Evaluation of proliferation and functional differentiation of LLC-PK1 cells on porous polymer membranes for the development of a bioartificial renal tubule device

To develop a bioartificial renal tubule system using renal tubular cells and porous polymer membrane hollow fibers, long-term maintenance of a confluent monolayer and the functionally differentiated condition of cells is essential. We examined the proliferation and functional differentiation of LLC-PK1 (Lewis-lung cancer porcine kidney 1) cells on two types of membranes: polysulfone and cellulose acetate. Cell proliferation was significantly higher on the polysulfone membrane than on the cellulose acetate membrane, and was enhanced by coating the membranes with various extracellular matrices. Confluent monolayer formation of cells was observed on matrix-coated polysulfone membrane but not on matrix-coated cellulose acetate membrane within 1 week. Cell proliferation continued for 3 weeks after confluent monolayer formation. Messenger RNA (mRNA) expression of glucose transporters, indicators of the functional differentiation of the LLC-PK1 cells, was observed in the polysulfone and cellulose acetate membrane groups, but was not observed in the nonporous polystyrene plate group under subconfluent conditions. Expression of glucose transporters mRNA was maintained for 3 weeks after confluent monolayer formation. Polysulfone membrane is more suitable than cellulose acetate membrane for a bioartificial renal tubule system with regard to LLC-PK1 cell proliferation. Extracellular matrix coating of the membrane further improves cell proliferation.     

Evaluation of Na+ active transport and morphological changes for bioartificial renal tubule cell device using Madin-Darby canine kidney cells

The function of current hemodialysis as an artificial kidney is insufficient because of the lack of reabsorptive function. In this study, we intend to develop a bioartificial renal tubule cell device (RTD) using tubular epithelial cells and artificial membranes and to evaluate the reabsorptive function of the confluent layers. Madin-Darby canine kidney (MDCK) cells were cultured on a nucleopore polycarbonate membrane for up to 4 weeks after confluence to examine the influence of the culture period on their ability to transport Na+ actively using Na+/K+ATPase (NKA). The results were (1) active Na+ transport of the cells averaged 24.8 mM/m(2) x 24 h during the initial 2 weeks after confluence and then decreased to about 4.2 mM/m(2) x 24 h during the next 2 weeks; (2) NKA localized on the basal-lateral sides of the cells during the initial 2 weeks, whereas it also localized on the apical side of the cells during the next 2 weeks; (3) long-term culture resulted in an increased number of upheaving cell mass, increased fatty droplets in the cells, and necrosis; and (4) scanning electron microscopy showed fewer microvilli 3-four weeks after confluence. It is concluded that the culture period is critical for developing RTD using cultured renal tubular epithelial cells.     

Evaluation of bioartificial renal tubule device prepared with human renal proximal tubular epithelial cells cultured in serum-free medium

Bioartificial renal tubule devices (BTD) use cell therapy to improve conditions commonly observed in recipients of artificial kidneys for treatment of kidney diseases. We previously reported significant improvement of the condition of acute kidney injury (AKI) animals after treatment with BTD prepared with lifespan-extended human renal proximal tubular cells (hRPTEC). However, a major obstacle to use of BTD for patients is their biological safety, because hRPTEC are cultured in medium containing fetal calf serum. To establish the biological safety of BTD, we prepared BTD with lifespan-extended hRPTEC cultured in a newly developed serum-free medium and compared these with BTD prepared with hRPTEC cultured in serum-containing conventional medium. Lifespan-extended hRPTEC cultured in serum-free medium (hRPTEC-SFM) can proliferate similar to hRPTEC cultured in serum-containing conventional medium (hRPTEC-CM). Comparison of leakage and of reabsorption of small molecules for BTD prepared with hRPTEC-SFM (BTD-SFM) with those for our previous BTD prepared with hRPTEC-CM (BTD-CM) showed transportation in these two types of BTD was almost identical. When AKI goats were treated with BTD-SFM for 26 h, increase of survival time and reduction of cytokine expression in blood cells were almost same as for AKI goats treated with BTD-CM. Quantification of the expression of some genes of hRPTEC in BTD revealed significant changes during BTD treatment for AKI goats. In conclusion, lifespan-extended hRPTEC-SFM work as well as hRPTEC-CM, and the biological safety of BTD for patients could be elevated without loss of function by preparation from hRPTEC-SFM.     

左肾静脉不全结扎对大鼠肾水通道蛋白1和水通道蛋白2mRNA表达的影响

目的：观察左肾静脉不全结扎大鼠肾ＡＱＰ１和ＡＱＰ２ｍＲＮＡ表达的变化,探讨肾静脉压增高引起肾小管重吸收减少的分子机制。方法：成年雄性Ｗｉｓｔａｒ大鼠随机分成左肾静脉不全结扎组（ＶＣＧ）和假手术组（ＳＯＧ）,用左肾脉不全结扎法复制精索静脉曲张,２月后进行Ｎｏｒｔｈｅｒｎｂｌｏｔ分析,结果与ＳＯＧ相比ＶＣＧ大鼠左肾ＡＱＰＩｍＲＮＡ表达强度明显减弱,ＡＱＰ２ｍＲＮＡ表达强度则无明显差异,睾丸组织无杂交信     

小鼠肾小管发育过程中纤维粘连蛋白与整合素α5的表达

背景：纤维粘连蛋白-整合素α5相互作用影响肾小管发育的体内研究较少。 目的：观察小鼠肾小管发育过程中纤维粘连蛋白及其受体整合素α5的表达。 方法：选取胚龄(E)12,14,16,18 d和生后日龄(P)1,3,7,14,21,28,40 d的小鼠。 结果与结论：免疫组织化学染色结果显示纤维粘连蛋白于E12 d时即表达于输尿管芽的基底膜处,随后表达于发育各个时期的肾小管基膜;整合素α5于E12 d时开始表达,表达部位是肾小管的上皮细胞。体视学测量结果显示纤维粘连蛋白表达的面密度值和整合素α5表达的体密度值都随肾脏的发育逐渐增大。Western blot结果显示纤维粘连蛋白和整合素α5蛋白的水平都随肾脏的发育逐渐增加。可见纤维粘连蛋白和整合素α5在小鼠肾小管的发育和成熟过程中的表达具有一定的时空性,对肾小管的发育起一定的作用。     

Nucleotide inhibition of phosphate transport in the renal proximal tubule

The observation that NAD inhibits sodium-dependent phosphate (P) uptake by the luminal brush border membrane (BBM) of the proximal tubule prompted us to examine the specificity and mechanism of this process. Addition of 10(-5) M NAD to the perfusate of isolated perfused rabbit proximal straight tubules inhibited lumen-to-bath P flux by approximately 50%. ADP-ribose had an identical effect, whereas nicotinamide had no effect. ADP and 5'-AMP (10(-5) M) also inhibited P flux. Na-dependent uptake of 32P by rabbit BBM vesicles was inhibited by 0.1-0.3 mM NAD, ADP-ribose, ADP, ATP, 5'-AMP, and GDP, which were preincubated with the vesicles for 30 min. The kinetics of inhibition showed an apparent increase in the Km for P but no change in Vmax. These findings are consistent with "competitive inhibition." The nucleotides inhibited P uptake even when BBM alkaline phosphatase was inhibited by 96% with 10 mM theophylline. Evidence of nonspecific phosphatase activity was present, since incubation of BBM with 0.1 mM solution of nucleotides for 30 min resulted in an elevation of free P in the medium of approximately 0.15-0.22 mM. Correction of 32P specific activity for this change resulted in values for Km and Vmax that were not significantly different from control. The "competitive inhibition" could thus be ascribed to an isotope-dilution effect. There was no evidence to suggest that NAD caused ADP-ribosylation of the luminal membrane. These studies indicate that adenine and guanine nucleotides do not inhibit P transport by a direct action on the luminal membrane of the proximal tubule but do inhibit lumen-to-bath P flux in isolated perfused proximal tubules at concentrations of 10(-5) M. Since there is no direct inhibitory effect of these compounds at the level of the BBM, it is possible that they inhibit P transport by altering some event subsequent to the transfer of P across the luminal membrane.     

Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

 Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK₁ and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK₁ clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl₂. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds. Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997     

Low O2 and high CO2 in LLC-PK1 cells culture mimics renal ischemia-induced apoptosis

Ischemia, absence or loss of blood flow in organs always presents as a dual phenomenon: tissue oxygen deficit and CO(2) excess (hypercapnia). Commonly hypoxic cell culture models kept CO(2) at normal nonischemic values. We report a study of apoptosis in an in vitro model of renal hypoxia that mimics in vivo tissue gas atmosphere composition determined during experimental ischemia in rat kidney (low O(2) plus high CO(2)). Renal tubular LLC-PK1 cell were transiently exposed to hypoxia, to hypercapnia or to both conditions (simulated ischemia). Exposure to simulated ischemic atmosphere, but not to low O(2) or high CO(2) alone, induced cell apoptosis in vitro. This suggests that ischemia-induced apoptosis in vivo would be dependent on the natural, joint action of hypoxia and hypercapnia. This should be taken into account in cell culture studies that would like to mimic in vivo ischemic conditions.     

水通道蛋白在鼠肺发育过程中的变化及意义

目的： 探讨水通道蛋白（AQPs）mRNA及蛋白表达在鼠肺发育过程中的变化及意义。方法: 以胎鼠、新生鼠、幼鼠和成年大鼠为研究对象,采用逆转录-聚合酶链反应（RT-PCR）和免疫组织化学（SABC）方法分别检测几种主要水通道AQP1、AQP3、AQP4和AQP5 mRNA及蛋白在肺脏的表达和分布;同时测定肺发育过程中一些相关指标,进行对比分析。结果: 大鼠肺发育连续不断,从胎鼠至新生鼠期增长最快,以后增速减慢,为一个动态的过程。肺AQPs mRNA在胎鼠时均开始微量转录,仅出现AQP1蛋白表达,出生后AQPs mRNA及蛋白均快速增加,呈增量表达至成年期。相关性分析显示,AQPs变化与肺发育指标变化存在显著正相关（P<0.05）。结论: AQPs可能参与了大鼠肺发育的诸多重要生理过程。     

Transcellular transport of calcium and inorganic phosphate in the small intestinal epithelium

Transport mechanisms involved in the small intestinal handling of inorganic phosphate and calcium have been studied by different in vitro methods during the last few years. In concordance with studies on intact epithelial preparations, studies with brush-border and basal-lateral membrane vesicles isolated from the small intestinal epithelial cell revealed that transcellular calcium and inorganic phosphate fluxes are coupled to transcellular sodium flux, i.e., secondary active via coupling to the primary active sodium flux. A sodium-coupled mechanism in the brush-border membrane leads to cellular accumulation of inorganic phosphate. A sodium-coupled mechanism leads to extrusion of calcium from the cell into the serosal interstitium. A primary active transport mediated by the Ca-ATPase and located in the basal-lateral membrane also exists for calcium. Regulation of transcellular phosphate and calcium flux proceeds via altered influx rates at the luminal cell pole.     

Transcellular and intercellular transport of anions in the kidney tubules of dogs

Fractional reabsorption of 4 anions was measured in anesthetized dogs either during inhibition of bicarbonate-dependent intercellular NaCl transport by acetazolamide or man-nitol, or during inhibition of transcellular NaCl reabsorption in the diluting segment by ethacrynic acid or ouabain. When administered subsequent to ethacrynic acid, acetazolamide reduced fractional reabsorption of SCN, Br, CI and I by 0.28±0.03, 0.28±0.02, 0.27±0.03 and 0.31±0.03. Mannitol given after ethacrynic acid reduced fractional reabsorptions by 0.23±0.04, 0.20±0.04, 0.20±0.05 and 0.20±0.05, respectively. Thus, the bicarbonate-dependent reabsorption system does not discriminate between these anions. Ethacrynic acid reduced fractional reabsorption of SCN, Br and CI by 0.28±0.05, 0.24±0.03, 0.22±0.03 in one group, by 0.32±0.04, 0.34±0.03, 0.31±0.04 in another group, with significantly smaller reductions for I, 0.07±0.03, in both groups. Ouabain reduced fractional reabsorption of Br, CI and I by 0.48±0.04, 0.46±0.04 and 0.24±0.03, respectively. Thus, anion permeability or transport affinity for bromide, chloride and iodide are equal both for inter- and transcellular transport, while iodide transport is slow along the transcellular route. No specific transport mechanism for chloride was detected.     

Expression of parathyroid hormone receptors in MDCK and LLC-PK1 cells

Parathyroid hormone (PTH) inhibits renal proximal tubular phosphate (P_i) and bicarbonate reabsorption by regulating the activity of apical Na/P_i cotransport and Na/H exchange. Two renal epithelial cell lines [proximal tubular, LLC-PK₁; distal tubular, Madin-Darby canine kidney, (MDCK) cells] were stably transfected with complementary deoxyribonucleic acids (cDNAs) encoding a cloned PTH receptor in order to examine the polarity of transfected receptor function and whether or not intrinsic P_i transport is regulated by the transfected PTH receptor. The receptors are functionally coupled to the stimulation of adenosine 35 cyclic monophosphate (cAMP) production at both cell surfaces in LLC-PK₁ cells, whereas this response is primarily limited to the basolateral surface in MDCK cells. Immunocytochemistry suggests an apical and basolateral localization of the transfected PTH receptor in LLC-PK₁ cells and only a basolateral localization in MDCK cells. PTH activation of the transfected receptors is not coupled to the regulation of intrinsic P_i transport in either LLC-PK₁ or MDCK cells.     

Characteristics of aquaporin 1, 3, and 5 expression during early murine salivary gland development

Aquaporins (AQPs) are essential to coordinate the transit of water and ions through the cell membrane. In salivary glands (SGs), AQPs have been associated with saliva formation, facilitating water absorption through the epithelium during the formation of hypotonic saliva, which is then secreted into the oral cavity. Different members of the AQP family have been suggested to play distinct roles during embryonic development, highlighted by their specific expression patterns. Here, we have investigated the expression patterns of AQP-1, AQP-3 and AQP-5 by immunofluorescence at key stages of salivary gland development, utilising cultured mouse embryonic submandibular (SMG) and sublingual (SLG) glands. The expression of AQPs was compared to a mitotic marker, phospho-histone 3 (PH3), a myoepithelial marker, smooth muscle actin (SMA), and a vascular marker, CD31. Qualitative analysis revealed that AQP-1 and AQP-3 were primarily expressed during the earlier phases of SG morphogenesis and were associated with cells undergoing mitotic processes (PH3-positive). AQP-5, in contrast, was not associated to mitotic figures, but was predominantly expressed during late stages of SG morphogenesis. Our results highlight that AQPs are expressed from early stages of SG morphogenesis and exhibit complimentary expression patterns that may contribute to the morphogenesis of salivary glands.     

腰痹舒干预腰椎间盘退变模型兔髓核组织水通道蛋白1、水通道蛋白3的表达

BACKGROUND: The aging and lesions of the intervertebral disc are closely related tothe lack of nutritional blood supply to the disc. Aquaporin plays an importantrole in the nutritional supply to the intervertebral discs, but the specificmechanism has not been fully defined.     

Smad 6和Smad 7基因腺相关病毒载体的构建及其表达

目的 :构建Smad 6和Smad 7基因腺相关病毒 (AAV)载体 ,并转染到人肾小管上皮细胞中表达。方法 :利用基因重组技术 ,采用BamHI和XhoI双酶分别将pcDNA3中的目的基因Smad6 /flag和Smad 7/flag融合基因片断切出 ,再克隆到质粒pAAV MCS中 ,构建重组质粒pAA Smad 6 /flag和pAA Smad 7/flag。采用磷酸钙沉淀法 ,以重组质粒或pAAV LacZ以及pAAV RC、pHelper共转染HEK2 93细胞 ,产生均有传染性的病毒颗粒。再将此病毒颗粒转染到培养的人肾小管细胞中 ,用免疫组化法检测其Smad 6和Smad 7转移基因的表达。结果 :成功地构建Smad 6和Smad 7基因的腺相关病毒载体 ,并可在肾小管上皮细胞中表达Smad 6和Smad 7。结论 :肾小管细胞能稳定、高效表达外源Smad 6和Smad 7,为今后建立Smad 6和Smad 7AAV基因治疗体内肾纤维化的模型奠定了良好的基础     

米非司酮对人羊膜上皮细胞水通道蛋白8表达的影响

目的观察米非司酮对人羊膜上皮细胞水通道蛋白8（AQP8）表达的影响,探讨药物调控羊膜上皮细胞AQP8表达的可行性。方法将体外培养的人羊膜上皮WISH细胞分为不同浓度米非司酮作用48h组和单一浓度米非司酮作用不同时间组,前者培养基中分别加入终浓度为0．1、1、5、10、15μmol／L的米非司酮,培养48h;后者培养基中加入终浓度为10μmol／L的米非司酮,分别培养6、12、24、36、48h,同时2组均设加入含0．01％二甲基亚砜（DMSO）完全培养液的对照组。采用免疫荧光细胞化学方法检测10μmol／L米非司酮作用48h后细胞AQP8蛋白的分布;RT-PCR和蛋白质印迹技术分别检测不同浓度米非司酮作用组和米非司酮作用不同时间组各组细胞AQP8mRNA和蛋白的表达水平。结果免疫荧光细胞化学检测显示,米非司酮作用后WISH细胞细胞质内及细胞膜上AQP8蛋白黄绿色荧光信号较对照组均明显减弱。在不同浓度米非司酮作用组中,除0．1μmol／L组外,其余各组细胞的AQP8mRNA和蛋白表达水平分别与对照组相比均明显减弱（均p〈0．01）,而0．1、1、5、10μmol／L组细胞的AQP8mRNA表达水平和1、5、10μmol／L组细胞的AQP8蛋白表达水平分别与15pmol／L组比较,差异均有统计学意义（均P〈0．05）。在米非司酮作用不同时间组中,除6h组外,其余各组细胞的AQP8mRNA和蛋白表达水平分别与对照组相比均明显减弱（均P〈0．01）,而6、12、24、36h组细胞的AQP8mRNA和蛋白表达水平分别与48h组比较,差异均有统计学意义（均P〈0．05）。结论米非司酮对人羊膜上皮细胞AQP8mRNA和蛋白的表达具有抑制作用。     

Role of aquaporin 3 in development, subtypes and activation of dendritic cells

Dendritic cells (DCs) uptake soluble antigens and large volumes of fluid through macropinocytosis and migrate for antigen presentation. Aquaporin 3 (AQP3), a water and glycerol transporting protein, is highly expressed in immature DCs. To elucidate the role of AQP3 in DC function, we investigated subtype and activation of DCs in AQP3 knock-out (AQP3(-/-)) mice. Depletion of AQP3 did not affect the development of bone marrow-derived DCs (BM-DCs) by GM-CSF or the Flt3 ligand and the level of expression of CD86 on unstimulated and LPS-stimulated BM-DCs. In addition, the percentage of CD86(+) cells among splenic cDCs after LPS treatment in both in vitro and in vivo conditions was similar in wild type and AQP3(-/-) mice. However, the frequency of CD4(+) cDCs in the spleen of AQP3(-/-) mice was significantly lower than that of wild type mice. There was higher expression of CD103 in the CD8(+) subpopulation of splenic cDCs from AQP3(-/-) mice than wild type mice. In the dermis, more CD103-expressing cells were detected in AQP3(-/-) mice than in wild type mice and the LPS-induced decrease of CD103(+) dermal DCs was impaired in AQP3(-/-) mice. AQP3 depletion did not affect the uptake of either albumin or dextran by CD11c(+) splenic DCs. However, HgCl(2), which is an AQP inhibitor, significantly inhibited the uptake of albumin but not dextran by CD11c(+) splenic DCs. These results suggest that AQP3 may play a role in modulating DC population and migration.