal cells was observed in response to exposure to bacterial components, which might explain the increased inflammatory response in the presence of leukocytes. The synthesis of IL-1α in the epithelial cells of the renal tubules in this inflammatory model leads to its accumulation in the nuclei, which has been reduced by the TLR4 antagonist polymyxin. TLR2 agonists also led to increased levels of IL-1α. The elevation in the content of IL-1α in nuclei was accompanied by increased acetylation of nuclear proteins, which has been reduced to control values after exposure to protective agents (Trolox, mitochondria-targeted antioxidant SkQR1 or LiCl). The high level of acetylation of histones is probably regulated by proinflammatory cytokines, and to some extent it is a marker of inflammation, which can indirectly be reduced by protective agents.

Effects of shiga toxin 2 on cellular regeneration mechanisms in primary and three-dimensional cultures of human renal tubular epithelial cells

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes post-diarrheal Hemolytic Uremic Syndrome (HUS), which is one of the most common causes of acute renal failure in children in Argentine. The aim of the present work was to study the effects of Shiga toxin type 2 (Stx2) on regenerative mechanisms of primary cultures of human cortical renal tubular epithelial cells (HRTEC) and three-dimensional (3D) cultures of HRTEC. Primary cultures of HRTEC were able to develop tubular structures when grown in matrigel, which showed epithelial cells surrounding a central lumen resembling the original renal tubules. Exposure to Stx2 inhibited tubulogenesis in 3D-HRTEC cultures. Moreover, a significant increase in apoptosis, and decrease in cell proliferation was observed in tubular structures of 3D-HRTEC exposed to Stx2. A significant reduction in cell migration and vimentin expression levels was observed in HRTEC primary cultures exposed to Stx2, demonstrating that the holotoxin affected HRTEC dedifferentiation. Furthermore, a decreased number of cells expressing CD133 progenitor marker was found in HRTEC cultures treated with Stx2. The CD133 positive cells also expressed the Stx receptor globotriaosylceramide, which may explain their sensitivity to Stx2. In conclusion, Stx2 affects the regenerative processes of human renal tubular epithelial cells in vitro, by inhibiting cell dedifferentiation mechanisms, as well as tubules restoration. The development of 3D-HRTEC cultures that resemble original human renal proximal tubules is a novel in vitro model to study renal epithelial repair mechanisms after injury.

Tuberin regulates reactive oxygen species in renal proximal cells, kidney from rodents, and kidney from patients with tuberous sclerosis complex

Reactive oxygen species (ROS) are an important endogenous source of DNA damage and oxidative stress in all cell types. Deficiency in tuberin resulted in increased oxidative DNA damage in renal cells. In this study, the role of tuberin in the regulating of ROS and NADPH oxidases was investigated. Formation of ROS and activity of NADPH oxidases were significantly higher in mouse embryonic fibroblasts and in primary culture of rat renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. In addition, expression of NADPH oxidase (Nox)1, Nox2, and Nox4 (Nox isoforms) was higher in mouse embryonic fibroblasts and renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. Furthermore, activity levels of NADPH oxidases and protein expression of all Nox isoforms were higher in the renal cortex of rat deficient in tuberin. However, treatment of tuberin‐deficient cells with rapamycin showed significant decrease in protein expression of all Nox. Significant increase in protein kinase C βII expression was detected in tuberin‐deficient cells, whereas inhibition of protein kinase C βII by bisindolylmaleimide I resulted in decreased protein expression of all Nox isoforms. In addition, treatment of mice deficient in tuberin with rapamycin resulted in significant decrease in all Nox protein expression. Moreover, protein and mRNA expression of all Nox were highly expressed in tumor kidney tissue of patients with tuberous sclerosis complex compared to control kidney tissue of normal subjects. These data provide the first evidence that tuberin plays a novel role in regulating ROS generation, NADPH oxidase activity, and Nox expression that may potentially be involved in development of kidney tumor in patients with tuberous sclerosis complex.

Albuminuria confers renal resistance to loop diuretics via the stimulation of NLRP3 inflammasome/prostaglandin signaling in thick ascending limb.

Renal resistance to loop diuretics is a frequent complication in a number of kidney disease patients with elusive mechanism. Employing human renal biopsy specimens, albumin overload mouse model, and primary cultures of mouse renal tubular cells, albuminuria effect on NKCC2 expression and function and the underlying mechanisms were investigated. In the renal biopsy specimens of albuminuric patients, we found that NKCC2 was significantly downregulated with a negative correlation with albuminuria severity as examined by immunohistochemistry. Meanwhile, NLRP3 and mPGES-1 were stimulated in NKCC2 positive tubules (thick ascending limb, TAL) paralleled with increased urinary PGE2 excretion. To examine the role of albuminuria in the downregulation of NKCC2 and the potential role of NLRP3/prostaglandin signaling in NKCC2 downregulation, an albumin overload mouse model was employed. Interestingly, we discovered that albuminuria downregulated NKCC2 protein expression in murine kidney and impaired the renal response to loop diuretic furosemide. Specifically, albuminuria suppressed NKCC2 expression and function through NLRP3/prostaglandin dependent signaling in TAL. In primary cultures of renal tubular cells, albumin directly reduced NKCC2 but enhanced NLRP3, COX-2, and mPGES-1 expression. These novel findings demonstrated that albuminuria is of importance in mediating the renal resistance to loop diuretics via NLRP3/prostaglandin signaling-dependent NKCC2 downregulation in TAL. This may also offer novel, effective targets for dealing with the resistance of loop diuretics in proteinuric renal diseases.

Albuminuria enhances NHE3 and NCC via stimulation of mitochondrial oxidative stress/angiotensin II axis.

Imbalance of salt and water is a frequent and challenging complication of kidney disease, whose pathogenic mechanisms remain elusive. Employing an albumin overload mouse model, we discovered that albuminuria enhanced the expression of NHE3 and NCC but not other transporters in murine kidney in line with the stimulation of angiotensinogen (AGT)/angiotensin converting enzyme (ACE)/angiotensin (Ang) II cascade. In primary cultures of renal tubular cells, albumin directly stimulated AGT/ACE/Ang II and upregulated NHE3 and NCC expression. Blocking Ang II production with an ACE inhibitor normalized the upregulation of NHE3 and NCC in cells. Interestingly, albumin overload significantly reduced mitochondrial superoxide dismutase (SOD2), and administration of a SOD2 mimic (MnTBAP) normalized the expression of NHE3, NCC, and the components of AGT/ACE pathway affected by albuminuria, indicating a key role of mitochondria-derived oxidative stress in modulating renin-angiotensin system (RAS) and renal sodium transporters. In addition, the functional data showing the reduced urinary excretion of Na and Cl and enhanced response to specific NCC inhibitor further supported the regulatory results of sodium transporters following albumin overload. More importantly, the upregulation of NHE3 and NCC and activation of ACE/Ang II signaling pathway were also observed in albuminuric patient kidneys, suggesting that our animal model accurately replicates the human condition. Taken together, these novel findings demonstrated that albuminuria is of importance in resetting renal salt handling via mitochondrial oxidative stress-initiated stimulation of ACE/Ang II cascade. This may also offer novel, effective therapeutic targets for dealing with salt and water imbalance in proteinuric renal diseases.

Requirement of clusterin expression for prosurvival autophagy in hypoxic kidney tubular epithelial cells.

Cellular autophagy is a prosurvival mechanism in the kidney against ischemia-reperfusion injury (IRI), but the molecular pathways that activate the autophagy in ischemic kidneys are not fully understood. Clusterin (CLU) is a chaperone-like protein, and its expression is associated with kidney resistance to IRI. The present study investigated the role of CLU in prosurvival autophagy in the kidney. Renal IRI was induced in mice by clamping renal pedicles at 32°C for 45 min. Hypoxia in renal tubular epithelial cell (TEC) cultures was induced by exposure to a 1% O2 atmosphere. Autophagy was determined by either light chain 3-BII expression with Western blot analysis or light chain 3-green fluorescent protein aggregation with confocal microscopy. Cell apoptosis was determined by flow cytometric analysis. The unfolded protein response was determined by PCR array. Here, we showed that autophagy was significantly activated by IRI in wild-type (WT) but not CLU-deficient kidneys. Similarly, autophagy was activated by hypoxia in human proximal TECs (HKC-8) and WT mouse primary TECs but was impaired in CLU-null TECs. Hypoxia-activated autophagy was CLU dependent and positively correlated with cell survival, and inhibition of autophagy significantly promoted cell death in both HKC-8 and mouse WT/CLU-expressing TECs but not in CLU-null TECs. Further experiments showed that CLU-dependent prosurvival autophagy was associated with activation of the unfolded protein response in hypoxic kidney cells. In conclusion, these data suggest that activation of prosurvival autophagy by hypoxia in kidney cells requires CLU expression and may be a key cytoprotective mechanism of CLU in the protection of the kidney from hypoxia/ischemia-mediated injury.

Potential signal pathway between all-trans retinoic acid and LMX1B in hypoxia-induced renal tubular epithelial cell injury

All-trans retinoic acid (ATRA), an active metabolite of vitamin A, exerts various effects on physiological processes such as cell growth, differentiation, apoptosis and inflammation. LMX1B, a developmental LIM-homeodomain transcription factor, is widely expressed in vertebrate embryos, and it takes part in the development of diverse structures such as limbs, kidneys, eyes, brains, etc. Renal tubular epithelial cell culture was performed, and mRNA and protein expression of some factors were detected. We recently demonstrated that ATRA up-regulated the LMX1B, and down-regulated the transforming growth factor-β1, collagen IV and fibronectin in a hypoxia/reoxygenation (H-R) injury system in renal tubular epithelial cells (RTEC). In conclusion, ATRA acts as a positive regulator of LMX1B in H-R RTEC.

Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3.

Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity.

Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105+). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression.

Oxidative stress causes imbalance of renal renin angiotensin system (RAS) components and hypertension in obese Zucker rats.

BackgroundOxidative stress plays an important role in the pathogenesis of hypertension, especially in obesity‐related hypertension. The natriuretic and antinatriuretic components of the renal renin angiotensin system (RAS) maintain sodium homeostasis and blood pressure. Here, we test the hypothesis that increased oxidative stress leads to the imbalance of RAS components and hypertension in obese Zucker rats.Methods and ResultsLean and obese rats received vehicle or tempol, a superoxide dismutase mimetic in the drinking water for 4 weeks. Compared with vehicle‐treated lean rats, vehicle‐treated obese rats exhibited higher blood pressure and increased renal oxidative stress, accompanied by increased diuretic and natriuretic responses to AT1R antagonist (Candesartan) and AT2R agonist (CGP‐42112A) and reduced diuretic and natriuretic response to MasR agonist (Ang‐[1 to 7]). Moreover, obese rats had higher ACE, AT1R and AT2R, lower ACE2 and MasR expressions in the kidney. All of the above‐mentioned abnormalities were reversed to some degree by tempol treatment. In primary cultures of renal proximal tubular (RPT) cells from lean and obese rats, tempol treatment also increased AT2R, ACE2, and MasR expressions but decreased AT1R and ACE expressions in obese rats.ConclusionsTaken together, our study indicated that the imbalance of renal RAS components was associated with increased oxidative stress in obese rats. Furthermore, antioxidant treatment with tempol reversed the imbalance of renal RAS components and led to diuresis and natriuresis, which, at least in part, explains the blood pressure‐lowering effect of antioxidant supplementation in obesity‐related hypertension.

Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury.

Background & objectives:Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury. One of the targets to protect against injury is ATP-dependent potassium (KATP) channels. These channels could be involved in EPO induced ischaemic preconditoning like a protective effect. We evaluated the cell cytoprotective effects of EPO in relation to KATP channel activation in the renal tubular cell culture model under hypoxic/normoxic conditions.Methods:Dose and time dependent effects of EPO, KATP channel blocker glibenclamide and KATP channel opener diazoxide on cellular proliferation were evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] under normoxic and hypoxic conditions in human renal proximal tubular cell line (CRL-2830). Evaluation of the dose and time dependent effects of EPO, glibenclamide and diazoxide on apoptosis was done by caspase-3 activity levels. Hypoxia inducible factor-1 alpha (HIF-1 α) mRNA levels were measured by semi-quantative reverse transcription polymerase chain reaction (RT)-PCR. Kir 6.1 protein expresion was evalutaed by Western blot.Results:Glibenclamide treatment decreased the number of living cells in a time and dose dependent manner, whereas EPO and diazoxide treatments increased. Glibenclamide (100 μM) treatment significantly blocked the anti-apoptotic effects of EPO (10 IU/ml) under both normoxic and hypoxic conditions. EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (P<0.01) whereas glibenclamide decreased (P<0.05) HIF-1 α mRNA expression. Glibenclamide significantly (P<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group.Interpretation & conclusions:Our results showed that the cell proliferative, cytoprotective and anti-apoptotic effects of EPO were associated with KATP channels in the renal tubular cell culture model under hypoxic/normal conditions.

Heparan Sulfate-Bound Dimeric IL-2: A Potential Mediator of Renal Ischemic Injury.

Interleukin-2 (IL-2) is a multifaceted cytokine that exhibits immunostimulatory and immunosuppressive properties. While primarily known as a monomer, a dimeric form of IL-2 has been identified in fish optic neurons, and this dimeric form was reported to be toxic to oligodendrocytes. Given this observation, we asked whether dimeric IL-2 is found in mammalian tissues. To this end, murine and human aorta and kidney tissues were homogenized and assessed by Western blot analysis for the presence of IL-2. Dimeric (30 kD) IL-2 was identified in each tissue. To determine whether dimeric IL-2 is retained by heparan sulfate, we digested the tissues with heparinase. Dimeric IL-2 was liberated, suggesting that dimeric IL-2 is bound, at least in part, by heparan sulfate. To ascertain whether dimeric IL-2 in murine or human tissues is cytotoxic, we treated cultures of renal tubular epithelial cells with increasing concentrations of dimeric IL-2 that was purified by electroelution from zinc-stained preparatory gels. Signs of cytotoxicity, including membrane blebbing and lactate dehydrogenase release, were apparent within 15 minutes of dimer addition. Commercial IL-2, isolated in an identical fashion to dimeric IL-2, was not cytotoxic. Systemic administration to mice of 10 ug of dimeric IL-2 induced vacuolization of renal tubular epithelium, a morphology typically seen with ischemic injury. Finally, murine kidneys subjected to 60 minutes of ischemia, as compared to sham controls, expressed greatly increased amounts of dimeric IL-2 in both tissue homogenates and heparinase digests. These results suggest that dimeric IL-2 may contribute to acute tubular necrosis and, in turn, renal dysfunction, induced as a consequence of severe hypotension, dehydration, sepsis, or reperfusion injury.

Carbon monoxide potently prevents ischemia-induced high-mobility group box 1 translocation and release and protects against lethal renal ischemia–reperfusion injury

High-mobility group box 1 (HMGB1) is a chromatin-binding nuclear molecule that has potent proinflammatory effects once released by damaged cells. In some disease models, carbon monoxide (CO) exhibits anti-inflammatory and protective properties. Here, we investigated whether the protective effect of CO on renal ischemia-reperfusion injury is associated with the inhibition of HMGB1 translocation and release. A renal ischemia-reperfusion injury model was established with a 100% mortality rate in untreated mice. Pretreatment with the CO-releasing molecule-2 (CORM-2) resulted in 100% survival, maximal preservation of renal function, a marked reduction in pathological damage, and blunted upregulation of TLR4, RAGE, TNF-α, IL-1β, IL-6, and MCP1 mRNA. Interestingly, CORM-2 pretreatment almost completely inhibited ischemia-induced HMGB1 nucleocytoplasmic shuttling and release. This inhibition was associated with a decrease in nuclear histone acetyltransferase activity. Indeed, CORM-2 pretreatment inhibited the acetylation and release of HMGB1 during hypoxic culture of primary mouse renal tubular epithelia cells in vitro. Using the same renal ischemia-reperfusion injury model, neutralization of HMGB1 was protective, and administration of exogenous HMGB1 largely reversed the protective effect of CORM-2 on kidney ischemia-reperfusion injury. Thus, CORM-2-delivered CO protects against lethal renal ischemia-reperfusion injury. This protection is correlated with the prevention of HMGB1 nuclear-cytoplasmic translocation and release.