经保存的大鼠胚胎后肾同种腹腔内移植模型的建立

目的建立胚胎后肾大网膜内移植大鼠模型,并探讨其在受者体内生长、发育情况。方法取孕16d(E16)和17d(E17)的SD大鼠胚胎,切取胚胎后肾,以组氨酸色氨酸酮戊二酸盐液(HTK液)保存3d,然后移植到切除单侧肾脏的成年SD大鼠的大网膜内,另设未经保存的E16胚胎后肾直接移植对照组。术后给予环孢素A皮下注射,术后3～4周后开腹观察器官形成情况;术后8周,切除受者自体肾脏,观察移植后肾的组织学形态,测定后肾功能。结果移植后3周,移植后肾肾单位、集合管及输尿管的结构正常,组织中少有淋巴细胞浸润,电镜显示移植后肾发育的肾血管球细胞及基底膜、近端肾小管、远端肾小管和集合管上皮细胞超微结构正常。移植后8周,移植后肾的湿重、体积、分泌尿量及内生肌酐清除率与对照组比较,差异无统计学意义(P>0.05)。结论E16、E17胚胎后肾大网膜内移植,并辅以环孢素A皮下注射,可以形成器官,并发挥功能。     

猪后肾帽状间充质细胞发育为肾小球足细胞的过程

目的:探讨中国实验用小型猪肾小球足细胞的发育过程。方法:应用过碘酸-希夫氏染色观察中国实验用小型猪胚胎不同时间点(胚胎28d至出生后21d,以周为单位,共17个时间点)肾小球发育过程中的形态学变化。应用免疫荧光技术检测猪胚肾不同阶段(帽状间充质、肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球、成熟肾小球)足细胞的发育过程。结果:猪在胚胎第28天(E28d)后肾已开始发育,可见典型的帽状间充质、肾小囊体、逗号形体和"S"形体;E35d可见肾小球形成,包括近皮质的不成熟肾小球以及近髓质的成熟肾小球。胚肾组织免疫荧光染色显示:胚肾早期足细胞标志物WT1表达于Six2阳性的后肾帽状间充质细胞,相继表达于肾小囊体、整个逗号形体、逗号形体尾部以及"S"形体下端,最终局限于肾小球足细胞。结论:中国实验用小型猪的足细胞来源于Six2阳性的后肾帽状间充质细胞,经过肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球阶段,发育成为成熟肾小球的足细胞。     

胚肾发育过程中诱骗受体2表达与细胞衰老的关系

目的通过研究诱骗受体2(DcR2)在小鼠胚肾发育过程中的定位和表达,探讨DcR2在胚肾发育中与细胞衰老的关系。 方法分别选取胚龄为12.5 d、16.5 d、20.5 d和出生后8w小鼠的肾脏组织,使用过碘酸雪夫(PAS)染色观察肾组织形态,定量RT-PCR检测肾组织DcR2 mRNA表达水平,免疫组织化学染色观察DcR2的表达分布,免疫荧光共染检测DcR2与近端肾小管标志绒毛蛋白villin、远端肾小管标志水通道蛋白2(AQP-2)、衰老标志P16、胞核形态标志物核纤层蛋白B1(LaminB1)、增殖标志Ki-67和增殖细胞核抗原(PCNA)的共表达关系。 结果随着胚肾的发育,胚肾组织中DcR2 mRNA及蛋白表达逐渐增多,且明显高于成年肾脏;DcR2特异性表达于肾小管,且与villin共表达,但不与AQP-2共表达;DcR2阳性细胞高表达P16,却低表达LaminB1、Ki-67和PCNA。 结论DcR2特异性表达于胚肾近端肾小管细胞,且表达水平随胚龄增长而增多。此外,DcR2阳性细胞具有衰老相关表型,提示DcR2可能在胚肾发育过程中通过调控细胞衰老而具有重要作用。     

猪肾小球内皮细胞与系膜细胞的发育过程及相互关系

目的:明确中国实验用小型猪肾小球内皮细胞与系膜细胞的发育过程及相互关系。方法:采集不同时间点(胚胎28～112d及出生后1d、7d、14d、21d)中国实验用小型猪肾组织,应用免疫荧光技术检测胚肾发育不同阶段(帽状间充质、肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球及成熟肾小球)内皮细胞标志物CD31与系膜细胞标志物α-SMA的表达情况。结果:内皮细胞标志物CD31在胚肾早期呈散在性分布,继而围绕肾小囊体和逗号形体呈"环抱"状分布,然后进入"S"形体下端的血管裂隙内聚集形成无管腔的"前毛细血管束",最后表达于成熟肾小球毛细血管内皮细胞。系膜细胞标志物α-SMA在早期胚肾、肾小囊体和逗号形体阶段均无表达;"S"形体早期分布于"S"形体周围,后期进入"S"形体下端的血管裂隙;毛细血管袢期聚集在肾小球血管极根部;随着肾小球发育逐渐向肾小球内延伸,最终表达于成熟肾小球系膜区。CD31与α-SMA双重染色的结果显示,在毛细血管袢期CD31阳性的内皮细胞聚集形成无管腔的前毛细血管束,而α-SMA阳性的系膜细胞聚集在肾小球血管极的根部;随着肾小球发育,α-SMA阳性的系膜细胞逐渐由血管极根部向肾小球内迁移,同时CD31阳性的内皮细胞逐渐形成带有管腔的毛细血管丛。结论:中国实验用小型猪肾小球内皮细胞的发育开始于后肾间充质阶段,系膜细胞的发育开始于"S"形体阶段,即肾小球系膜细胞发育在内皮细胞之后;在肾小球血管丛形成过程中,内皮细胞与系膜细胞间可能存在重要的相互作用。     

肾组织水通道蛋白表达与肾病综合征水肿的关系

目的 了解肾组织局部水通道蛋白（AQP）表达与肾病综合征（NS）水肿的关系。 方法 NS患者分为水肿组（14例）及无水肿组（8例）,以非NS患者为对照组（10例）。以免疫组织化学方法检测各组患者肾组织AQP1、2、4表达,分析其与患者水肿程度的关系。 结果 NS患者AQP1主要表达在近端肾小管,其中水肿组免疫组化阳性指数为0.0373±0.0110,表达量显著低于非水肿组的0.0510±0.0120和对照组的0.0574±0.0100,差异均有统计学意义;而后两组间差异无统计学意义。AQP1在各组肾小球也有表达,水肿组阳性指数为0.0106±0.0037,显著高于非水肿组的0.0021±0.0013和对照组的0.0020±0.0012,差异均有统计学意义;而后两组间差异无统计学意义。AQP2主要表达在集合管系统,其中水肿组阳性指数为0.0498±0.0081,非水肿组为0.0370±0.0072,均显著高于对照组的0.0255±0.0103,差异均有统计学意义;而水肿组与非水肿组间差异也有统计学意义。AQP4在肾脏皮质集合管未见表达。 结论 不同AQP亚型在NS患者肾组织表达有差异。AQP2在NS水肿发生中起着重要作用,而AQP1的表达异常也参与水肿的发生。     

复方金钱草减轻小鼠草酸钙结晶肾损伤的实验研究

目的：探讨复方金钱草对小鼠草酸钙结晶肾损伤的作用及可能的作用机制.方法：24只C57 BL/6小鼠按随机数字表法随机分为对照组（A组）、单纯结晶组（B组）、复方金钱草干预组（C组）,每组8只.B组：给予100 mg/kg乙醛酸盐;C组：给予100 mg/kg乙醛酸盐＋20 ml/kg复方金钱草浸膏;A组：给予100 mg/kg的生理盐水,在连续给药7 d后,检测各组小鼠血生化指标,肾组织钙含量水平,光镜下观察各组小鼠肾组织切片中草酸钙结晶体分布及组织病理改变,免疫组化及免疫荧光染色对TRPV5、Calbindin-D28进行定位分析,Western Blot法检测各组肾组织中TRPV5、Calbindin-D28k的含量.结果：与单纯结晶组相比,复方金钱草干预组小鼠血清尿素氮（BUN）及肌酐（Scr）水平、肾组织钙含量显著下降（P〈0.05）,肾组织草酸钙结晶沉积及肾脏病理组织损伤均显著减轻（P〈0.05）,但肾脏TRPV5及Calbindin-D28k表达量均强于单纯结晶组（P〈0.05）.结论：复方金钱草可能通过上调肾脏TRPV5及Calbindin-D28k表达量从而减少了肾小管内钙盐结晶体的形成,起到了保护肾脏的作用.     

水通道蛋白在多囊肾病小鼠肾囊泡上皮细胞的表达与调控

目的 探讨水通道蛋白(AQP)在多囊肾病囊泡上皮细胞的表达和调控。 方法 采用免疫荧光染色和Western印迹法分别检测不同亚型的水通道蛋白AQP1、AQP2、AQP3和AQP4在小鼠常染色体隐性遗传病jck多囊肾小鼠肾脏的表达定位和表达调控。 结果 8周龄jck纯合子小鼠的肾脏占体质量的百分率约是同窝野生型小鼠肾脏的4倍,肾脏组织出现多发、大小不一囊泡,囊泡上皮细胞呈扁平状,肾脏间质可见纤维化。jck小鼠血尿素水平为（42.6±6.7） mmol/L,约是野生型小鼠血尿素水平[（8.4±1.9） mmol/L]的5倍（P < 0.01）。免疫荧光定位分析结果表明AQP1 在近曲小管上皮细胞顶膜和基底膜表达,也表达于髓袢降支细段和直小血管降支,肾囊泡上皮细胞未见AQP1表达。AQP2在集合管和肾囊泡上皮细胞顶膜表达,AQP3和AQP4在集合管和囊泡上皮细胞基底膜表达。Western印迹分析结果表明,jck肾脏AQP2、AQP3和AQP4蛋白表达水平与野生型肾脏相似,但AQP1在jck肾脏的表达水平显著低于其在野生型肾脏的表达水平（P < 0.01）。 结论 jck多囊肾小鼠肾囊泡上皮表达AQP2、AQP3和AQP4,提示肾囊泡来源于肾集合管,水通道蛋白可能在肾囊泡生长过程中起重要作用。     

肾小管坏死后修复与胚胎发育过程中细胞极性形成的比较

目的:细胞极性是肾小管上皮细胞发挥生理功能的结构基础。本研究的目的是明确成体大鼠肾小管坏死后修复过程中与胚胎大鼠肾小管发育过程中细胞极性形成过程的一致性。方法:通过皮下注射硫酸庆大霉素制作出生后8周雄性Wistar大鼠急性肾小管坏死后自然修复的动物模型,分别在注射庆大霉素后第7天(用药5d,停药2d)、第14天和第28天采集肾脏标本;另外采集妊娠20d大鼠的胚胎肾脏标本。观察成体大鼠肾小管坏死后修复再生过程中与胚胎大鼠肾小管发育过程中肾小管的形态学变化;以Na+-K+-ATP酶作为细胞极性的标志物,采用免疫荧光染色技术对比观察成体大鼠肾小管坏死后修复再生过程中与胚胎大鼠肾小管发育过程中细胞极性形成的过程。结果:(1)成体大鼠注射庆大霉素后第7天,肾小管上皮细胞坏死、脱落,肾小管基底膜裸露,管腔内有大量脱落的上皮细胞碎片;第14天,肾小管基底膜出现新再生的肾小管上皮细胞;第28天,大多数肾小管结构基本恢复正常。(2)成体大鼠注射庆大霉素后第7天,裸露的肾小管基底膜没有Na+-K+-ATP酶的表达;第14天,新再生的肾小管上皮细胞有Na+-K+-ATP酶的表达,但此时Na+-K+-ATP酶没有明显的极性分布,胞膜和胞浆均有表达;第28天,Na+-K+-ATP酶呈极性分布,主要表达在肾小管上皮细胞的侧基底膜。(3)胚胎大鼠肾小管发育过程是从S形体发育成为不成熟的肾小管,再发育成为成熟的肾小管。(4)胚胎大鼠肾小管发育过程中,Na+-K+-ATP酶的表达从没有极性到有极性分布,即Na+-K+-ATP酶从肾小管上皮细胞的胞膜和胞浆均有表达到只局限在侧基底膜表达。结论:成体大鼠肾小管坏死后修复过程中细胞极性形成的过程与胚胎大鼠肾小管发育过程中细胞极性形成的过程是一致的,提示大鼠肾小管坏死后修复再生的过程类似肾小管胚胎发育的过程。     

原发性肾病综合征患者尿水通道蛋白异常及其意义

目的：探讨原发性肾病综合征患者尿液水通道蛋白含量变化与其在肾组织中表达量的相关性.方法：54例经肾活检病理证实为原发性肾小球疾病的患者分为3组,即非NS组（A组）、NS不伴水肿组（B组）、NS伴水肿组（C组）,应用ELISA法检测患者尿液AQP1及AQP2含量,免疫组化法检测3组患者肾组织AQP1及AQP2的表达.结果：（1）C组尿液AQP1含量为（43.078±17.923）μg,较A组的（39.189±12.448）μg及B组的（41.492±14.766）μg有升高,但差异无统计学意义;C组（45.309±16.921）μg、B组（38.621±13.187）μg尿AQP2含量显著高于A组的（30.320±9.528）μg;（2）AQP1主要表达于近端小管,C组AQP1阳性面积百分比为（0.414±0.201）%,显著低于A组的（0.683±0.311）%及B组的（0.652±0.300）%,A、B组间表达差异无统计学意义;AQP2主要表达于集合管,C组AQP2阳性面积百分比为（0.823±0.002）%,显著高于A组的（0.512±0.213）%及B组的（0.665±0.228）%,A、B两组间表达也差异有统计学意义;（3）尿AQP1含量与AQP1阳性面积百分比无显著相关性;B、C两组尿AQP2含量与AQP2阳性面积百分比呈显著正相关.结论：尿AQP2含量能反映其在肾组织中表达的多寡,但尿AQP1含量与其在肾组织的表达无明显相关.     

消酐散对肾气虚模型大鼠肾组织AQP2表达的影响

目的：探讨消酐散对肾气虚模型大鼠肾功能、肾脏集合管上皮主细胞水通道蛋白2（aquaporin2,AQP2）表达变化。方法：应用丁胺卡那霉素复制肾气虚模型,采用消酐散治疗,于21 d后检测肾功能,采用免疫组化、Western blot方法、RT-PCR方法测定肾组织AQP2蛋白及mRNA表达的变化。结果：消酐散治疗组大鼠肾功能与模型组比较明显改善（P〈0.05）;AQP2的表达显著上调（P〈0.01）。结论：消酐散具有明显的改善肾气虚模型大鼠肾功能的作用;其机制可能与上调肾气虚模型大鼠肾小管上皮细胞AQP2表达有关。     

Renal concentrating and diluting function in deficiency of specific aquaporin genes

Aquaporins (AQP) are water-transporting proteins expressed in many fluid-transporting epithelia and endothelia. In kidney, AQP1 is expressed in plasma membranes of proximal tubule, thin descending limb of Henle and descending vasa recta, AQP2 in collecting duct luminal membrane, AQP3 and AQP4 in collecting duct basolateral membrane, AQP6 in intercalated cells, and AQP7 in the S3 segment of proximal tubule. Human mutations in AQP2 cause hereditary non-X-linked nephrogenic diabetes insipidus. Transgenic mice lacking the renal aquaporins have been useful in defining their role. Mice deficient in AQP1 are polyuric and unable to form a concentrated urine because of defective proximal tubule fluid absorption and countercurrent multiplication. Mice lacking AQP3 are markedly polyuric due to low water permeability across the cortical and outer medullary collecting duct. However, mice lacking AQP4, which is expressed mainly in inner medullary collecting duct, manifest only a mild defect in maximum urinary concentrating ability. The aquaporin null mice have normal urinary diluting ability. From many renal and extrarenal phenotype studies of aquaporin null mice, we conclude that aquaporins are important for rapid near-isosmolar transepithelial fluid absorption/secretion and for rapid vectorial water movement driven by osmotic gradients. The renal phenotype in aquaporin null mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents.     

Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract

Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. It has recently been shown that a 1341-bp fragment of the 5' flanking region containing the Ksp-cadherin gene promoter can recapitulate the complete expression pattern of the gene in the developing kidney and GU tract. Similar to the endogenous Ksp-cadherin gene, transgenes containing 1341 bp of the 5' flanking region are expressed in developing nephrons, ureteric bud, mesonephric tubules, Wolffian duct, and Müllerian duct. In adult mice, the expression is restricted to renal tubules. In the current study, Ksp1.3/Cre transgenic mice carrying 1329 bp of the Ksp-cadherin 5' flanking region linked to the Cre recombinase gene were produced. Adult transgenic mice expressed Cre recombinase in renal tubules, especially collecting ducts and thick ascending limbs of Henle's loops. Transgenic embryos expressed Cre recombinase in the branching ureteric bud, developing renal tubules, and sex ducts. Ksp1.3/Cre transgenic mice were crossed with mice carrying a lacZ reporter gene that is activated by Cre/lox-mediated genetic recombination. Bitransgenic progeny expressed lacZ exclusively in renal tubules, mesonephric tubules, ureteric bud, developing ureter, and Wolffian duct. These results demonstrate that Ksp1.3/Cre transgenic mice express Cre recombinase exclusively in the kidney and developing GU tract and can mediate epithelial-specific Cre/lox recombination in these tissues. Ksp1.3/Cre transgenic mice should be useful for cell lineage studies and kidney-specific gene targeting.     

In Vitro Differentiation of MSC into Cells with a Renal Tubular Epithelial-Like Phenotype

Bone marrow mesenchymal stem (stromal) cells (MSCs) are shown to differentiate into different renal lineages in in vivo injury models. Nevertheless, the in vitro differentiation of MSCs into a renal tubular epithelial lineage has not been investigated. We hypothesize that the injured renal epithelial cells express renotypic factors that may influence the differentiation of MSCs into a renal tubular epithelial lineage. MSCs were cocultured for up to seven days with injured or uninjured murine cortical tubular renal epithelial cells (MCTs), which are separated by a physical barrier; following the coculture, MSCs were examined for the expression of two renal tubular epithelial-specific markers, kidney-specific cadherin (Ksp-cadherin) and aquaporin-1 (AQP1). MSCs differentiated into a tubular epithelial-like phenotype, as shown by the appearance of Ksp-cadherin and AQP1 by day 7 when cocultured with injured MCTs. Further, MSCs showed tubulogenic characteristics when cocultured in a three-dimensional matrix. Nonetheless, MSCs cultured with the conditioned medium from injured MCTs, cocultured with ureteric bud cells, or treated with nephrogenic factors did not differentiate into renal epithelial cells. Based on our findings, we conclude that MSCs can differentiate into a renal epithelial lineage independent of cell fusion when cocultured with injured renal cells.     

BMP receptor ALK3 controls collecting system development

The molecular signals that regulate growth and branching of the ureteric bud during formation of the renal collecting system are largely undefined. Members of the bone morphogenetic protein (BMP) family signal through the type I BMP receptor ALK3 to inhibit ureteric bud and collecting duct cell morphogenesis in vitro. We investigated the function of the BMP signaling pathway in vivo by generating a murine model of ALK3 deficiency restricted to the ureteric bud lineage (Alk3(UB-/-) mice). At the onset of branching morphogenesis, Alk3(UB-/-) kidneys are characterized by an abnormal primary (1 degrees ) ureteric bud branch pattern and an increased number of ureteric bud branches. However, during later stages of renal development, Alk3(UB-/-) kidneys have fewer ureteric bud branches and collecting ducts than wild-type kidneys. Postnatal Alk3(UB-/-) mice exhibit a dysplastic renal phenotype characterized by hypoplasia of the renal medulla, a decreased number of medullary collecting ducts, and abnormal expression of beta-catenin and c-MYC in medullary tubules. In summary, normal kidney development requires ALK3-dependent BMP signaling, which controls ureteric bud branching.     

Role of aquaporin water channels in kidney function studied using transgenic mice

Several aquaporin (AQP) water transporting proteins are expressed in mammalian kidney: AQP1 in plasma membranes of proximal tubule, thin descending limb of Henle, and descending vasa recta; AQP2 in collecting duct luminal membrane; AQP3 and AQP4 in collecting duct basolateral membrane; AQP6 in intercalated cells; and AQP7 in the S3 segment of proximal tubule. To define the role of aquaporins in renal physiology, we have generated and characterized transgenic null mice deficient in AQP1, AQP3, and AQP4, individually and in combinations, as well as AQP2 mutant mice, in which the T126M mutation causing human nephrogenic diabetes insipidus was introduced. AQP1-deficient mice are polyuric and unable to concentrate their urine in response to water deprivation or vasopressin administration. AQP1 deletion greatly reduces osmotic water permeability in proximal tubule, thin descending limb of Henle, and descending vasa recta, resulting in defective proximal tubule fluid absorption and medullary countercurrent exchange. Mice lacking AQP3 have low basolateral membrane water permeability in cortical collecting duct and excrete large quantities of dilute urine. Mice lacking AQP4 have low water permeability in inner medullary collecting duct, but manifest only a mild defect in maximum urinary concentrating ability. These data, taken together with phenotype analyses of brain, lung, and gastrointestinal organs, support the paradigm that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. The renal phenotype data in aquaporin knockout mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents. Received: March 19, 2001 / Accepted: March 22, 2001     

Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation

Metanephric organ culture has been used to determine whether embryonic kidney tubules can be stimulated by cAMP to form cysts. Under basal culture conditions, wild-type kidneys from embryonic day 13.5 to 15.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-d period. Treatment of these kidneys with 8-Br-cAMP or the cAMP agonist forskolin induced the formation of dilated tubules within 1 h, which enlarged over several days and resulted in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Tubule dilation was reversible upon withdrawal of 8-Br-cAMP and was inhibited by the cAMP-dependent protein kinase inhibitor H89 and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)172. For further testing of the role of CFTR, metanephric cultures were prepared from mice with a targeted mutation of the Cftr gene. In contrast to kidneys from wild-type mice, those from Cftr -/- mice showed no evidence of tubular dilation in response to 8-Br-cAMP, indicating that CFTR Cl(-) channels are functional in embryonic kidneys and are required for cAMP-driven tubule expansion. A requirement for transepithelial Cl(-) transport was demonstrated by inhibiting the basolateral Na(+),K(+),2Cl(-) co-transporter with bumetanide, which effectively blocked all cAMP-stimulated tubular dilation. For determination of whether cystic dilation occurs to a greater extent in PKD kidneys in response to cAMP, Pkd1(m1Bei) -/- embryonic kidneys were treated with 8-Br-cAMP and were found to form rapidly CFTR- and Na(+),K(+),2Cl(-) co-transporter-dependent cysts that were three- to six-fold larger than those of wild-type kidneys. These results suggest that cAMP can stimulate fluid secretion early in renal tubule development during the time when renal cysts first appear in PKD kidneys and that PKD-deficient renal tubules are predisposed to abnormally increased cyst expansion in response to elevated levels of cAMP.     

Induction of collecting duct morphogenesis in vitro by heparin-binding epidermal growth factor-like growth factor

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family of growth factors, is synthesized as a membrane-an-chored precursor (proHB-EGF) that is capable of stimulating adjacent cells in a juxtacrine manner. ProHB-EGF is cleaved in a protein kinase C-dependent process, to yield the soluble form. It was observed that HB-EGF acts as a morphogen for the collecting duct system in developing kidneys. HB-EGF protein was expressed in the ureteric bud of embryonic kidneys. Cultured mouse ureteric bud cells (UBC) produced HB-EGF via protein kinase C activation. After stimulation with phorbol ester (12-O-tetradecanoylphorbol-13-acetate) or recombinant soluble HB-EGF, UBC cultured in three-dimensional collagen gels formed short tubules with varied abundant branches. When proHB-EGF-transfected UBC were stimulated with 12-O-tetradecanoylphorbol-13-acetate and cultured in collagen gels, they exhibited linear growth, forming long tubular structures with few branches at the time of appearance of proHB-EGF on the cell surface. The structures exhibited a strong resemblance to the early branching ureteric bud of embryonic kidneys. When UBC were cultured in the presence of transforming growth factor-beta and soluble HB-EGF, they formed long tubules and few branches, similar to the structures observed in proHB-EGF-transfected UBC. These cells exhibited apical-basolateral polarization and expression of the water channel aquaporin-2. These findings indicate that soluble HB-EGF and proHB-EGF induce branching tubulogenesis in UBC in different ways. Juxtacrine activation by proHB-EGF or the synergic action of soluble HB-EGF with transforming growth factor-beta is important for well balanced morphogenesis of the collecting duct system.     

A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract

Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed exclusively in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. Transgenic mice carrying 3425 bp of the Ksp-cadherin 5' flanking region linked to a lacZ reporter gene express beta-galactosidase exclusively in the kidney, although the expression pattern is incomplete (Am J Physiol 277: F599-F610, 1999). To further define the region that mediates tissue-specific expression, transgenic mice carrying 1341 bp or 324 bp of the 5' flanking region linked to a green fluorescent protein (GFP) reporter gene were produced. Transgenic mice carrying 1341 bp of the 5' flanking region expressed GFP in all embryonic tissues that endogenously express Ksp-cadherin, including the ureteric bud, Wolffian duct, Müllerian duct, and developing tubules in the mesonephros and metanephros. In the adult kidney, GFP was highly expressed in thick ascending limbs of Henle's loops and collecting ducts and weakly expressed in proximal tubules and Bowman's capsules. Transgenic mice carrying 324 bp of the 5' flanking region exhibited expression exclusively in tubular epithelial cells in the developing kidney and GU tract. Immunoblot analysis showed that the expression of GFP was restricted to the kidney in adult mice. Taken together, these results demonstrate that 324 bp of the Ksp-cadherin 5' flanking region is sufficient to direct epithelial-specific expression in the developing kidney and GU tract. Transgenic mice that express GFP in the mesonephros, metanephros, ureteric bud, and sex ducts may be useful for cell lineage studies.     

Calbindin-D9k and parvalbumin are exclusively located along basolateral membranes in rat distal nephron.

There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.