Connecting tubules develop from the tip of the ureteric bud in the human kidney.

The connecting tubule (CNT) is a unique segment of the nephron connecting the metanephric mesenchyme (MM)-derived distal convoluted tubule (DCT) and ureteric bud (UB)-derived collecting duct (CD). Views on the cellular origin of the CNT in the human kidney are controversial. It was suggested that in mice, the connecting segment arises from the distal compartment of the renal vesicle (RV). However, there are several differences in embryonic development between the mouse and human kidney. The aim of our study was to establish the possible origin of the CNT in the human kidney. We analysed the expression of markers defining distinct cells of the CNT CD in foetal and adult human kidneys by immunohistochemistry. Based on microscopic observation, we suggest that CNT differentiates from the outgrowth of cells of the UB tip, and therefore the CNT is an integral part of the CD system. In the adult kidney, the CNT and CD consist of functionally and morphologically similar cells expressing α- and ß-intercalated cell (IC) and principal cell (PC) markers, indicating their common origin.

Roscovitine blocks collecting duct cyst growth in Cep164-deficient kidneys.

Nephronophthisis is an autosomal recessive kidney disease with high genetic heterogeneity. Understanding the functions of the individual genes contributing to this disease is critical for delineating the pathomechanisms of this disorder. Here, we investigated kidney function of a novel gene associated with nephronophthisis, CEP164, coding a centriolar distal appendage protein, using a Cep164 knockout mouse model. Collecting duct-specific deletion of Cep164 abolished primary cilia from the collecting duct epithelium and led to rapid postnatal cyst growth in the kidneys. Cell cycle and biochemical studies revealed that tubular hyperproliferation is the primary mechanism that drives cystogenesis in the kidneys of these mice. Administration of roscovitine, a cell cycle inhibitor, blocked cyst growth in the cortical collecting ducts and preserved kidney parenchyma in Cep164 knockout mice. Thus, our findings provide evidence that therapeutic modulation of cell cycle activity can be an effective approach to prevent cyst progression in the kidney.

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development.

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.

Prolonged prenatal hypoxia selectively disrupts collecting duct patterning and postnatal function in male mouse offspring.

KEY POINTS: In the present study, we investigated whether hypoxia during late pregnancy impairs kidney development in mouse offspring, and also whether this has long-lasting consequences affecting kidney function in adulthood. Hypoxia disrupted growth of the kidney, particularly the collecting duct network, in juvenile male offspring. By mid-late adulthood, these mice developed early signs of kidney disease, notably a compromised response to water deprivation. Female offspring showed no obvious signs of impaired kidney development and did not develop kidney disease, suggesting an underlying protection mechanism from the hypoxia insult. These results help us better understand the long-lasting impact of gestational hypoxia on kidney development and the increased risk of chronic kidney disease. ABSTRACT: Prenatal hypoxia is a common perturbation to arise during pregnancy, and can lead to adverse health outcomes in later life. The long-lasting impact of prenatal hypoxia on postnatal kidney development and maturation of the renal tubules, particularly the collecting duct system, is relatively unknown. In the present study, we used a model of moderate chronic maternal hypoxia throughout late gestation (12% O2 exposure from embryonic day 14.5 until birth). Histological analyses revealed marked changes in the tubular architecture of male hypoxia-exposed neonates as early as postnatal day 7, with disrupted medullary development and altered expression of Ctnnb1 and Crabp2 (encoding a retinoic acid binding protein). Kidneys of the RARElacZ line offspring exposed to hypoxia showed reduced ß-galactosidase activity, indicating reduced retinoic acid-directed transcriptional activation. Wild-type male mice exposed to hypoxia had an early decline in urine concentrating capacity, evident at 4 months of age. At 12 months of age, hypoxia-exposed male mice displayed a compromised response to a water deprivation challenge, which was was correlated with an altered cellular composition of the collecting duct and diminished expression of aquaporin 2. There were no differences in the tubular structures or urine concentrating capacity between the control and hypoxia-exposed female offspring at any age. The findings of the present study suggest that prenatal hypoxia selectively disrupts collecting duct patterning through altered Wnt/ß-catenin and retinoic acid signalling and this results in impaired function in male mouse offspring in later life.

Asymmetric BMP4 signalling improves the realism of kidney organoids.

We present a strategy for increasing the anatomical realism of organoids by applying asymmetric cues to mimic spatial information that is present in natural embryonic development, and demonstrate it using mouse kidney organoids. Existing methods for making kidney organoids in mice yield developing nephrons arranged around a symmetrical collecting duct tree that has no ureter. We use transplant experiments to demonstrate plasticity in the fate choice between collecting duct and ureter, and show that an environment rich in BMP4 promotes differentiation of early collecting ducts into uroplakin-positive, unbranched, ureter-like epithelial tubules. Further, we show that application of BMP4-releasing beads in one place in an organoid can break the symmetry of the system, causing a nearby collecting duct to develop into a uroplakin-positive, broad, unbranched, ureter-like 'trunk' from one end of which true collecting duct branches radiate and induce nephron development in an arrangement similar to natural kidneys. The idea of using local symmetry-breaking cues to improve the realism of organoids may have applications to organoid systems other than the kidney.

[THE ROLE OF THE PI3-KINASE IN THE FAST NONGENOMIC ALDOSTERONE EFFECTS IN THE PRINCIPAL CELLS OF THE CORTICAL COLLECTING DUCT OF THE RAT KIDNEY IN THE POSTNATAL ONTOGENESIS].

The involvement of wortmannin (10 -5 M), phosphatidyl inositol 3-kinase (PI3K) blocker, in the implementation of the rapid nongenomic aldosterone (10 nM) effects on the intracellular sodium (Na i +) and the principal cell volume of cortical collecting duct (CCD) of 10-day and adult rat kidney CCD was studied. Using fluorescence microscopy with intracellular dye Na Green and Calcein we found that wortmannin weakened the effect of aldosterone on the Na i + at low sodium in the extracellular medium (14 mM NaCl), and slowed the rate of reduction of the principal cell volume in the presence of aldosterone at the hypoosmotic shock (240/140 mOsm) since 10 days of age. The findings suggest the participation of phosphatidylinositol pathway in the fast nongenomic aldosterone effects (seconds and minutes) at the early stage of postnatal ontogenesis.

ß1 integrin NPXY motifs regulate kidney collecting-duct development and maintenance by induced-fit interactions with cytosolic proteins.

Loss of ß1 integrin expression inhibits renal collecting-system development. Two highly conserved NPXY motifs in the distal ß1 tail regulate integrin function by associating with phosphtyrosine binding (PTB) proteins, such as talin and kindlin. Here, we define the roles of these two tyrosines in collecting-system development and delineate the structural determinants of the distal ß1 tail using nuclear magnetic resonance (NMR). Mice carrying alanine mutations have moderate renal collecting-system developmental abnormalities relative to ß1-null mice. Phenylalanine mutations did not affect renal collecting-system development but increased susceptibility to renal injury. NMR spectra in bicelles showed the distal ß1 tail is disordered and does not interact with the model membrane surface. Alanine or phenylalanine mutations did not alter ß1 structure or interactions between α and ß1 subunit transmembrane/cytoplasmic domains; however, they did decrease talin and kindlin binding. Thus, these studies highlight the fact that the functional roles of the NPXY motifs are organ dependent. Moreover, the ß1 cytoplasmic tail, in the context of the adjacent transmembrane domain in bicelles, is significantly different from the more ordered, membrane-associated ß3 integrin tail. Finally, tyrosine mutations of ß1 NPXY motifs induce phenotypes by disrupting their interactions with critical integrin binding proteins like talins and kindlins.

Remodeling of the fetal collecting duct epithelium.

Congenital urinary tract obstruction induces changes to the renal collecting duct epithelium, including alteration and depletion of intercalated cells. To study the effects of obstruction on the ontogeny of intercalated cell development, we examined normal and obstructed human fetal and postnatal kidneys. In the normal human fetal kidney, intercalated cells originated in the medullary collecting duct at 8 weeks gestation and remained most abundant in the inner medulla throughout gestation. In the cortex, intercalated cells were rare at 18 and 26 weeks gestation and observed at low abundance at 36 weeks gestation. Although early intercalated cells exhibit an immature phenotype, Type A intercalated cells predominated in the inner and outer medullae at 26 and 36 weeks gestation with other intercalated cell subtypes observed rarely. Postnatally, the collecting duct epithelium underwent a remodeling whereby intercalated cells become abundant in the cortex yet absent from the inner medulla. In 18-week obstructed kidneys with mild to moderate injury, the intercalated cells became more abundant and differentiated than the equivalent age-matched normal kidney. In contrast, more severely injured ducts of the late obstructed kidney exhibited a significant reduction in intercalated cells. These studies characterize the normal ontogeny of human intercalated cell development and suggest that obstruction induces premature remodeling and differentiation of the fetal collecting duct epithelium.

Met and the epidermal growth factor receptor act cooperatively to regulate final nephron number and maintain collecting duct morphology.

Ureteric bud (UB) branching during kidney development determines the final number of nephrons. Although hepatocyte growth factor and its receptor Met have been shown to stimulate branching morphogenesis in explanted embryonic kidneys, loss of Met expression is lethal during early embryogenesis without obvious kidney abnormalities. Met(fl/fl);HoxB7-Cre mice, which lack Met expression selectively in the UB, were generated and found to have a reduction in final nephron number. These mice have increased Egf receptor expression in both the embryonic and adult kidney, and exogenous Egf can partially rescue the branching defect seen in kidney explants. Met(fl/fl);HoxB7-Cre;wa-2/wa-2 mice, which lack normal Egfr and Met signaling, exhibit small kidneys with a marked decrease in UB branching at E14.5 as well as a reduction in final glomerular number. These mice developed progressive interstitial fibrosis surrounding collecting ducts with kidney failure and death by 3-4 weeks of age. Thus, in support of previous in vitro findings, Met and the Egf receptor can act cooperatively to regulate UB branching and mediate maintenance of the normal adult collecting duct.

[The role of protein kinase C in the establishment of the mechanism of vasopressin antidiuretic action in the rat kidney during mammalian postnatal development].

The kidney of immaturely born mammals in early postnatal development is insensitive to the effect of the antidiuretic hormone, vasopressin. It has been demonstrated that water permeability of the epithelial cells in the collecting ducts of a rat kidney increases during development; in this process, the response to desmopressin, an agonist of vasopressin V2 receptors, appears at the age of 20 days. The observed increase in water permeability is connected with an increased content of the water channel's proteins aquaporins AQP2 and AQP3 in the plasma membrane. The calcium-dependent protein kinase C isoforms are the likely components of the vasopressin signal's transduction and are possibly involved in the mechanisms underlying the maturation of sensitivity to this hormone. The contents of three protein kinase C isoforms (alpha, delta, and zeta) in rats at different periods of their postnatal development were estimated using Western blot hybridization. It has been shown that the contents of protein kinase C isoforms alpha and delta increase with development, whereas the content of isoform zeta remains constant. The most likely participant of the mechanism providing for maturation of the cell's hormonal competence for vasopressin is the calcium-dependent protein kinase Ca, because it's content in the plasma membrane is maximal on days 20-24, which coincides with the time when the vasopressin action appears.

Kidney regeneration by xeno-embryonic nephrogenesis.

Establishment of a functional whole kidney de novo has not received much attention because of the formidable challenges and the slow pace of advances in this field of research. This situation has changed recently with publication of data revealing the catastrophic nature of Medicaid costs for dialysis-related diseases. An innovative approach is needed in our search for therapies for kidney diseases and to provide a substitute for dialysis as soon as possible. Regenerative medicine offers great hope for realizing this goal. We established a system by which human mesenchymal stem cells can differentiate into a functional renal unit using a program of nephrogenesis in a developing xeno-embryo. In this article, recent research in the field of developing whole kidneys is reviewed, and possible therapeutic applications for kidney diseases are proposed in combination with our knowledge of the emerging field of kidney stem cell biology.

The formation of pores in the basal lamina of regenerated renal tubules.

Little information is available concerning the generation of renal tubules, but this information is urgently needed in regenerative medicine for the future treatment of acute and chronic renal failures. Of major interests are the integration of stem/progenitor cells, the cellular development and the tubular growth in a spatial environment. In this regard, we investigated the basal aspect of renal tubules generated at the interphase of an artificial interstitium. Stem/progenitor cells derived from neonatal rabbit kidney were mounted inside a specific tissue holder and covered by layers of polyester fleece. The tissue was then kept in a perfusion culture container for 13 days in chemically defined IMDM containing aldosterone (1 x 10(-7)m) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryo-sections labeled with soybean agglutinin (SBA) and tissue-specific antibodies indicating that collecting duct tubules were developed. Scanning electron microscopy (SEM) revealed that the generated tubules were completely covered by a basal lamina. Most interestingly, the matrix was not consistently composed, but exhibited three categories of pores. The most frequently found pore type had an apparent diameter of 133+/-26 nm followed by a medium-sized pore type of 317+/-35 nm. Another category of pores with a diameter of 605+/-101 nm was rather rarely found. All of the pores were evenly distributed and not restricted to particular sites. The newly detected pores are not related to culture artifacts, since they were also detected in collecting duct tubules of the neonatal rabbit kidney. It remains to be evaluated whether these pores support physiological transport functions or if they indicate the site where extracellular matrix proteins are inserted into newly synthesized basal lamina.

Potassium transport in the maturing kidney.

The distal nephron and colon are the primary sites of regulation of potassium (K(+)) homeostasis, responsible for maintaining a zero balance in adults and net positive balance in growing infants and children. Distal nephron segments can either secrete or reabsorb K(+) depending on the metabolic needs of the organism. In the healthy adult kidney, K(+) secretion predominates over K(+) absorption. Baseline K(+) secretion occurs via the apical low-conductance secretory K(+) (SK) channel, whereas the maxi-K channel mediates flow-stimulated net urinary K(+) secretion. The K(+) retention characteristic of the neonatal kidney appears to be due not only to the absence of apical secretory K(+) channels in the distal nephron but also to a predominance of apical H-K-adenosine triphosphatase (ATPase), which presumably mediates K(+) absorption. Both luminal and peritubular factors regulate the balance between K(+) secretion and absorption. Perturbation in any of these factors can lead to K(+) imbalance. In turn, these factors may serve as effective targets for the treatment of both hyper-and hypokalemia. The purpose of this review is to present an overview of recent advances in our understanding of mechanisms of K(+) transport in the maturing kidney.

Postnatal expression of KLF12 in the inner medullary collecting ducts of kidney and its trans-activation of UT-A1 urea transporter promoter.

Maturation of the inner medulla of the kidney occurs after birth and is vital for mammals to acquire maximal urinary concentrating ability. During this process, expression of several kidney transporters and channels involved in urine concentrating mechanisms is known to be regulated. We previously isolated KLF15 as a transcription factor that regulates the expression of the ClC-K1 chloride channel. We have now found that another KLF transcription factor, KLF12, is expressed in the kidney from around 15 days after birth. To gain insight into its involvement in the maturation process of the inner medulla, we first determined the expression site of KLF12 within the kidney by in situ hybridization. By comparing the AQP2 immunolocalization in sequential sections, KLF12 was found to be expressed in the collecting ducts. Because expression of the urea transporter UT-A1 and amiloride-sensitive epithelial sodium channels ENaC is known to be tightly regulated in the collecting ducts after birth, we tested whether KLF12 has a regulatory role in the promoter activities of these genes. KLF12 is able to increase UT-A1 but not ENaC promoter activity through the binding to CACCC motif. These results suggest that KLF12 is involved in the maturation processes of collecting ducts after birth, and that UT-A1 is a target gene of KLF12.

Novel cystogenic role of basic fibroblast growth factor in developing rodent kidneys.

Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor that is accumulated in human dysplastic and cystic renal diseases. Previous studies have shown that bFGF can modulate the growth of developing renal tubules; however, its role in the pathogenesis of renal cyst formation is not clearly understood. Here, we tested the hypothesis that overexpression of bFGF in developing rodent kidneys induces cyst formation in vivo. We used two different adenoviral-mediated gene-transferring approaches to overexpress bFGF in developing rodent kidneys. Initially, metanephric kidney (MK) explants harvested from embryonic day 15 Sprague-Dawley rats were infected with adenoviral vectors (rAd) encoding human bFGF or LacZ genes and transplanted under the renal capsule of adult female rats. Subsequently, to determine whether bFGF could induce renal cysts in developing kidneys with an intact renal collecting system, we injected rAd-bFGF or LacZ vectors in the retroorbital plexus of newborn mice. Basic FGF induced a more efficient integration of the MK explants into the host kidneys and increased the vascularization and proliferation of developing tubules, leading to tubular dilatation and rapid formation of renal cysts. In addition, we successfully expressed human bFGF in the kidney of newborn mice in vivo and induced tubular dilatation and renal cysts. In contrast, mice injected with rAd-lacZ did not develop tubular dilatation or renal cysts. To the best of our knowledge, these experiments show for the first time that overexpression of bFGF in developing rodent kidneys can induce the formation of renal cysts in vivo.

Development of water transport in the collecting duct.

The ability of the immature kidney to concentrate urine is lower than in adults. This can lead to severe water and electrolyte disorders, especially in premature babies. Resistance to AVP and lower tonicity of the medullary interstitium seem to be the major factors limiting urine concentration in newborns. AVP-stimulated cAMP generation is impaired. This is the result of inhibition of the production by PGE(2) acting through EP3 receptors and increased degradation by phosphodiesterase IV. The expression of aquaporin-2 (AQP2) in the immature kidney is low; however, under conditions of water deprivation and after stimulation with DDAVP, it rises to adult levels. The expression of AQP3 and AQP4 is intact at birth and does not seem to contribute to the hyporesponsiveness to AVP. Low sodium transport by thick ascending loops of Henle, immaturity of the medullary architecture, and adaptations in the transport of urea contribute to the lower tonicity of the medullary interstitium. This paper reviews the alterations in the AVP signal transduction pathway in the immature kidney.

Ontogeny of renal sodium transport.

One of the main functions of the adult kidney is to maintain a constant extracellular fluid balance. The adult kidney does this, by and large, by filtering a massive quantity of fluid and reabsorbing the solutes needed to maintain volume and electrolyte homeostasis, while leaving the waste products to be excreted in the urine. One of the most precisely regulated functions of the adult kidney is to maintain sodium balance. The challenge of the neonatal kidney is even greater. It must maintain a positive salt balance for growth while the neonate is fed a diet that is very low in sodium. This review focuses on how the neonatal kidney reabsorbs NaCl with a special emphasis on the differences between the neonatal and adult kidney.

Renal potassium handling in healthy and sick newborns.

Growing infants must maintain a state of positive K+ balance, a task accomplished, in large part, by the kidney. The distal nephron is uniquely adapted to retain total body K+ early in life. The magnitude and direction of net K+ transport in the cortical collecting duct (CCD), the segment responsible for the final renal regulation of K+ balance in the adult, reflect the balance of opposing fluxes of K+ secretion and K+ absorption. Evidence now indicates that the low capacity of the neonatal CCD for K+ secretion is due, at least in part, to a relative paucity of conducting K+ channels in the urinary membrane. A relative excess of K+ absorption in this nephron segment may further reduce net urinary K+ secretion. Under conditions prevailing in vivo, the balance of fluxes in the CCD likely contributes to the relative K+ retention characteristic of the neonatal kidney.

Developmental regulation of expression of renal potassium secretory channels.

PURPOSE OF REVIEW: Somatic growth is associated with an increase in total body K content. K homeostasis is regulated, in large part, by urinary K excretion. Within the adult kidney and specifically the cortical collecting duct, K secretion is accomplished by the passive diffusion of cell K into the urinary fluid down a favorable electrochemical gradient through K selective channels. The purpose of this review is to summarize the results of recent studies that provide insight into how the cortical collecting duct is uniquely adapted for K retention early in life. RECENT FINDINGS: Electrophysiological analyses have identified two types of apical K channels in the mammalian cortical collecting duct. The prevalence of the secretory K channel and its high open probability at the resting membrane potential in the adult has led to the belief that this channel mediates baseline K secretion. The Ca and stretch-activated maxi-K channel has been proposed to mediate flow-stimulated K secretion. In contrast to the high rates of K secretion observed in adult cortical collecting ducts microperfused in vitro, segments isolated from neonatal animals show no significant net K transport until after the third week of postnatal life. The temporal delay between expression of conducting secretory K channels (baseline K secretion) and maxi-K channels (flow-stimulated K secretion) in the maturing cortical collecting duct reflect unique developmental programs regulating the transcription and/or translation of ROMK (rat outer medullary K channel) and slo, the molecular correlates of the secretory K and maxi-K channels, respectively. SUMMARY: The K retention characteristic of the neonatal kidney is due, in part, to a paucity of distinct K channels mediating baseline and flow-stimulated K secretion in the collecting duct. The signals directing the developmental regulation of channel expression are as yet unknown.

[Vasopressin-dependent water permeability of the basolateral membrane of the kidney outer medullary collecting duct in postnatal ontogenesis in rats]. / Vazopressinzavisimaia vodnaia pronitsaemost' bazolateral'noi poverkhnosti sobiratel'nykh trubok naruzhnogo mozgovogo veshchestva pochki krysy v postnatal'nom ontogeneze.

Kidneys of new-born animals are resistant to arginine vasopressin (AVP). The ability of the hormone to regulate water permeability of the collecting duct can be seen from weaning period, probably due to the maturation of the intracellular signaling pathway. The purpose of the present work was to investigate the effect of V2 receptor agonist dDAVP on the water permeability of OMCD basolateral membrane in 10-, 22- and 60-day old Wistar rats. We also estimated ontogenetic gene expression of AQP2, AQP3, AQP4 and V2 receptor. Osmotic water permeability (Pf) of the basolateral membrane of microdissected OMCD was measured under control conditions and after incubation with the agonist V2 receptor desmopressin (dDAVP; 10(-7) M). Water permeability in 10- and 22-day old rats under control conditions were significantly higher than in adults. Desmopressin stimulated significant increase of this parameter in 22-day old pups (Pf = = 125 +/- 4.85; Pf = 174 +/- 8.2 microns/s, p < 0.001) and adult rats (Pf = 100.5 +/- 7.38; Pf = 178.8 +/- 9.54 microns/s, p < 0.001). Osmotic water permeability of the OMCD basolateral membrane in 10-day old rats does not depend on dDAVP (Pf = 172.5 +/- 23.8; Pf = 164.8 +/- 34 microns/s). With the RT-PCR, we observed a gradual increase of AQP2 and V2 receptor genes expression during postnatal ontogenesis. The gene expression of AQP3 and AQP4 remained unchanged during postnatal ontogenesis. In general, the water permeability of the OMCD basolateral membrane of rats can be stimulated by AVP since the 22nd day of postnatal life. The water permeability of the OMCD basolateral membrane under control conditions gradually decreased during postnatal development, while gene expression of AQP3 and AQP4 was unchanged. The mechanism of this decrease remains to be established.