In developing mouse kidneys, orientation of loop of Henle growth is adaptive and guided by long-range cues from medullary collecting ducts.

The path taken by the loop of Henle, from renal cortex to medulla and back, is critical to the ability of the kidney to concentrate urine and recover water. Unlike most developing tubules, which navigate as blind-ended cylinders, the loop of Henle extends as a sharply bent loop, the apex of which leads the double tubes behind it in a 'V' shape. Here, we show that, in normal kidney development, loops of Henle extend towards the centroid of the kidney with an accuracy that increases the longer they extend. Using cultured kidney rudiments, and manipulations that rotate or remove portions of the organ, we show that loop orientation depends on long-range cues from the medulla rather than either the orientation of the parent nephron or local cues in the cortex. The loops appear to be attracted to the most mature branch point of the collecting duct system but, if this is removed, they will head towards the most mature collecting duct branch available to them. Our results demonstrate the adaptive nature of guidance of this unusual example of a growing epithelium, and set the stage for later work devoted to understanding the molecules and mechanisms that underlie it.

A Sall1-NuRD interaction regulates multipotent nephron progenitors and is required for loop of Henle formation.

The formation of the proper number of nephrons requires a tightly regulated balance between renal progenitor cell self-renewal and differentiation. The molecular pathways that regulate the transition from renal progenitor to renal vesicle are not well understood. Here, we show that Sall1interacts with the nucleosome remodeling and deacetylase complex (NuRD) to inhibit premature differentiation of nephron progenitor cells. Disruption of Sall1-NuRD in vivo in knock-in mice (ΔSRM) resulted in accelerated differentiation of nephron progenitors and bilateral renal hypoplasia. Transcriptional profiling of mutant kidneys revealed a striking pattern in which genes of the glomerular and proximal tubule lineages were either unchanged or upregulated, and those in the loop of Henle and distal tubule lineages were downregulated. These global changes in gene expression were accompanied by a significant decrease in THP-, NKCC2- and AQP1-positive loop of Henle nephron segments in mutant ΔSRM kidneys. These findings highlight an important function of Sall1-NuRD interaction in the regulation of Six2-positive multipotent renal progenitor cells and formation of the loop of Henle.

Role of Prox1 in the Transforming Ascending Thin Limb of Henle's Loop during Mouse Kidney Development.

The homeobox transcription factor Prox1 is critical to the development of many embryonic organs and tissues, although current understanding of its expression in the developing renal medulla is limited. We examined the functional role of Prox1 during mouse kidney development with particular emphasis on the developing loop of Henle. Our data show that Prox1 is expressed in the transdifferentiating region from the NKCC2-positive thick ascending limb, into the CLC-K1-positive ascending thin limb of Henle's loop beginning at embryonic day 18. From 1 to 14 days of age, Prox1-positive cells gradually disappeared from the papillary tip, and remained in the initial part of inner medulla after 21 days. In this transforming area, no Prox1 was observed in cells undergoing apoptosis but was expressed strongly in the remaining cells, which differentiated into ascending thin limb epithelial cells. In vitro and in vivo approaches showed that Prox1 expression increases where the osmolality is near optimal range, but decreases at below- or above-optimal ranges. Renal hypoosmolality induced by furosemide (NKCC2 inhibitor) inhibited Prox1 expression and delayed maturation of the ascending limb of Henle's loop. Together, these studies suggest that Prox1 appears to be a critical stage specific regulator of specifying ascending thin limb cell fate and that its expression is regulated by osmolality.

Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis.

MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle's loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -ß1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.

An improved method of renal tissue engineering, by combining renal dissociation and reaggregation with a low-volume culture technique, results in development of engineered kidneys complete with loops of Henle.

BACKGROUND: Tissue engineering of functional kidney tissue is an important goal for clinical restoration of renal function in patients damaged by infectious, toxicological, or genetic disease. One promising approach is the use of the self-organizing abilities of embryonic kidney cells to arrange themselves, from a simply reaggregated cell suspension, into engineered organs similar to fetal kidneys. The previous state-of-the-art method for this results in the formation of a branched collecting duct tree, immature nephrons (S-shaped bodies) beside and connected to it, and supportive stroma. It does not, though, result in the significant formation of morphologically detectable loops of Henle - anatomical features of the nephron that are critical to physiological function. METHODS: We have combined the best existing technique for renal tissue engineering from cell suspensions with a low-volume culture technique that allows intact kidney rudiments to make loops of Henle to test whether engineered kidneys can produce these loops. RESULTS: The result is the formation of loops of Henle in engineered cultured 'fetal kidneys', very similar in both morphology and in number to those formed by intact organ rudiments. CONCLUSION: This brings the engineering technique one important step closer to production of a fully realistic organ.

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 thick ascending limb chloride transport by low-protein diet and dexamethasone.

Prenatal administration of dexamethasone and a low-protein diet has been shown to result in hypertension in the offspring when they are adults. The cause for the hypertension is unknown. The purpose of this study was to examine whether there was prenatal programming of thick ascending limb transport. Rats were administered either dexamethasone for 4 days (0.2 mg/kg body wt) by intraperitoneal injection daily between the 15th and 18th day of gestation, or they were fed a low-protein diet (6% protein) or an isocaloric normal protein diet (20% protein) from day 12 gestation until birth. The offspring were studied as adults. Prenatal dexamethasone and dietary protein deprivation resulted in an increase in blood pressure. Offspring of mothers fed a low-protein diet had an increase in medullary but not cortical bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) protein abundance (P < 0.01). There was not a statistically significant increase in medullary NKCC2 by prenatal dexamethasone (P = 0.07). Both prenatal administration of dexamethasone and a low-protein diet resulted in an increase in medullary thick ascending limb chloride transport compared with control (298 +/- 33 pmoles x mm(-1) x min(-1), 280 +/- 26 pmoles x mm(-1) x min(-1), and 191 +/- 21 pmoles x mm(-1) x min(-1), respectively P < 0.05). There was a higher lumen-positive transepithelial potential difference in the prenatal dexamethasone and low-protein group compared with control as well. Administration of furosemide for 24 h resulted in a decrease in blood pressure in the low-protein group but not the control group. This study demonstrates that insults administered to the fetus can program altered sodium transport. Increased tubular sodium transport is a likely cause for the hypertension by prenatal programming.

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.

Sulfated HNK-1 epitope in developing and mature kidney: a new marker for thin ascending loop of Henle and tubular injury in acute tubular necrosis.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.

Differential distribution of the vasopressin V receptor along the rat nephron during renal ontogeny and maturation.

BACKGROUND: Ontogeny and cellular distribution of vasopressin receptors in the kidney are key factors determining the role of vasopressin in renal physiology. Expression of vasopressin V(2) receptor (V(2)R) mRNA and the immunoreactive protein in rat kidney were investigated. METHODS: An antiserum directed to epitope TLD25 of the rat V(2)R sequence was characterized by Western blotting. Expression of V(2)R mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR), and on protein level by immunohistochemistry. RESULTS: Specificity of the antiserum was documented by Western blots from cells expressing a fusion protein of V(2)R and GFP. Using lysates of rat kidney and of native cell lines expressing V(2)R but not V(1)R, our antiserum to peptide TLD25 revealed a major band of 55 kD corresponding to the monomeric form of V(2)R, and a band of 110 kD most likely representing the homodimeric form of the receptor. This highly specific antiserum allowed us to localize the V(2)R in thick ascending limbs, distal convoluted and connecting tubules, and in collecting ducts. During ontogeny, immunoreactivity was first observed at the luminal membrane on prenatal day 20, emerging at the basolateral side from postnatal day 5 on. RT-PCR demonstrated V(2)R transcripts from prenatal day 18 to gradually increasing thereafter. CONCLUSION: Expression of V(2)R is first detectable in the late embryonic stage of rat ontogeny starting from day E18 and gradually increasing with kidney maturation. In the adult kidney, V(2)R is differentially distributed in the various nephron segments.

Upregulation of renal BSC1 and TSC in prenatally programmed hypertension.

Prenatal factors, especially intrauterine growth retardation, have been shown to correlate with the risk of essential hypertension in adult life, but the mechanisms are unknown. An experimental model of prenatal programming of hypertension in the rat, induced by a maternal low-protein diet during pregnancy, was employed to study the role of renal Na reabsorption in the pathogenesis. The abundance of the apical Na transporter type III Na/H exchanger (NHE3), bumetanide-sensitive Na-K-2Cl cotransporter (BSC1), thiazide-sensitive Na-Cl cotransporter (TSC), and the amiloride-sensitive epithelial Na channel (ENaC) was determined by semiquantitative immunoblotting in kidneys from the offspring at 4 wk of age, before hypertension became manifest. There were no significant differences between the experimental and control rats in the abundance of NHE3 or any of the ENaC subunits. In contrast, the quantity of BSC1 in the experimental group was increased to 302% of control (P < 0.001) and that of TSC to 157% of control (P < 0.05). Determination of specific mRNA levels by ELISA-linked RT-PCR revealed a significantly increased BSC1 mRNA at 1 day (P < 0.01), 4 wk (P < 0.01), and 8 wk (P < 0.001) of age, and a significantly increased TSC mRNA at 4 wk of age (P < 0.05) in the experimental group. The results suggest that prenatal programming of hypertension involves transcriptional upregulation of Na transport in thick ascending limb and distal convoluted tubule.

Expression of urea transporters in the developing rat kidney.

Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). Because newborn rats are not capable of producing concentrated urine, we examined the time of expression and the distribution of UT-A and UT-B in the developing rat kidney by light and electron microscopic immunocytochemistry. Kidneys from 16-, 18-, and 20-day-old fetuses, 1-, 4-, 7-, 14-, and 21-day-old pups, and adult animals were studied. In the adult kidney, UT-A was expressed intensely in the inner medullary collecting duct (IMCD) and terminal portion of the short-loop descending thin limb (DTL) and weakly in long-loop DTL in the outer part of the inner medulla. UT-A immunoreactivity was not present in the fetal kidney but was observed in the IMCD and DTL in 1-day-old pups. The intensity of UT-A immunostaining in the IMCD gradually increased during postnatal development. In 4- and 7-day-old pups, UT-A immunoreactivity was present in the DTL at the border between the outer and inner medulla. In 14- and 21-day-old pups, strong UT-A immunostaining was observed in the terminal part of short-loop DTL in the outer medulla, and weak labeling remained in long-loop DTL descending into the outer part of the inner medulla. In the adult kidney, there was intense staining for UT-B in descending vasa recta (DVR) and weak labeling of glomeruli. In the developing kidney, UT-B was first observed in the DVR of a 20-day-old fetus. After birth there was a striking increase in the number of UT-B-positive DVR, in association with the formation of vascular bundles. The intensity of immunostaining remained strong in the outer medulla but gradually decreased in the inner medulla. We conclude that the expression of urea transporters in short-loop DTL and DVR coincides with the development of the ability to produce a concentrated urine.

[Structural organization of the tubular component of the renal countercurrent system]. / Osobennosti strukturnoi organizatsii kanal'tsevogo komponenta protivotochnoi sistemy pochki.

The time course of the developing tubules of the dog kidney in the prenatal period of ontogenesis was studied by the methods of microscopy and three-dimensional reconstruction. It was established that the structural functional unit of the osmoregulatory system is a cone-shaped fascicle whose central axis is a collecting tubule. Henle's loops of three juxtamedullary nephrons and accompanying blood vessels are grouped concentrically along the axis. The tubules are grouped in such a manner that the most central position is occupied by the ascending portions of the loop while the descending ones lie somewhat outwards.