中国实验用小型猪肾小管的发育及各节段标志物的表达

目的:明确解剖和生理上与人非常接近的中国实验用小型猪肾脏发育过程中肾小管的形态学变化和肾小管各节段的特异性标志物。方法:采用高碘酸-希夫(PAS)染色和免疫荧光染色技术,系统观察中国实验用小型猪妊娠28～112d(E28d～E112d)和出生后1d、7d、14d、21d(P1d～21d),共17个不同时间点猪肾小管的发育及肾小管特异性标志物雪莲花凝集素(LTL)、水通道蛋白1(AQP1)、钙结合蛋白(calbindin)-D28k在肾小管不同节段的表达。结果:(1)中国实验用小型猪E28d可见后肾间充质和输尿管芽,即后肾已经开始发育;但这时还没有肾小管。E35d可见不同节段的肾小管,即肾小管已开始发育。从E35d～P14d(E112d仔猪出生),肾皮质均有生肾区存在,即不断有新的肾单位发生;P21d生肾区消失,即不再有新的肾单位产生。(2)①LTL在E28d表达在输尿管芽,E35d开始在近端小管表达,以刷状缘表达最为明显;表达由弱到强,由点状到线状。②AQP1在E28d未见表达,E35d开始表达;AQP1表达在近端小管和髓袢的降支细段,主要表达在细胞膜,尤其在管腔侧的表达更为明显。③Calbindin-D28k在E28d表达在输尿管芽,E35d开始表达在远端小管和集合管;Calbindin-D28k主要表达在细胞质,随着肾小管发育,表达逐渐增强。(3)发现集合管来源于输尿管芽,发源于输尿管芽的集合管从被膜下的生肾区一直延伸到肾髓质。结论:中国实验用小型猪妊娠35d可以见到不同节段肾小管。LTL、AQP1、Calbindin-D28k可以分别作为猪近端小管、髓袢、远端小管和集合管的标志物。     

miRNAs in mammalian ureteric bud development

The collecting duct network and the urothelium of the ureter of the metanephric kidney are derived from the ureteric bud epithelium, initially an outgrowth from the caudal end of the Wolffian duct at the onset of the metanephric kidney development. The tips of the ureteric bud epithelium undergo reiterative branching morphogenesis, which generates more tips and trunks, whereas the ureteric trunks grow and differentiate into principal cells and intercalated cells of the collecting ducts that regulate body water and acid–base homeostasis. microRNAs (miRNAs) are a family of small non-coding RNAs that regulate a diversity of biological processes including organogenesis, mostly by negatively regulating their target gene expression. In this review, I will summarize the current knowledge on the critical roles of miRNAs expressed in the ureteric bud epithelium in ureteric bud morphogenesis and differentiation, including ureteric bud branching morphogenesis, collecting duct terminal differentiation, cystogenesis of the collecting ducts, and ureter development.     

Immunolocalization of fibroblast growth factor-1 and -2 in the embryonic rat kidney

Summary: Fibroblast growth factors (FGF) regulate cell proliferation, migration, differentiation and angiogenesis during morphogenesis in many different tissues. Recent evidence indicates that exogenous FGF-2 stimulates mesenchymal condensation in cultured rat metanephroi, a crucial epithelial-mesenchymal induction event in the developing nephron. the aim of the present investigation was to determine the in vivo distribution of FGF-1 and FGF-2 in developing rat metanephroi at embryonic days 14, 15, 16, 18 and 20. Avidin-biotin enhanced indirect immunohistochemistry was used to demonstrate that both FGF-1 and FGF-2 were co-localized in metanephroi at all ages studied. High levels of FGF-1 and FGF-2 were present in ureteric bud branches and in developing distal tubules. Fibroblast growth factor-1 and FGF-2 were colocalized in developing nephron elements, from vesicles to S-shaped bodies, and in the mesangium of capillary loop and maturing stage glomeruli. Both growth factors were present in the mesenchyme of the nephrogenic zone and in the interstitium of the developing cortex. However, immunostaining for FGF was not evident in mesenchymal condensates, endothelial cells, medullary interstitial cells, or in the thin undifferentiated epithelium of the immature loop of Henle. These findings indicate that the expression of both FGF-1 and FGF-2 is tightly regulated in the embryonic kidney and suggest a role for these molecules in kidney development.     

Role of fibroblast growth factor receptor signaling in kidney development

Fibroblast growth factor receptors (Fgfrs) are expressed in the ureteric bud and metanephric mesenchyme of the developing kidney. Furthermore, in vitro and in vivo studies have shown that exogenous fibroblast growth factors (Fgfs) increase growth and maturation of the metanephric mesenchyme and ureteric bud. Deletion of fgf7, fgf10, and fgfr2IIIb (the receptor isoform that binds Fgf7 and Fgf10) in mice lead to smaller kidneys with fewer collecting ducts and nephrons. Overexpression of a dominant negative receptor isoform in transgenic mice has revealed more striking defects including renal aplasia or severe dysplasia. Moreover, deletion of many fgf ligands and receptors in mice results in early embryonic lethality, making it difficult to determine their roles in kidney development. Recently, conditional targeting approaches revealed that deletion of fgf8 from the metanephric mesenchyme interrupts nephron formation. Furthermore, deletion of fgfr2 from the ureteric bud resulted in both ureteric bud branching and stromal mesenchymal patterning defects. Deletion of both fgfr1 and fgfr2 in the metanephric mesenchyme resulted in renal aplasia, characterized by defects in metanephric mesenchyme formation and initial ureteric bud elongation and branching. Thus, Fgfr signaling is critical for growth and patterning of all renal lineages at early and later stages of kidney development.     

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.     

Stem cells in the embryonic kidney

The mammalian kidney, the metanephros, is formed by a reciprocally inductive interaction between two precursor tissues, the metanephric mesenchyme and the ureteric bud. The ureteric bud induces the metanephric mesenchyme to differentiate into the epithelia of glomeruli and renal tubules. Multipotent renal progenitors that form colonies upon Wnt4 stimulation and strongly express Sall1 exist in the metanephric mesenchyme; these cells can partially reconstitute a three-dimensional structure in an organ culture setting. Six2 maintains this mesenchymal progenitor population by opposing Wnt4-mediated epithelialization. Upon epithelial tube formation, Notch2 is required for the differentiation of proximal nephron structures (podocyte and proximal tubules). In addition, the induction methods of the intermediate mesoderm, the precursor of the metanephric mesenchyme, begin to be elucidated. If derivation of metanephric mesenchyme becomes possible, we will be closer to the generation and manipulation of multiple cell lineages in the kidney.     

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.     

Lessons learned about adult kidney stem cells from the malpighian tubules of Drosophila

All multicellular organisms have a specialized organ to concentrate and excrete wastes from the body. The kidneys in vertebrates and the malpighian tubules in Drosophila accomplish these functions. Mammals and Drosophila share some similar features during renal tubular development. Vertebrate kidneys are derived through the mutual induction of the ureteric bud and metanephric mesoderm, whereas the malpighian tubules of Drosophila develop from the hindgut primordium and visceral mesoderm. The vertebrate kidney also has the capacity to recover and regenerate following episodes of acute injury. Previous studies suggest that stem cells and progenitor cells may be involved in the repair and regeneration of injured renal tissue. However, studies differ as to the source of the regenerating renal cells. Recently, multipotent stem cells in Drosophila malpighian tubules were identified, and it was demonstrated that several differentiated cells in the malpighian tubules arise from these stem cells. In this article, the current understanding of kidney development and stem cell fate in mammal and Drosophila is compared. Furthermore, the potential application of the adult renal stem cells in kidney repair and the treatment of kidney cancers are discussed.     

Essential roles of Sall1 in kidney development

SALL1 is a mammalian homologue of the Drosophila region-specific homeotic gene spalt (sal) and heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We isolated a mouse homologue of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period with kidney agenesis. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud and homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth. Therefore, Sall1 is essential for ureteric bud invasion, the initial key step for metanephros development. We also generated mice in which a green fluorescent protein (GFP) gene was inserted into the Sall1 locus and we isolated the GFP-positive population from embryonic kidneys of these mice by fluorescence-activated cell sorting (FACS). We then compared gene expression profiles in the GFP-positive and -negative population using microarray analysis, followed by in situ hybridization. We detected many genes known to be important for metanephros development, and genes expressed abundantly in the metanephric mesenchyme. We also found groups of genes which are not known to be expressed in the metanephric mesenchyme. Thus a combination of microarray technology and Sall1-GFP mice is useful for systematic identification of genes expressed in the developing kidney.     

Galectin-3 modulates ureteric bud branching in organ culture of the developing mouse kidney

Galectin-3 is a mammalian beta-galactoside-specific lectin with functions in cell growth, adhesion, and neoplastic transformation. On the basis of expression patterns in humans, it is proposed that galectin-3 modulates fetal collecting duct growth. This article provides evidence that galectin-3 can modulate branching morphogenesis of the mouse ureteric bud/collecting duct lineage. With the use of immunohistochemistry, galectin-3 was not detected in early metanephrogenesis but was upregulated later in fetal kidney maturation when the protein was prominent in basal domains of medullary collecting ducts. Addition of galectin-3 to embryonic days 11 and 12 whole metanephric cultures inhibited ureteric bud branching, whereas galectin-1 did not perturb morphogenesis, nor did a galectin-3 mutant lacking wild-type high-affinity binding to extended oligosaccharides. Exogenous galectin-3 retarded conversion of renal mesenchyme to nephrons in whole metanephric explants but did not affect nephron induction by spinal cord in isolated renal mesenchymes. Finally, addition of a blocking antiserum to galectin-3 caused dilation and distortion of developing epithelia in embryonic day 12 metanephroi cultured for 1 wk. The upregulation of galectin-3 protein during kidney maturation, predominantly at sites where it could mediate cell/matrix interactions, seems to modulate growth of the ureteric tree.     

Cell proliferation in the loop of henle in the developing rat kidney.

In the developing rat kidney, there is no separation of the medulla into an outer and inner zone. At the time of birth, ascending limbs with immature distal tubule epithelium are present throughout the renal medulla, all loops of Henle resemble the short loop of adult animals, and there are no ascending thin limbs. It was demonstrated previously that immature thick ascending limbs in the renal papilla are transformed into ascending thin limbs by apoptotic deletion of cells and transformation of the remaining cells into a thin squamous epithelium. However, it is not known whether this is the only source of ascending thin limb cells or whether cell proliferation occurs in the segment undergoing transformation. This study was designed to address these questions and to identify sites of cell proliferation in the loop of Henle. Rat pups, 1, 3, 5, 7, and 14 d old, received a single injection of 5-bromo-2'-deoxyuridine (BrdU) 18 h before preservation of kidneys for immunohistochemistry. Thick ascending and descending limbs were identified by labeling with antibodies against the serotonin receptor, 5-HT(1A), and aquaporin-1, respectively. Proliferating cells were identified with an antibody against BrdU. BrdU-positive cells in descending and ascending limbs of the loop of Henle were counted and expressed as percentages of the total number of aquaporin-1-positive and 5-HT(1A)-positive cells in the different segments. In the developing kidney, numerous BrdU-positive nuclei were observed in the nephrogenic zone. Outside of this location, BrdU-positive tubule cells were most prevalent in medullary rays in the inner cortex and in the outer medulla. BrdU-labeled cells were rare in the papillary portion of the loop of Henle and were not observed in the lower half of the papilla after 3 d of age. BrdU-labeled nuclei were not observed in segments undergoing transformation or in newly formed ascending thin limb epithelium. It was concluded that the growth zone for the loop of Henle is located around the corticomedullary junction, and the ascending thin limb is mainly, if not exclusively, derived from cells of the thick ascending limb.     

The vulnerability of the thin descending limbs of Henle's loop in the isolated perfused rat kidney

In the isolated rat kidney perfused without erythrocytes, the medullary thick ascending limb shows extensive injury. Damage to the thin limbs of Henle's loop has been mentioned only briefly. Thin limbs were examined in the isolated perfused kidney under a variety of conditions that alter oxygenation and active transport in the medulla and are known to affect injury to the medullary thick ascending limb. The thin descending limbs of short loops were preserved in all experimental groups, but those of the long loop showed necrosis that was restricted to the proximal portion, where the epithelium is more complex. In oxygenated kidneys, necrosis involved 41% +/- 5% (mean +/- SE) of the medullary thick ascending limbs and 10% +/- 3% of the proximal portion of long loops of thin descending limbs. Under hypoxic conditions, necrosis involved 90% +/- 3% of the medullary thick ascending limbs and 70% +/- 5% of the proximal portion of long loops of thin descending limbs (P less than 0.0001 compared with oxygenated kidneys). Ouabain and absence of filtration completely prevented necrosis of both nephron segments. Thus, the proximal portions of long loops of thin descending limbs, in resemblance to medullary thick ascending limbs, are especially susceptible to transport-dependent hypoxic injury.     

Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental dysgenesis.

BACKGROUND: A disintegrin and metalloproteinase with thrombospondin motifs 1, Adamts-1, is important for the development and function of the kidney. Mice lacking this protein present with renal lesions comprising enlarged calyces, and reduced cortex and medulla layers. Our current findings are consistent with the defect occurring due to a developmental dysgenesis. METHODS: We generated Adamts-1 null mice, and further investigated their kidney phenotype in a time course study ranging from E18.5 to 12 months of age. Immunohistochemistry was used to assess the localization of type IV collagen, TGF-beta and F4/80-positive macrophages in the kidneys of Adamts-1 null mice compared to wild-type control animals. The expression of Adamts-1 mRNA was determined in metanephric kidney explants by in situ hybridization. RESULTS: Adamts-1 null mice have a gross kidney defect. At day 18.5 of gestation, the Adamts-1 null kidney has a normal appearance but at birth when the kidney begins to function, the defect becomes evident. During development of the kidney Adamts-1 expression was specifically detected in the developing loops of Henle, as well as in the proximal and distal convoluted tubules. Expression was not detected in the ureter, ureteric bud or its derivatives as had been previously suggested. At 6 months and 1 year of age, the Adamts-1 null mice displayed interstitial fibrosis in the cortical and medullary regions of the kidney. At 1 year of age, the Adamts-1 null mice displayed mild interstitial matrix expansion associated with increased collagen type IV expression, without apparent tubular dilatation, compared to wild-type animals. Immunohistochemical analysis demonstrated TGF-beta protein localized to infiltrating macrophages and glomeruli of Adamts-1 null mice. CONCLUSIONS: Adamts-1 is required for the normal development of the kidney. The defect observed in its absence results from a dysgenic malformation affecting the medulla that becomes apparent at birth, once the kidneys start to function.     

Aquaporin-1 is not expressed in descending thin limbs of short-loop nephrons

In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1-positive proximal tubules to aquaporin-1-negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.     

Renal-specific oxidoreductase biphasic expression under high glucose ambience during fetal versus neonatal development

BACKGROUND: Renal-specific oxidoreductase (RSOR) has been recently identified in mice kidneys of diabetic animals, and it is developmentally regulated. Its expression during fetal, neonatal, and postnatal periods was assessed under high glucose ambience. METHODS: Whole-mount immunofluorescence and confocal microscopy were performed to assess the effect of high glucose on the morphogenesis of mice fetal kidneys. RSOR mRNA and protein expression was assessed by competitive polymerase chain reaction (PCR) and immunoprecipitation methods in embryonic kidneys (day E13 to E17) subjected to high glucose ambience and by Northern and Western blot analyses of kidneys of newborn and 1-week-old mice with hyperglycemia. The spatiotemporal changes in the RSOR expression were assessed by in situ hybridization analyses and immunofluorescence microscopy. In addition, the extent of apoptosis in the kidneys was determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. RESULTS: Whole-mount microscopy of the embryonic metanephroi revealed a dose-dependent disruption in the ureteric bud iterations with reduced population of the nascent nephrons. Both gene and protein expressions were reduced in day E13 to E17 metanephroi, while increased in kidneys of newborn and 1-week-old mice. In day E13 and day E15 kidneys, the RSOR was expressed in the ureteric bud branches and some of the immature tubules, and its expression was reduced with high glucose treatment. In day E17 kidneys the RSOR was expressed in the tubules of the deeper cortex, and its expression was marginally decreased. In newborn kidneys, this enzyme was expressed in the subcortical tubules and it spread to the entire width of the renal cortex in hyperglycemic state. In 1-week-old mice kidneys, the RSOR was localized to the entire cortex, and in animals with blood glucose above 300 mg/dL, its intensity increased with extension of expression into the outer medullary tubules. A dose-dependent fulminant apoptosis was observed in day E13 to E17 kidneys subjected to high glucose ambience. In newborn and 1-week-old mice control kidneys, the apoptosis was minimal although slightly increased during hyperglycemia. CONCLUSION: High glucose has a differential effect on the RSOR expression in kidneys during the embryonic versus neonatal/postnatal period. This may partly be related to the differential degree of apoptosis, a process reflective of oxidant stress that is seen in diabetic milieu, which as previously has been shown to adversely effect the modulators of fetal development and thereby the morphogenesis of the kidney and RSOR expression.     

Mechanisms to concentrate the urine: an opinion

PURPOSE OF REVIEW: Our goal is to suggest how the renal concentrating mechanism is regulated in vivo. RECENT FINDINGS: The majority of descending thin limbs of the loop of Henle lack aquaporin-1 water channels, and loops of Henle in the inner medulla lack urea transporters. SUMMARY: Lack of water permeability in the descending thin limbs of the loop of Henle offers several advantages. First, since much less water is added to the outer medullary interstitial compartment, inhibitory control mechanisms on sodium and chloride reabsorption from the medullary thick ascending of loop of Henle initiated by water addition from the medullary collecting duct can be effective. Second, recycling of urea is efficient, as little urea will be washed out of the medulla. Third, delivery of a larger volume of filtrate to the medullary thick ascending limb of the loop of Henle permits both an appreciable reabsorption of sodium along with only a small fall in the luminal concentration of sodium in each of these liters. Hence there need be only a small lumen positive voltage in the medullary thick ascending limb of the loop of Henle. The absence of urea transporters in the loop of Henle in the inner medulla is required for a passive mechanism of sodium and chloride reabsorption in the inner medulla. Control of urea reabsorption from the medullary collecting duct is needed to prevent excessive oliguria in electrolyte-poor urine.     

Adult kidney tubular cell population showing phenotypic plasticity, tubulogenic capacity, and integration capability into developing kidney

Using in vivo bromodeoxyuridine (BrdU) labeling, a tubular cell population (label-retaining tubular cells [LRTC]) was identified recently in normal adult kidneys, which contributes actively to the regeneration process of the kidney after injury. Here, these LRTC are characterized in vitro. The LRTC population was isolated from BrdU-treated rat kidney by FACS. Both LRTC and non-LRTC underwent proliferation and maintained an epithelial phenotype in the presence of tubulogenic growth factors such as EGF, TGF-alpha, IGF-I, and hepatocyte growth factor. It is interesting that LRTC also proliferated without epithelial markers expression in the presence of soluble factors derived from an embryonic kidney metanephric mesenchyme cell line. The type of extracellular matrix strongly influenced the phenotype of LRTC. Furthermore, in three-dimensional collagen gel culture, LRTC formed tubule-like or tubulocystic structures in response to growth factors (hepatocyte growth factor and fibroblast growth factor) that are known to induce kidney cell tubulogenesis in vitro and/or participate in renal regeneration in vivo. In contrast, non-LRTC did not form these structures. When transplanted into the metanephric kidney, LRTC but not non-LRTC were integrated into epithelial components of nephron, including the proximal tubular cells and the ureteric bud. They also differentiated into fibroblast-like cells. Collectively, these findings suggest that LRTC are an adult kidney tubular cell population that shows phenotypic plasticity, tubulogenic capacity, and integration capability into the developing kidney.