Protection Effects And Molecular Mechanisms Of K_ATP Channel Openers On Uric Acid-induced Renal Injury

Hyperuricemia is a common feature of renal disease of all etiologies; as glomerularfiltration rate falls, the serum uric acid increases due to reduced renal excretion [1].Herein, uric acid has long been regarded as a marker of reduced kidney function.However, some studies suggest that elevated uric acid levels might contribute to thedevelopment and progression of renal disease [2]. In the days before treatment of goutwas available, as many as25%of subjects died from renal failure. In addition,25%had albuminuria,50to65%had decreased inulin clearances,70to80%had decreasedrenal plasma flow, and95%had histologic evidence of chronic renal injury [3]. Thesefindings demonstrate that hyperuricemia can induce, aggravate and predict renalinjury [1,4-7]. In turn, renal injury can reduce renal urate excretion. The pathogenesisof hyperuricemia and renal injury is progressive, and develops into a vicious cycle.Renal disease that progresses to end-stage renal disease (ESRD) imposes a greatburden on the affected individual and on society, which mainly bears the cost ofESRD (currently more than$10billion to treat about333,000patients annually in theU.S.)[8]. Although currently available treatments can prevent hyperuricemiaefficaciously, drugs able to ameliorate renal injury induced by hyperuricemia are stilllacking. Renoprotection drugs: renin–angiotensin system inhibitors do not stop the progression of renal disease in patients with proteinuria [9,10]; loop diuretics mightslightly but significantly increase the serum uric acid levels [8,11]. Angiotensin IIreceptor blockers (ARBs), such as losartan and pratosartan, increase excretion of uricacid and decrease the serum levels of uric acid (SUA levels) in both healthy andhypertensive individuals through the inhibition of the urate transporter1(URAT1). Butthe effects of various ARBs on the disposition of uric acid might be different. Somedrugs do not affect the SUA levels [12-14].Previous studies from our laboratory, using in vitro and in vivo experiments, haveshown that iptakalim, a new KCO, prevents elevated blood pressure, improvesendothelial dysfunction and renal disorders induced by hyperuricemia; the ability ofthe kidney to eliminate uric acid is improved, and consequently, SUA levels arereduced effectively [15]. A great many clinical studies have demonstrated thatendothelial dysfunction is a common finding in patients with cardiovascular and renaldisease. Underlying mechanisms that participate in endothelial dysfunction includeimpaired NO production, increased endothelin (ET) generation and overexpression ofadhesion molecules. Iptakalim protects against endothelial dysfunction through thepreferential activation of the SUR2B/Kir6.1subtypes of KATPexpressed inendothelium without activation of SUR1/Kir6.2. Endothelial protection of iptakalim isinvolved in preventing pathological elevation of ET-1, enhancing NO production andremarkably suppressing intercellular adhesive molecule-1(ICAM-1), vascular celladhesive molecule-1(VCAM-1) and MCP-1gene over-expression [16]. It'sdemonstrated that iptakalim is a promising antihypertensive drug suitable forhyperuricemic individuals with hypertension and renal injury. Moreover, iptakalimexerts protective effects on renal injury induced by hypertension, ischemia reperfusionthrough its endothelium protection [17,18]. Yet, the little information regarding thedirect effects of iptakalim on the main cell types in the kidney remains available.The nephron consists of glomeruli and renal tubule. The mature glomeruluscontains at least four cell types: capillary endothelial cells, visceral epithelial cells(podocytes), parietal epithelial cells, and mesangial cells [19]. Each of main cell typesin the kidney is important in the pathogenesis and progression of renal disease [20-22]. Physiological and molecular studies have shown that renal K_ATPchannels play animportant role in renal potassium homeostasis and renal microvascular tone underphysiological conditions [23]. The structure and function of renal K_ATPchannelsmay be changed under pathological conditions[24-25]. At the molecular level, theK_ATPchannel is an octameric complex, composed of two subunits: a pore-formingsubunit of the Kir6.x subfamily and a sulfonylurea receptor (SUR). Differentcombinations of Kir6.x and SUR yield the tissue-specific K_ATPchannel subtypes,which have different electrophysiological and pharmacological characters. SUR2Bcombine with Kir6.1to form functional K_ATPchannels in the renal tubular epithelium,glomerular mesangium, and the endothelium and the smooth muscle of blood vessels[26-29].Iptakalim, a new KCO, was preferential activation of the SUR2B/Kir6.1subtypes of K_ATPexpressed in endothelium without activation of SUR1/Kir6.2[30].Herein, we hypothesized that targeting renal K_ATPchannels with iptakalim may exertprotective effects on the kidney. Natakalim exhibited good selectivity onSUR2B/Kir6.1, not SUR2A/Kir6.2or Kir6.2/SUR1channels. Diazoxide had noselectivity on SUR1, SUR2A and SUR2B. Diazoxide was more effective in activatingSUR2A/Kir6.2channels, and mild effective in activating SUR2B/Kir6.1, lesseffective in activating SUR1/Kir6.2. Natakalim and diazoxide were very usedful forstudying the mechnisms of iptakalim protecting important renal cells.Based on the earlier investigations, we first observed the effects of iptakalim on therenal glomerulus endothelial, mesangial and renal tubular epithelial cells impaired byuric acid, and then compared with the effects of other KCOs (natakalim and diaoxide).In order to analyses the target of iptakalim, the inhibition of glibenclamide was alsoobserved.We would provide the experiment evidence for iptakalim developing intothe anti-injuries of kidney drug and explore the clinical indication of iptakalimthrough this research.Part1The protective effects of ATP-sensitive potassium channel openers againstthe injury of renal cells induced by high concentration of uric acidThe effects of iptakalim on the survival rates of renal cells injuried by uric acidwere investigated by MTT method, and its pharmacological characteristics were further explored.1.1The effects of uric acid on the survival rates of renal tubular epithelial,mesangial and glomerular endothelial cellsAfter treatment with uric acid at concentrations of0,75,150,300,600or1200mg/l for24h in renal glomerular endothelial, mesangial and tubular epithelialcells, we determined the cell viability by MTT assay.600mg/l uric acid could damageglomerular endothelial cells (P<0.05).1200mg/l uric acid significantly decreased thesurvival rates of three types of renal cells (P<0.01).1.2The effects of iptakalim, natakalim, diazoxide or glibenclamide on thesurvival rates of renal tubular epithelial, mesangial and glomerularendothelial cells48h of incubation with10μM iptakalim, natakalim, diazoxide or glibenclamidedid not affect the survival rates of renal glomerular endothelial, mesangial and tubularepithelial cells.1.3The protective effects of ATP-sensitive potassium channel openers against theinjury of renal cells induced by high concentration of uric acidOn treatment with uric acid1200mg/l for24h, survivied renal cells rateremarkably decreased (P<0.01).24h of pretreatment with increasing concentrations ofiptakalim or natakalim (0.1-100μM) notably elevated the survival rates of the renalglomerular endothelial, mesangial cells in comparison with those of the model group(P<0.01-0.05). Preincubation with10-100μM iptakalim or natakalim for24hsignificantly attenuated uric acid-induced cytotoxicity in the renal tubular epithelialcells (P<0.05).10μM iptakalim or natakalim could inhibit the death of renalglomerular endothelial, mesangial and tubular epithelial cells. These effects ofiptakalim or natakalim could be markedly reversed by coadministration withglibenclamide, a selective K_ATPchannel blocker, at10μM.24h of pretreatment withincreasing concentrations of diazoxide (0.1-100μM) notably elevated the survivalrates of the renal glomerular endothelial, mesangial cells in comparison with those ofthe model group (P<0.01-0.05). Preapplication of renal tubular epithelial cells with0.01-100μM diazoxide for24h didn't affect the cell survival rates.10μM diazoxide could inhibit the death of renal glomerular endothelial, mesangial cells. These effectsof diazoxide could be markedly reversed by coadministration with glibenclamide, aselective K_ATPchannel blocker, at10μM.Part2The mechanism of ATP-sensitive potassium channel openers protectingimportant renal cells2.1The regulatory effects of ATP-sensitive potassium channel openers on theprotein expression of Kir6.1and SUR2B in the renal cellsPCR was performed in the presence of specific primers for Kir6.1and SUR2B.Reaction products corresponding to the expected fragment size for Kir6.1and SUR2Bwere detected in the renal tubular epithelial, mesangial and glomerular endothelialcells. Each of these products was confirmed by DNA sequence analysis. Western blotand immunostaining were used to determine the protein expression of Kir6.1andSUR2B.After treatment of1200mg/l uric acid for24h in renal glomerular endothelial,mesangial and renal tubular epithelial cells, the protein expression of Kir6.1andSUR2B was increased (P<0.05), while that of Kir6.1and SUR2B was decreased inpresence of iptakalim or natakalim at the concentration of10μM (P<0.05). Thesedecreases in the expression of Kir6.1and SUR2B were clearly prevented by10μMglibenclamide.10μM diazoxide down regulated the the protein expression of Kir6.1and SUR2B in renal glomerular endothelial, mesangial cells. These effects ofdiazoxide were inhibited by10μM glibenclamide.2.2Effects of ATP-sensitive potassium channel openers treatment on theH₂O₂-induced [Ca2+]imobilizationInvestigated the regulatory effects of iptakalim on cellular function in renal cellsvia detecting the intracellular Ca2+, and then compared with the effects of other KCOs(natakalim and diaoxide). In order to analyses the target of iptakalim, the inhibition ofglibenclamide was also observed.2.2.1The effects of H₂O₂on the intracellular Ca2+in the important renal cellsAfter treatment with H₂O₂at concentrations of0,0.003%,0.03%,0.3%,3%or30%in renal glomerular endothelial, mesangial and tubular epithelial cells, we observed intracellular Ca2+with a multimode benchtop microplate reader.0.3%H₂O₂significantly increased intracellular Ca2+of three types of renal cells (P<0.01).2.2.2The effects of iptakalim, natakalim, diazoxide or glibenclamide on theintracellular Ca2+in the important renal cells10μM iptakalim, natakalim, diazoxide or glibenclamide did not affect theintracellular Ca2+in renal glomerular endothelial, mesangial and tubular epithelialcells.2.2.3Effects of ATP-sensitive potassium channel openers treatment on theH₂O₂-induced [Ca2+]i mobilizationExposure of renal glomerular endothelial, mesangial and renal tubular epithelialcells to0.3%H₂O₂caused a significant increase in the level of intracellular Ca2+(P<0.01). Pretreatment of10μM iptakalim or natakalim significantly reduced theintracellular free Ca2+concentration in renal glomerular endothelial, mesangial andtubular epithelial cells (P<0.01-0.05). These effects of iptakalim or natakalim wereinhibited by10μM glibenclamide. Pretreatment of10μM diazoxide significantlyreduced the intracellular free Ca2+concentration in renal glomerular endothelial,mesangial cells (P<0.01-0.05). These effects of diazoxide were inhibited by10μMglibenclamide. Diazoxide didn't affect the intracellular free Ca2+concentration inrenal tubular epithelial cells.2.3Effects of ATP-sensitive potassium channel openers on the NO releaseInvestigated the regulatory effects of iptakalim on cellular function in renal cellsvia detecting the production of cellular NO, and then compared with the effects ofother KCOs (natakalim and diaoxide). In order to analyses the target of iptakalim, theinhibition of glibenclamide was also observed.10μM diazoxide or glibenclamide did not affect the production of NO in renalglomerular endothelial, mesangial and tubular epithelial cells.10μM iptakalim,natakalim did not affect the production of NO in renal glomerular endothelial,mesangial cells. Pretreatment of10μM iptakalim or natakalim significantly downregulated the NO generation.Exposure of renal glomerular endothelial, mesangial and renal tubular epithelial cells to0.3%H₂O₂caused a significant decrease in NO production (P<0.01).Pretreatment of10μM iptakalim, natakalim or diazoxide significantly upregulated theNO generation impaired by0.3%H₂O₂(P<0.05) and this facilitatory effect ofiptakalim or natakalim was reversed by10μM glibenclamide in glomerularendothelial cells. Iptakalim, natakalim or diazoxide displayed no obvious effects onother types of cells.2.4Effects of ATP-sensitive potassium channel openers on the uric acid-inducedmonocytic adhesion to renal glomerular endothelial cells.Investigated the regulatory effects of iptakalim on cellular function in renal cellsvia detecting the uric acid-induced monocytic adhesion to renal glomerularendothelial cells, and then compared with the effects of other KCOs (natakalim anddiaoxide). In order to analyses the target of iptakalim, the inhibition of glibenclamidewas also observed.Monocytic adhesion to renal glomerular endothelial cells was notably promotedby1200mg/l uric acid (P<0.01). Pretreatment of10μM iptakalim, natakalim ordiazoxide for24h significantly reduced the monocytic adhesion in comparison withthat of the model group (P<0.05). It was showed that the inhibitory effects ofiptakalim, natakalim or diazoxide were antagonized by10μM glibenclamide.2.5Effects of ATP-sensitive potassium channel openers on the MCP-1production induced by uric acid in renal glomerular endothelial cells.Investigated the regulatory effects of iptakalim on cellular function in renal cellsvia detecting the MCP-1production (induced by uric acid) in renal glomerularendothelial cells, and then compared with the effects of other KCOs (natakalim anddiaoxide). In order to analyses the target of iptakalim, the inhibition of glibenclamidewas also observed.There was marked up-regulation of MCP-1protein synthesis by renal glomerularendothelial cells after24h exposure to1200mg/l uric acid (P<0.05). Preincubationwith10μM iptakalim, natakalim or diazoxide significantly decreased MCP-1production (P<0.05). A further experiment showed that10μM glibenclamidesuppressed the down-regulation of MCP-1protein synthesis induced by iptakalim, natakalim or diazoxide.2.6The effects of ATP-sensitive potassium channel openers on the NFκBtranslocation in renal cellsImmunofluorescence staining for NFκB were performed to determine whetherNFκB translocation. After stimulation with uric acid, translocation of NFκBp65from cytoplasms to nuclei of renal glomerular endothelial cells were observed, whilethat of NFκBp65was supressed in presence of iptakalim, natkalim or diazoxide at theconcentration of10μM. This inhibition of NFκB translocation was clearly preventedby10μM glibenclamide.As mentioned above, renal injury induced by uric acid was closely related tocellular damage of glomerular mesangium, endothelium and tubular epithelium.Iptakalim exhibited some selectivity for K_ATPchannels expressed in the renal cells. Inthese three main cell types to form nephron, iptakalim preferentialy activatedSUR2B/Kir6.1subtype of K_ATPchannels in the glomerular mesangium, endothelium,and mildly opened SUR2B/Kir6.1subtype of K_ATPchannels in the tubular epithelium.Renal cell protection of iptakalim was involved in preventing pathological elevationof intracellular Ca2+, enhancing NO production, remarkably suppressing NFκBtranslocation and inhibiting the production of MCP-1and adhesion of mononuclearcells. The inhibitory effects of iptakalim on the up-regulation of Kir6.1and SUR2Bprotein expression induced by high uric acid in the renal cells were clearly preventedby10μM glibenclamide.Considering all the findings described, iptakalim could be developed into a newtherapeutic strategy for protecting against renal injury induced by hyperuricemia withhypertension because of iptakalim's anti-hypertension, endothelial protection andprotection on renal cells. Meanwhile this article provides the experimental clues forinvestigating strategy for the treatment of renal damage.