Conserved and Divergent Features of Mesenchymal Progenitor Cell Types within the Cortical Nephrogenic Niche of the Human and Mouse Kidney.

Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2+ nephron progenitor cells (NPCs) and Foxd1+ interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2+ NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2+ NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.

Inversin modulates the cortical actin network during mitosis.

Mutations in inversin cause nephronophthisis type II, an autosomal recessive form of polycystic kidney disease associated with situs inversus, dilatation, and kidney cyst formation. Since cyst formation may represent a planar polarity defect, we investigated whether inversin plays a role in cell division. In developing nephrons from inv-/- mouse embryos we observed heterogeneity of nuclear size, increased cell membrane perimeters, cells with double cilia, and increased frequency of binuclear cells. Depletion of inversin by siRNA in cultured mammalian cells leads to an increase in bi- or multinucleated cells. While spindle assembly, contractile ring formation, or furrow ingression appears normal in the absence of inversin, mitotic cell rounding and the underlying rearrangement of the cortical actin cytoskeleton are perturbed. We find that inversin loss causes extensive filopodia formation in both interphase and mitotic cells. These cells also fail to round up in metaphase. The resultant spindle positioning defects lead to asymmetric division plane formation and cell division. In a cell motility assay, fibroblasts isolated from inv-/- mouse embryos migrate at half the speed of wild-type fibroblasts. Together these data suggest that inversin is a regulator of cortical actin required for cell rounding and spindle positioning during mitosis. Furthermore, cell division defects resulting from improper spindle position and perturbed actin organization contribute to altered nephron morphogenesis in the absence of inversin.

Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development.

Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.

Prenatal programming of rat cortical collecting tubule sodium transport.

Prenatal insults have been shown to lead to elevated blood pressure in offspring when they are studied as adults. Prenatal administration of dexamethasone and dietary protein deprivation have demonstrated that there is an increase in transporter abundance for a number of nephron segments but not the subunits of the epithelial sodium channel (ENaC) in the cortical collecting duct. Recent studies have shown that aldosterone is elevated in offspring of protein-deprived mothers when studied as adults, but the physiological importance of the increase in serum aldosterone is unknown. As an indirect measure of ENaC activity, we compared the natriuretic response to benzamil in offspring of mothers who ate a low-protein diet (6%) with those who ate a normal diet (20%) for the last half of pregnancy. The natriuretic response to benzamil was greater in the 6% group (821.1 ± 161.0 µmol/24 h) compared with the 20% group (279.1 ± 137.0 µmol/24 h), consistent with greater ENaC activity in vivo (P < 0.05). In this study, we also directly studied cortical collecting tubule function from adult rats using in vitro microperfusion. There was no difference in basal or vasopressin-stimulated osmotic water permeability. However, while cortical collecting ducts of adult offspring whose mothers ate a 20% protein diet had no sodium transport (-1.9 ± 3.1 pmol·mm(-1)·min(-1)), the offspring of rats that ate a 6% protein diet during the last half of pregnancy had a net sodium flux of 10.7 ± 2.6 pmol·mm(-1)·min(-1) (P = 0.01) in tubules perfused in vitro. Sodium transport was measured using ion-selective electrodes, a novel technique allowing measurement of sodium in nanoliter quantities of fluid. Thus we directly demonstrate that there is prenatal programming of cortical collecting duct sodium transport.

Hox10 genes function in kidney development in the differentiation and integration of the cortical stroma.

Organogenesis requires the differentiation and integration of distinct populations of cells to form a functional organ. In the kidney, reciprocal interactions between the ureter and the nephrogenic mesenchyme are required for organ formation. Additionally, the differentiation and integration of stromal cells are also necessary for the proper development of this organ. Much remains to be understood regarding the origin of cortical stromal cells and the pathways involved in their formation and function. By generating triple mutants in the Hox10 paralogous group genes, we demonstrate that Hox10 genes play a critical role in the developing kidney. Careful examination of control kidneys show that Foxd1-expressing stromal precursor cells are first observed in a cap-like pattern anterior to the metanephric mesenchyme and these cells subsequently integrate posteriorly into the kidney periphery as development proceeds. While the initial cap-like pattern of Foxd1-expressing cortical stromal cells is unaffected in Hox10 mutants, these cells fail to become properly integrated into the kidney, and do not differentiate to form the kidney capsule. Consistent with loss of cortical stromal cell function, Hox10 mutant kidneys display reduced and aberrant ureter branching, decreased nephrogenesis. These data therefore provide critical novel insights into the cellular and genetic mechanisms governing cortical cell development during kidney organogenesis. These results, combined with previous evidence demonstrating that Hox11 genes are necessary for patterning the metanephric mesenchyme, support a model whereby distinct populations in the nephrogenic cord are regulated by unique Hox codes, and that differential Hox function along the AP axis of the nephrogenic cord is critical for the differentiation and integration of these cell types during kidney organogenesis.

Na+,K+-ATPase activity and subunit protein expression: ontogeny and effects of exogenous and endogenous steroids on the cerebral cortex and renal cortex of sheep.

We examined the effects of development, exogenous, and endogenous glucocorticoids on Na(+),K(+)-ATPase activity and subunit protein expression in ovine cerebral cortices and renal cortices. Ewes at 60%, 80%, and 90% gestation, newborns, and adults received 4 dexamethasone or placebo injections. Cerebral cortex Na(+),K(+)-ATPase activity was higher (P < .05) in placebo-treated newborns than fetuses of placebo-treated ewes and adults, α(1)-expression was higher at 90% gestation than the other ages; α(2)-expression was higher in newborns than fetuses; α(3)-expression was higher in newborns than 60% gestation; ß(1)-expression was higher in newborns than the other ages, and ß(2)-expression higher at 60% than 80% and 90% gestation, and in adults. Renal cortex Na(+),K(+)-ATPase activity was higher in placebo-treated adults and newborns than fetuses. Cerebral cortex Na(+),K(+)-ATPase activity was higher in dexamethasone- than placebo-treated adults, and α(1)-expression higher in fetuses of dexamethasone- than placebo-treated ewes at 60% and 80% gestation. Renal cortex Na(+),K(+)-ATPase activity and α(1)-expression were higher in fetuses of dexamethasone- than placebo-treated ewes at each gestational age, and ß(1)-expression was higher in fetuses of dexamethasone- than placebo-treated ewes at 90% gestation and in dexamethasone- than placebo-treated adults. Cerebral cortex Na(+),K(+)-ATPase activity, α(1)-expression, ß(1)-expression, and renal cortex α(1)-expression correlated directly with increases in fetal cortisol. In conclusion, Na(+),K(+)-ATPase activity and subunit expression exhibit specific developmental patterns in brain and kidney; exogenous glucocorticoids regulate activity and subunit expression in brain and kidney at some ages; endogenous increases in fetal cortisol regulate cerebral Na(+),K(+)-ATPase, but exogenous glucocorticoids have a greater effect on renal than cerebral Na(+),K(+)-ATPase.

Chronic rejection triggers the development of an aggressive intragraft immune response through recapitulation of lymphoid organogenesis.

The unwarranted persistence of the immunoinflammatory process turns this critical component of the body's natural defenses into a destructive mechanism, which is involved in a wide range of diseases, including chronic rejection. Performing a comprehensive analysis of human kidney grafts explanted because of terminal chronic rejection, we observed that the inflammatory infiltrate becomes organized into an ectopic lymphoid tissue, which harbors the maturation of a local humoral immune response. Interestingly, intragraft humoral immune response appeared uncoupled from the systemic response because the repertoires of locally produced and circulating alloantibodies only minimally overlapped. The organization of the immune effectors within adult human inflamed tissues recapitulates the biological program recently identified in murine embryos during the ontogeny of secondary lymphoid organs. When this recapitulation was incomplete, intragraft B cell maturation was impeded, limiting the aggressiveness of the local humoral response. Identification of the molecular checkpoints critical for completion of the lymphoid neogenesis program should help develop innovative therapeutic strategies to fight chronic inflammation.

A novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization.

Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1-3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium-around 85 microliters-using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle.

The long-term label retaining population of the renal papilla arises through divergent regional growth of the kidney.

Long-term pulse chase experiments previously identified a sizable population of BrdU-retaining cells within the renal papilla. The origin of these cells has been unclear, and in this work we test the hypothesis that they become quiescent early during the course of kidney development and organ growth. Indeed, we find that BrdU-retaining cells of the papilla can be labeled only by pulsing with BrdU from embryonic (E) day 11.25 to postnatal (P) day 7, the approximate period of kidney development in the mouse. BrdU signal in the cortex and outer medulla is rapidly diluted by cellular proliferation during embryonic development and juvenile growth, whereas cells within the papilla differentiate and exit the cell cycle during organogenesis. Indeed, by E17.5, little or no active proliferation can be seen in the distal papilla, indicating maturation of this structure in a distal-to-proximal manner during organogenesis. We conclude that BrdU-retaining cells of the papilla represent a population of cells that quiesce during embryonic development and localize within a region of the kidney that matures early. We therefore propose that selective papillary retention of BrdU arises through a combination of regionalized slowing of, and exit from, the cell cycle within the papilla during the period of ongoing kidney development, and extensive proliferative growth of the juvenile kidney resulting in dilution of BrdU below the detection level in extra-papillary regions.

A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney.

The mammalian kidney is organized into a cortex where primary filtration occurs, and a medullary region composed of elongated tubular epithelia where urine is concentrated. We show that the cortico-medullary axis of kidney organization and function is regulated by Wnt7b signaling. The future collecting duct network specifically expresses Wnt7b. In the absence of Wnt7b, cortical epithelial development is normal but the medullary zone fails to form and urine fails to be concentrated normally. The analysis of cell division planes in the collecting duct epithelium of the emerging medullary zone indicates a bias along the longitudinal axis of the epithelium. By contrast, in Wnt7b mutants, cell division planes in this population are biased along the radial axis, suggesting that Wnt7b-mediated regulation of the cell cleavage plane contributes to the establishment of a cortico-medullary axis. The removal of beta-catenin from the underlying Wnt-responsive interstitium phenocopies the medullary deficiency of Wnt7b mutants, suggesting a paracrine role for Wnt7b action through the canonical Wnt pathway. Wnt7b signaling is also essential for the coordinated growth of the loop of Henle, a medullary extension of the nephron that elongates in parallel to the collecting duct epithelium. These findings demonstrate that Wnt7b is a key regulator of the tissue architecture that establishes a functional physiologically active mammalian kidney.

Na+, K+-ATPase activity and subunit isoform protein abundance: effects of antenatal glucocorticoids in the frontal cerebral cortex and renal cortex of ovine fetuses.

We examined the effects of single and multiple maternal glucocorticoid courses on cerebral cortical (CC) and renal cortical (RC) Na(+),K(+)-ATPase activity and protein isoform abundance in fetal sheep. Ewes received four dexamethasone or placebo injections in the single course (SC) groups, and the same treatment once a week for five-weeks in the multiple course (MC) groups. CC Na(+),K(+)-ATPase a(2)-abundance was higher (P<0.05) and beta(2)-abundance lower in the SC dexamethasone than placebo group, but Na(+),K(+)-ATPase activity did not change. CC Na(+),K(+)-ATPase activity, a(1)-, beta(1) -, and beta(2)-abundance were lower in the MC dexamethasone than placebo group, but a(2)- and a(3)-abundance did not change. Both dexamethasone courses did not affect CC cell number. RC Na(+),K(+)-ATPase activity, a(1)- and beta(1) -abundance were higher in the MC dexamethasone than placebo group, but did not change in the SC dexamethasone group. We conclude MC, but not a SC of dexamethasone, affect fetal cerebral and renal Na(+),K(+)-ATPase, and MC result in differential effects on Na(+),K(+)-ATPase in these organs.

Thyroid hormone regulates renocortical COX-2 and PGE2 expression in the late gestation fetal sheep.

Cyclooxygenase 2 (COX-2) is important for development of the fetal kidney. Precisely how renal COX-2 expression is regulated in fetal life is unclear. The hypothesis that thyroid hormone positively regulates COX-2 and PGE(2) levels in the late gestation fetal kidney cortex was tested. Sham, thyroidectomized (TX), and TX + thyroid hormone replacement (R) fetal sheep were studied. TX was performed at 120 days gestational age (dGA). TX + R fetuses were continuously infused with thyroxine from 3 days after surgery until study completion. Fetal kidney cortex was obtained at 137 dGA for measurement of renal cyclooxygenase type-2 (COX-2) protein and PGE(2) metabolites. Renocortical COX-2 and PGE(2) levels were significantly lower in TX compared with sham and TX + R fetuses. There were no differences between sham and TX + R fetuses. These findings demonstrate that thyroid hormone positively regulates renal COX-2 and PGE(2) expression in the late gestation fetal sheep kidney.

Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney.

During kidney development and in response to inductive signals, the metanephric mesenchyme aggregates, becomes polarized, and generates much of the epithelia of the nephron. As such, the metanephric mesenchyme is a renal progenitor cell population that must be replenished as epithelial derivatives are continuously generated. The molecular mechanisms that maintain the undifferentiated state of the metanephric mesenchymal precursor cells have not yet been identified. In this paper, we report that functional inactivation of the homeobox gene Six2 results in premature and ectopic differentiation of mesenchymal cells into epithelia and depletion of the progenitor cell population within the metanephric mesenchyme. Failure to renew the mesenchymal cells results in severe renal hypoplasia. Gain of Six2 function in cortical metanephric mesenchymal cells was sufficient to prevent their epithelial differentiation in an organ culture assay. We propose that in the developing kidney, Six2 activity is required for maintaining the mesenchymal progenitor population in an undifferentiated state by opposing the inductive signals emanating from the ureteric bud.

Upregulation of histidine decarboxylase expression in superficial cortical nephrons during pregnancy in mice and women.

Mechanisms regulating pregnancy-induced changes in renal function are incompletely understood. Few candidate genes have been identified and data suggest that alternate mechanisms remain to be elucidated. Our objective was to screen thousands of genes expressed in kidneys from mice throughout gestation to identify possible key regulators of renal function during pregnancy. Mouse complementary DNA microarrays were used to screen for differences in expression during pregnancy in C57BL/6 mice. Interesting candidate genes whose expression varied with pregnancy were further analyzed by reverse transcription-PCR and Northern blot. Expression was localized by in situ hybridization and immunohistochemistry. Follow-up immunohistochemical analyses in archival human kidney sections from the fetus, non-pregnant, and pregnant women were also performed. Histidine decarboxylase (HDC), the enzyme that synthesizes histamine, was markedly upregulated in the mouse kidney during pregnancy. HDC expression localized to proximal tubule cells of fetal and adult mice. Females showed strong expression in the juxtamedullary zone before pregnancy and upregulation in the superficial cortical zone (SCZ) by mid-gestation. Histamine colocalized with HDC. Male mice showed only low HDC expression. Similar expression patterns were observed in human kidneys. Our results show that HDC expression and histamine production are increased in the SCZ during pregnancy. If histamine acts as a vasodilator, we speculate that increasing production in the SCZ may increase renal blood flow to this zone and recruit superficial cortical nephrons during pregnancy.

Ontogeny and effects of thyroid hormone on beta1-adrenergic receptor mRNA expression in ovine fetal kidney cortex.

OBJECTIVE: Previous studies indicate that thyroidectomy (TX) decreases renin gene expression in ovine fetal renal cortex in late gestation. Fetal ovine renin-containing renocortical cells become increasingly responsive to beta-adrenergic stimulation as gestation proceeds. Increases in plasma thyroid hormone concentrations parallel this change, suggesting that there is a positive developmental relationship between the two. To examine this hypothesis, we determined the ontogeny of beta1-adrenergic receptor (beta1R) mRNA expression, and the effect of thyroid hormone on in vivo and in vitro expression in fetal sheep. METHODS: Renocortical tissue was obtained from naive, TX, and sham-operated fetuses to determine beta1R mRNA levels. Renin-containing renocortical cells from TX or sham fetuses were treated with isoproterenol (Iso) or forskolin (FSK) for analysis of cellular cyclic adenosine monophosphate (cAMP) levels. Renocortical cells from naive fetuses were treated with triiodothyronine (T3) to assess cellular beta1R mRNA levels. Fetal plasma thyroxine (T4) level was determined. RESULTS: Renocortical beta1R mRNA expression increased significantly between 100 and 140 days' gestational age (dGA), while TX attenuated this increase (P <.01). Renocortical cellular cAMP levels were higher in sham compared to TX fetuses following incubation with Iso or FSK (P <.05). Cells incubated with T3 exhibited significantly increased beta1R mRNA expression (P <.05). CONCLUSION: The data suggest that thyroid hormone may be involved in modulating ovine fetal renocortical beta1R gene expression during development. We speculate that the increased beta1R mRNA expression in renal cortical cells as development progresses may mediate the increases in renin gene response to beta-adrenergic stimulation in late gestation.

The spiny mouse (Acomys cahirinus) completes nephrogenesis before birth.

The spiny mouse is relatively mature at birth. We hypothesized that like other organs, the kidney may be more developed in the spiny mouse at birth, than in other rodents. If nephrogenesis is complete before birth, the spiny mouse may provide an excellent model with which to study the effects of an altered intrauterine environment on renal development. Due to its desert adaptation, the spiny mouse may have a reduced cortex-to-medulla ratio but an equivalent total nephron number to the C57/BL mouse. Kidneys were collected from fetal and neonatal spiny mice and sectioned for gross examination of metanephric development. Kidneys were collected from adult spiny mice (10 wk of age), and glomerular number, volume, and cortex-to-medulla ratios were determined using unbiased stereology. Nephrogenesis is complete in spiny mouse kidneys before birth. Metanephrogenesis begins at approximately day 18, and by day 38 of a 40-day gestation, the nephrogenic zone is no longer present. Spiny mice have a significantly (P < 0.001) lower total nephron number compared with C57/BL mice, although the total glomerular volume is similar. The cortex-to-medulla ratio of the spiny mouse is significantly (P < 0.01) smaller. The spiny mouse is the first rodent species shown to complete nephrogenesis before birth. This makes it an attractive candidate for the study of fetal and neonatal kidney development and function. The reduced total nephron number and cortex-to-medulla ratio in the spiny mouse may contribute to its ability to highly concentrate its urine under stressful conditions (i.e., dehydration).

Evaluation of absolute volume of human fetal kidney's cortex and medulla during gestation.

BACKGROUND: Human fetal kidney is quite different from the mature kidney, both macroscopically and hystologically. Lobulated surface of the human fetal kidney reflects its inner organisation. AIM: To determine the fetal kidneys' volume according to the gestational age, to establish periods of their maximal and minimal growth and to compare these values for various gestational ages. METHODS: Forty five human fetal kidneys aged from IV to X lunar months were analyzed. Kidneys were divided into nine groups according to their gestational age. The volumes of cortex and medulla were determined using stereological methods. The results were statistically analyzed and the periods of significant growth of these structures were marked. RESULTS: Fetal kidney's cortex and medulla grew continually with a very high coefficient of linear correlation with crown-rump length. The cortex/medulla ratio was minimal in the first half of V lunar month, when medulla grew most rapidly and it was maximal immediately before birth, when cortex achieved its maximum. CONCLUSION: This study was an effort to provide some parameters which would help in the future investigations of the development of human fetal kidney.

Temporal-spatial co-localisation of tissue transglutaminase (Tgase2) and matrix metalloproteinase-9 (MMP-9) with SBA-positive micro-fibres in the embryonic kidney cortex.

Growth of the kidney is a complex process piloted by the collecting duct (CD) ampullae. The dichotomous arborisation and consecutive elongation of this tubular element determines the exact site and time for the induction of nephrons in the overlaying mesenchymal cap condensates. The mechanism by which the CD ampullae find the correct orientation is currently unknown. Recently, we have demonstrated micro-fibres that originate from the basal aspect of the CD ampullae and extend through the mesenchyme to the organ capsule. The micro-fibres are assumed to be involved in the growth and arborisation process of the CD ampulla. Therefore, we have investigated the specific distribution of the micro-fibres during branching morphogenesis. We have also analysed whether the micro-fibres co-localise with extracellular matrix (ECM)-modulating enzymes and whether the co-localisation pattern changes during CD ampulla arborisation. Micro-fibres were detected in all stages of CD ampulla arborisation. Tissue transglutaminase (Tgase2) co-localised with soybean agglutinin (SBA)-positive micro-fibres, whose presence depended upon the degree of CD branching. Matrix metalloproteinase-9 (MMP-9) also co-localised with micro-fibres, but its expression pattern was different from that for Tgase2. Western blotting experiments demonstrated that Tgase2 and MMP-9 co-migrated with SBA-labelled proteins. Thus, the micro-fibres are developmentally modulated by enzymes of the ECM in embryonic kidney cortex. These findings illustrate the importance of micro-fibres in directing CD ampulla growth.

Morphometric index of the developing murine kidney.

Mammalian kidney morphogenesis begins when the ureteric bud (UB) induces surrounding metanephric mesenchyme to differentiate into nephrons, the functional units of the mature organ. Although several genes required for this process have been identified, the mechanisms that control final nephron number and the localization of distinct tubular segments to cortical and medullary zones of the kidney remain poorly understood. This finding is due, in part, to the lack of quantitative studies describing the acquisition of mature renal structure. We have analyzed the following parameters of the developing murine kidney throughout embryogenesis: nephron and UB tip number, distance between UB branch points and total kidney, and cortical and medullary volume. Results of this morphometric analysis reveal previously unrecognized changes in the pattern of UB growth and rate of nephrogenesis. In addition, this morphometric index provides a much-needed reference for accurately describing renal patterning defects exhibited by genetically altered mice.

Characterization of micro-fibers at the interface between the renal collecting duct ampulla and the cap condensate.

The development of renal histo-architecture substantially depends on the three-dimensional extension of the collecting duct (CD) ampulla, since under its influence, nephron induction takes place in the surrounding mesenchyme. Recently, micro-fibers were detected by soybean agglutinin (SBA), which line from the basal aspect of each CD ampulla through the mesenchyme towards the organ capsule in embryonic kidney. Their unique distribution suggests that they may play an important role in the control of CD ampulla growth and in forming the renal stem cell niche. A profound analysis of interstitial proteins between the CD ampulla and the nephrogenic mesenchyme is lacking. Consequently, the goal of the current investigation was to colocalize the micro-fibers detected by SBA with interstitial proteins. For this reason a detailed cell biological analysis of extracellular molecules at this site was carried out. Double labeling showed that the micro-fibers do not correspond to known collagens and other extracellular matrix molecules such as agrin, versican or MMP-9. In addition, it could be demonstrated that the micro-fibers do not contain epithelial or mesenchymal cell elements. Furthermore, two-dimensional electrophoresis with subsequent Western blotting yielded two different amino acid sequences (1: GHYADPTSPR; 2: NNGCCSSDYHA) obtained from SBA-labeled protein spots. Both amino acid sequences could not be assigned to known rodent proteins. The findings suggest that the SBA-labeled micro-fibers represent a new type of extracellular structure between the CD ampulla, the mesenchyme and the organ capsule.