Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency.

The prevailing view is that the ClC-Ka chloride channel (mouse Clc-k1) functions in the thin ascending limb to control urine concentration, whereas the ClC-Kb channel (mouse Clc-k2) functions in the thick ascending limb (TAL) to control salt reabsorption. Mutations of ClC-Kb cause classic Bartter syndrome, characterized by renal salt wasting, with perinatal to adolescent onset. We studied the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2D and advanced 3D imaging of optically cleared kidneys. We show that Clc-k1 and Clc-k2 were broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and Clc-k2 revealed that both participated in NKCC2- and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 caused tubular injury and impaired renal medulla and TAL development. Inducible deletion of Clc-k2 beginning after medulla maturation produced mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and Clc-k2 contributed to salt reabsorption in TAL and distal convoluted tubule (DCT) in neonates, potentially explaining the less-severe phenotypes in classic Bartter syndrome. As opposed to the current understanding that salt wasting in adult patients with Bartter syndrome is due to Clc-k2 deficiency in adult TAL, our results suggest that it originates mainly from defects occurring in the medulla and TAL during development.

Alteration of Cx37, Cx40, Cx43, Cx45, Panx1, and Renin Expression Patterns in Postnatal Kidneys of Dab1-/- (yotari) Mice.

Numerous evidence corroborates roles of gap junctions/hemichannels in proper kidney development. We analyzed how Dab1 gene functional silencing influences expression and localization of Cx37, Cx40, Cx43, Cx45, Panx1 and renin in postnatal kidneys of yotari mice, by using immunohistochemistry and electron microscopy. Dab1 Δ102/221 might lead to the activation of c-Src tyrosine kinase, causing the upregulation of Cx43 in the medulla of yotari mice. The expression of renin was more prominent in yotari mice (p < 0.001). Renin granules were unusually present inside the vascular walls of glomeruli capillaries, in proximal and distal convoluted tubules and in the medulla. Disfunction of Cx40 is likely responsible for increased atypically positioned renin cells which release renin in an uncontrolled fashion, but this doesn't rule out simultaneous involvement of other Cxs, such as Cx45 which was significantly increased in the yotari cortex. The decreased Cx37 expression in yotari medulla might contribute to hypertension reduction provoked by high renin expression. These findings imply the relevance of Cxs/Panx1 as markers of impaired kidney function (high renin) in yotari mice and that they have a role in the preservation of intercellular signaling and implicate connexopathies as the cause of premature death of yotari mice.

Inhibition of Endothelin system during the postnatal nephrogenic period in the rat. Its relationship with hypertension and renal disease in adulthood.

The aim of this work was to study the effect of a high sodium (HS) diet on blood pressure and renal function in male adult rats that have been treated with a dual Endothelin receptor antagonist (ERA) during their early postnatal period (day 1 to 20 of life). Male Sprague-Dawley rats were divided in four groups: CNS: control rats with normosodic diet; ERANS: ERA-treated rats with normosodic diet; CHS: control rats with high sodium diet; ERAHS: ERA-treated rats with HS diet. Systolic blood pressure (SBP) was recorded before and after the diet and 24-hour metabolic cage studies were performed. AQP2 and α-ENac expressions were measured by western blot and real time PCR in the renal medulla. Vasopressin (AVP) pathway was evaluated by measuring V2 receptor and adenylyl cyclase 6 (AC6) expression and cAMP production in the renal medulla. Pre-pro ET-1mRNA was also evaluated in the renal medulla. Only rats that had been treated with an ERA during their postnatal period increased their SBP after consumption of a HS diet, showing an impaired capacity to excrete sodium and water, i.e. developing salt sensitivity. This salt sensitivity would be mediated by an increase in renomedullary expression and activity of AQP2 and α-ENaC as a consequence of increased AC6 expression and cAMP production and/or a decreased ET-1 production in the renal medulla. The knowledge of the molecular mechanisms underlying the perinatal programming of salt sensitive hypertension will allow the development of reprogramming strategies in order to avoid this pathology.

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.

[Investigation of renal corticomedullary differentiation with age-related change on non-contrast-enhanced MRI].

OBJECTIVE: To evaluate the relationship between renal corticomedullary differentiation, renal cortical thickness and age-related changes with non-contrast-enhanced steady-state free precession(SSFP) magnetic resonance imaging (MRI) and spatially selective inversion recovery(IR) pulse technology as well as its applied value . METHODS: A total of 76 healthy volunteers had been recruited from August 2014 to June 2015 in First Hospital of China Medical University.All volunteers were divided into three groups: 2-40 years old, 41-60 years old, 61-80 years old. All 76 volunteers underwent non-contrast-enhanced steady-state free precession(SSFP) 3.0 T MRI scan using variable inversion times (TIs)(TI=1 000, 1 100, 1 200, 1 300, 1 400, 1 500, 1 600, 1 700 ms). The renal corticomedullary differentiation was observed and the signal intensity of renal cortex and medulla were measured respectively as well in order to calculate renal corticomedullary contrast ratio. Besides, renal cortical thickness and renal size were measured. RESULTS: All 76 volunteers were successfully performed all the sequences of MRI scan, including 152 useful imaging of kidney in total. The renal corticomedullary differentiation was clearly shown in all subjects. There was negative correlation between the optimal inversion time(TI) and age(r=-0.65, P<0.01). Similarly, negative correlation was observed between renal corticomedullary contrast ratio and age(r=-0.35, P<0.01). The mean renal cortical thickness of all subjects was (5.33±0.71)mm and there were statistically significant difference among those different groups, which was negative-related with age(r=-0.79, P<0.01). There was no statistically significant difference between sexuality and renal cortical thickness.Additionally, renal cortical thickness had no statistically significant difference in both sides of kidneys. CONCLUSION: The renal corticomedullary differentiation is depicted clearly by means of non-contrast-enhanced steady-state free precession MRI with spatially selective inversion recovery pulse technology. The optimal inversion time decreases along with the increase of age. In the meanwhile, the renal cortical thickness could be measured truthfully and accurately.

A Subpopulation of Label-Retaining Cells of the Kidney Papilla Regenerates Injured Kidney Medullary Tubules.

To determine whether adult kidney papillary label-retaining cells (pLRCs) are specialized precursors, we analyzed their transcription profile. Among genes overexpressed in pLRCs, we selected candidate genes to perform qPCR and immunodetection of their encoded proteins. We found that Zfyve27, which encodes protrudin, identified a subpopulation of pLRCs. With Zfyve27-CreERT2 transgenic and reporter mice we generated bitransgenic animals and performed cell-lineage analysis. Post tamoxifen, Zfyve27-CreERT2 marked cells preferentially located in the upper part of the papilla. These cells were low cycling and did not generate progeny even after long-term observation, thus they did not appear to contribute to kidney homeostasis. However, after kidney injury, but only if severe, they activated a program of proliferation, migration, and morphogenesis generating multiple and long tubular segments. Remarkably these regenerated tubules were located preferentially in the kidney medulla, indicating that repair of injury in the kidney is regionally specified. These results suggest that different parts of the kidney have different progenitor cell pools.

Age-related change in renal corticomedullary differentiation: evaluation with noncontrast-enhanced steady-state free precession (SSFP) MRI with spatially selective inversion pulse using variable inversion time.

PURPOSE: To evaluate age-related change in renal corticomedullary differentiation and renal cortical thickness by means of noncontrast-enhanced steady-state free precession (SSFP) magnetic resonance imaging (MRI) with spatially selective inversion recovery (IR) pulse. MATERIALS AND METHODS: The Institutional Review Board of our hospital approved this retrospective study and patient informed consent was waived. This study included 48 patients without renal diseases who underwent noncontrast-enhanced SSFP MRI with spatially selective IR pulse using variable inversion times (TIs) (700-1500 msec). The signal intensity of renal cortex and medulla were measured to calculate renal corticomedullary contrast ratio. Additionally, renal cortical thickness was measured. RESULTS: The renal corticomedullary junction was clearly depicted in all patients. The mean cortical thickness was 3.9 ± 0.83 mm. The mean corticomedullary contrast ratio was 4.7 ± 1.4. There was a negative correlation between optimal TI for the best visualization of renal corticomedullary differentiation and age (r = -0.378; P = 0.001). However, there was no significant correlation between renal corticomedullary contrast ratio and age (r = 0.187; P = 0.20). Similarly, no significant correlation was observed between renal cortical thickness and age (r = 0.054; P = 0.712). CONCLUSION: In the normal kidney, noncontrast-enhanced SSFP MRI with spatially selective IR pulse can be used to assess renal corticomedullary differentiation and cortical thickness without the influence of aging, although optimal TI values for the best visualization of renal corticomedullary junction were shortened with aging.

Postnatal development of the renal medulla; role of the renin-angiotensin system.

Adverse events during foetal development can predispose the individual for cardiovascular disease later in life, a correlation known as foetal programming of adult hypertension. The 'programming' events have been associated with the kidneys due to the significant role in extracellular volume control and long-term blood pressure regulation. Previously, nephron endowment and functional consequences of a low nephron number have been extensively investigated without achieving a full explanation of the underlying pathophysiological mechanisms. In this review, we will focus on mechanisms of postnatal development in the renal medulla with regard to the programming effects. The renin-angiotensin system is critically involved in mammalian kidney development and impaired signalling gives rise to developmental renal lesions that have been associated with hypertension later in life. A consistent finding in both experimental animal models and in human case reports is atrophy of the renal medulla with developmental lesions to both medullary nephron segments and vascular development with concomitant functional disturbances reaching into adulthood. A review of current knowledge of the role of the renin-angiotensin system for renal medullary development will be given.

SDF-1 activates papillary label-retaining cells during kidney repair from injury.

The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795-804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair.

Angiotensin II regulates growth of the developing papillas ex vivo.

We tested the hypothesis that lack of angiotensin (ANG) II production in angiotensinogen (AGT)-deficient mice or pharmacologic antagonism of ANG II AT(1) receptor (AT(1)R) impairs growth of the developing papillas ex vivo, thus contributing to the hypoplastic renal medulla phenotype observed in AGT- or AT(1)R-null mice. Papillas were dissected from Hoxb7(GFP+) or AGT(+/+), (+/-), (-/-) mouse metanephroi on postnatal day P3 and grown in three-dimentional collagen matrix gels in the presence of media (control), ANG II (10(-5) M), or the specific AT(1)R antagonist candesartan (10(-6) M) for 24 h. Percent reduction in papillary length was attenuated in AGT(+/+) and in AGT(+/-) compared with AGT(-/-) (-18.4 ± 1.3 vs. -32.2 ± 1.6%, P < 0.05, -22.8 ± 1.3 vs. -32.2 ± 1.6%, P < 0.05, respectively). ANG II blunted the decrease in papilla length observed in respective media-treated controls in Hoxb7(GFP+) (-1.5 ± 0.3 vs. -10.0 ± 1.4%, P < 0.05) or AGT(+/+), (+/-), and (-/-) papillas (-12.8 ± 0.7 vs. -18.4 ± 1.3%, P < 0.05, -16.8 ± 1.1 vs. -23 ± 1.2%, P < 0.05; -26.2 ± 1.6 vs. -32.2 ± 1.6%, P < 0.05, respectively). In contrast, percent decrease in the length of Hoxb7(GFP+) papillas in the presence of the AT(1)R antagonist candesartan was higher compared with control (-24.3 ± 2.1 vs. -10.5 ± 1.8%, P < 0.05). The number of proliferating phospho-histone H3 (pH3)-positive collecting duct cells was lower, whereas the number of caspase 3-positive cells undergoing apoptosis was higher in candesartan- vs. media-treated papillas (pH3: 12 ± 1.4 vs. 21 ± 2.1, P < 0.01; caspase 3: 3.8 ± 0.5 vs. 1.7 ± 0.2, P < 0.01). Using quantitative RT-PCR, we demonstrate that AT(1)R signaling regulates the expression of genes implicated in morphogenesis of the renal medulla. We conclude that AT(1)R prevents shrinkage of the developing papillas observed ex vivo via control of Wnt7b, FGF7, ß-catenin, calcineurin B1, and α3 integrin gene expression, collecting duct cell proliferation, and survival.

Development of the kidney medulla.

The mature renal medulla, the inner part of the kidney, consists of the medullary collecting ducts, loops of Henle, vasa recta and the interstitium. The unique spatial arrangement of these components is essential for the regulation of urine concentration and other specialized kidney functions. Thus, the proper and timely assembly of medulla constituents is a crucial morphogenetic event leading to the formation of a functioning metanephric kidney. Mechanisms that direct renal medulla formation are poorly understood. This review describes the current understanding of the key molecular and cellular mechanisms underlying morphological aspects of medulla formation. Given that hypoplasia of the renal medulla is a common manifestation of congenital obstructive nephropathy and other types of congenital anomalies of the kidney and urinary tract (CAKUT), better understanding of how disruptions in medulla formation are linked to CAKUT will enable improved diagnosis, treatment and prevention of CAKUT and their associated morbidity.

Developmental immunolocalization of heat shock protein 70 (HSP70) in epithelial cell of rat kidney.

During renal development the cells in the medulla are exposed to elevated and variable interstitial osmolality. Heat shock protein 70 (HSP70) is a major molecular chaperone and plays an important role in the protection of cells in the renal medulla from high osmolality. The purpose of this study was to establish the time of immunolocalization and distribution of HSP70 in developing and adult rat kidney. In addition, changes in HSP70 immunolocalization following the infusion of furosemide were investigated. In adult animals, the HSP70 was expressed in the medullary thin ascending limb of Henle's loop (ATL) and inner medullary collecting duct (IMCD). In developing kidney, HSP70 immunoreactivity was first detected in the IMCD of the papillary tip on postnatal day 1. From four to 14 days of age, HSP70 was detected in the ATL after transformation from thick ascending limb, beginning at the papillary tip and ascending to the border between the outer and inner medulla. The immunolocalization of HSP70 in both the ATL and IMCD gradually increased during two weeks. The gradual increase in HSP70 was associated with an increase in its mRNA abundance. However, furosemide infusion resulted in significantly reduced HSP70 immunolocalization in the IMCD and ATL. These data demonstrated that the expression of HSP70 was closely correlated with changes in interstitial osmolality during the development of the kidney. We suggest that HSP70 protects ATL and IMCD cells in the inner medulla from the stress of high osmolality and may be involved in the transformation of the ATL of the long loop of Henle during renal development.

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.

Prominin-2 is a novel marker of distal tubules and collecting ducts of the human and murine kidney.

Prominin-1 (CD133) and its paralogue, prominin-2, are pentaspan membrane glycoproteins that are strongly expressed in the kidney where they have been originally cloned from. Previously, we have described the localization of prominin-1 in proximal tubules of the nephron. The spatial distribution of prominin-2, however, has not yet been documented in the kidney. We therefore examined the expression of this molecule along distinct tubular segments of the human and murine nephron using in situ hybridization and immunohistochemistry. Our findings indicated that human prominin-2 transcripts and protein were confined to distal tubules of the nephron including the thick ascending limb of Henle's loop and the distal convoluted tubule, the connecting duct and to the collecting duct system. Therein, this glycoprotein was enriched at the basolateral plasma membrane of the tubular epithelial cells with exception of the thick ascending limb where it was also found in the apical domain. This is in contrast with the exclusive apical localization of prominin-1 in epithelial cells of proximal nephron tubules. The distribution of murine prominin-2 transcripts was reminiscent of its human orthologue. In addition, a marked enrichment in the epithelium covering the papilla and in the urothelium of the renal pelvis was noted in mice. Finally, our biochemical analysis revealed that prominin-2 was released into the clinically healthy human urine as a constituent of small membrane vesicles. Collectively our data show the distribution and subcellular localization of prominin-2 within the kidney in situ and its release into the urine. Urinary detection of this protein might offer novel diagnostic approaches for studying renal diseases affecting distal segments of the nephron.

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.

Postnatal adrenalectomy impairs urinary concentrating ability by increased COX-2 and leads to renal medullary injury.

We hypothesized that aldosterone promotes development of the renal medulla in the postnatal period and that cyclooxygenase-2 (COX-2) activity contributes to renal dysfunction after impaired aldosterone signaling. To test these hypotheses, rat pups underwent either sham operation or adrenalectomy at postnatal day 10. Adrenalectomized rats were divided into no steroid substitution (ADX), corticosterone replacement (ADX-C), and corticosterone and DOCA substitution (ADX-CD) groups that received subcutaneous pellets with steroids. Without replacement, pups failed to thrive and exhibited impaired urinary-concentrating ability. The renal medulla was significantly smaller, and the medullary interstitial osmolality was lower in the ADX group, whereas COX-2 and PGE2 tissue levels were significantly elevated compared with levels shown in sham animals. Substitution with DOCA and corticosterone corrected these changes, whereas corticosterone replacement alone improved survival but not weight gain and urinary-concentrating ability. Administration of a COX-2 inhibitor to ADX rats (parecoxib, 5 mg.kg(-1).day(-1), days 17-20) increased weight gain, urinary-concentrating ability, and papillary osmolality. After fluid deprivation, parecoxib attenuated weight loss and the increase in plasma Na+ concentration and osmolality. It is concluded that mineralocorticoid is required for normal postnatal development of the renal medulla. COX-2 contributes to impaired urine-concentrating ability, NaCl loss, and extracellular volume depletion in postnatal mineralocorticoid deficiency.

Glomerular eNOS gene expression during postnatal maturation and AT1 receptor inhibition.

Glomerular maturation increases from immature superficial to advanced juxtamedullary nephrons, while nephrogenesis continues postnatally in porcine kidneys. Endothelial NOS, eNOS, shows significant postnatal renal developmental regulation, perhaps mediated by Angiotensin II (AII). The objective was to compare eNOS mRNA gene expression between superficial and juxtamedullary glomeruli obtained from piglets and adult pigs utilizing laser capture microdissection during basal conditions and, to determine the role of the AII AT1 receptor, AT1, after chronic AT1 inhibition (AT1X) with candesartan. Superficial glomerular eNOS expression was lowest in newborns (NB) and at 7 days, and was highest in 14, 21 day old piglets and adults. Juxtamedullary glomerular eNOS, while similar in NB, 14, 21 day and adult, dipped to the lowest level at 7 days. Juxtamedullary glomerular eNOS expression in the NB was 7 fold greater than in superficial glomeruli. AT1X did not change eNOS expression in adult glomeruli. AT1X significantly reduced NB eNOS expression in both superficial, 90+/-10%, and juxtamedullary glomeruli, 89+/-5% respectively. In conclusion, eNOS gene expression demonstrates significant differences between NB superficial and juxtamedullary glomeruli, significant postnatal developmental regulation of both glomerular locations, and this expression may be mediated in the NB by AII via the AT1 receptor.

Sex-differential expression of ornithine aminotransferase in the mouse kidney.

The mouse kidney expresses the gene of ornithine aminotransferase (Oat). Previous works suggest that Oat is differentially expressed in female and male mouse kidney (Alonso E, Rubio V. Biochem J 259: 131-138, 1989; Levillain O, Diaz JJ, Blanchard O, Dechaud H. Endocrinology 146: 950-959, 2005; Manteuffel-Cymborowska M, Chmurzynska W, Peska M, Grzelakowska-Sztabert B. Int J Biochem Cell Biol 27: 287-295, 1995; Natesan S, Reddy SR. Comp Biochem Physiol B Biochem Mol Biol 130: 585-595, 2001; Yu H, Yoo PK, Aguirre CC, Tsoa RW, Kern RM, Grody WW, Cederbaum SD, Iyer RK. J Histochem Cytochem 51: 1151-1160, 2003). This study was designed to provide a detailed description of the sexual dimorphism of Oat expression in the mouse kidney and to test the influence of sex hormones on its regulation. Experiments were performed on male and female Swiss OF1 mice during their postnatal development, at adulthood, and in orchidectomized and ovariectomized mice. Kidneys, dissected renal zones, and mitochondria were used to analyze OAT mRNA and protein levels and measure OAT activity. The results revealed that before puberty, Oat expression was similar between female and male kidneys whereas from puberty until adulthood Oat expression increased in the female kidney, becoming approximately 2.5-fold higher than in the male kidney. This sex-differential expression of Oat was associated with a sex-specific distribution of Oat along the corticopapillary axis and within the nephron. OAT was three- to fourfold more expressed in the female than the male cortex. In males, Oat was highly expressed in the medulla, mainly in the thick ascending limbs. Renal Oat distribution in orchidectomized mice resembled that in the females. Ovariectomy did not influence Oat expression. Sex differences are explained by the physiological increase in plasma testosterone in males. Expression of medium-chain acyl-CoA synthetase protein confirmed this finding. We report sexual dimorphism of Oat expression in the mouse kidney and show that Oat is naturally downregulated in the presence of testosterone.

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.

Glucocorticoid impairs growth of kidney outer medulla and accelerates loop of Henle differentiation and urinary concentrating capacity in rat kidney development.

In the rat, urinary concentrating ability develops progressively during the third postnatal (P) week and nearly reaches adult level at weaning (P21) governed by a rise in circulating glucocorticoid. Elevated extracellular osmolality can lead to growth arrest of epithelial cells. We tested the hypothesis that supranormal exposure of rat pups to glucocorticoid before the endogenous surge enhances urinary concentrating ability but inhibits renomedullary cell proliferation. Proliferating-cell nuclear antigen (PCNA)-positive cells shifted from the nephrogenic zone in the first postnatal week to Tamm-Horsfall-positive thick ascending limb (TAL) cells at the corticomedullary junction at P10-14. Renal PCNA protein abundance was stable in the suckling period and decreased 10-fold after weaning. Renal PCNA protein abundance decreased in response to dexamethasone (DEXA; 100 microg x kg(-1) x day(-1), P8-12). Prolonged administration of DEXA (P1-P11) reduced selectively the area and thickness of the outer medulla and the number of PCNA-positive cells. DEXA (P8-12) increased urinary and papillary osmolality in normohydrated and water-deprived pups and led to osmotic equilibrium between interstitium and urine, whereas apoptotic and GADD153-positive cells increased in the inner medulla. TAL-associated NaCl transporters Na-K-2Cl cotransporter, Na-K-ATPase-alpha(1), Na/H exchanger type 3, and ROMK increased significantly at weaning and in response to DEXA. We conclude that a low level of circulating glucocorticoid is permissive for proliferation of Henle's loop and the outer medulla before weaning. A reduced papillary tonicity is a crucial factor for the reduced capacity to concentrate urine during postnatal kidney development. We speculate that supranormal exposure to glucocorticoid in the suckling period can alter kidney medullary structure and function permanently.