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Background/aims: The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity., Methods: In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies., Results: The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture., Conclusions: We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

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.

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.

Large-conductance Ca(+)-activated K(+) (BKCa) channels are widely distributed in a variety of cells and play a pivotal and specific role in many pathophysiological conditions. However, the function of BK(Ca) channels in the kidney cortex and hippocampus during the postnatal development has not received attention. In this study, to elucidate the role of BK(Ca) channels during the development, it is essential to establish the location and quantitation of expression of BK(Ca). The expressions of BK(Ca) were detected in the kidney and hippocampus on postnatal days (P) 1, 3, 5, 7, 14, 21, 28, and 49 by immunohistochemical and Western blot analysis. Our results showed that expressions of BK(Ca) channels were found in tubules and corpuscles at all time points. The expression was also observed at all developmental stages of the renal corpuscles, such as comma-shaped body, S-shaped body, renal corpuscles of stage III, and renal corpuscles of stage IV. During the development, the expression of BK(Ca) channels was decreased and the most prominent change of BK(Ca) protein level appeared between P14 and P21. In contrast, BK(Ca) channels were expressed in all regions of the hippocampus at every time point with the level increasing during the early development (P1 to P14). The findings of the present study suggest that BKCa channels play an important role during the postnatal development in both the renal cortex and hippocampus.

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., (© 2013 Wiley Periodicals, Inc.)

Nephrogenesis is mainly characterized by the interaction of two distinct renal constituents, the ureteric bud and the metanephric mesenchyme. In this paper we describe by means of light and electron microscopic techniques the morphological events that take place during the early stages of cap mesenchymal formation. Samples of normal renal tissue were excised from newborn NOD mice and processed by standard light and electron microscopy techniques. In all samples examined we detected the presence of several cap mesenchymal aggregates in different stages of differentiation. They varied from small solid nodules with few ovoid cells to bigger pine-cone-like aggregates, characterized by a peculiar distribution and morphology of their cellular constituents. Our data highlight, for the first time, the presence of a specific cap mesenchymal structure, the pine-cone body and show, at ultrastructural level, how each cap aggregate epithelializes proceeding in stages from a condensed mesenchymal aggregate to the renal vesicle, through the intermediate "pine-cone body" stage.

TRPC6, a member of the TRPC family, attracts much attention from the public because of its relationship with the disease. In both the brain and kidney, TRPC6 serves a variety of functions. The aim of the present study was to observe the expression and effects of TRPC6 in renal cortex and hippocampus during early postnatal development of the mouse. In the present study, immunohistochemistry and Western blotting were used to detect the expression of TRPC6 in the mouse kidney and hippocampus of postnatal day 1, 3, 5, 7, 14, 21, 28 and 49 (P1, P3, P5, P7, P14, P21, P28 and P49). Results showed that the expression of TRPC6 was increased in the mouse hippocampus, and there was a significant increase between P7 and P14 during the postnatal development. Meanwhile, the expression of TRPC6 was also detected in glomerulus and tubules, and a decreased expression was found during postnatal maturation of mouse renal cortex. From these in vivo experiments, we concluded that the expression of TRPC6 was active in the developing mouse kidney cortex, and followed a loss of expression with the development of kidney. Meanwhile, an increased expression was found in the hippocampus with the development. Together, these data suggested that the developmental changes in TRPC6 expression might be required for proper postnatal kidney cortex development, and played a critical role in the hippocampus during development, which formed the basis for understanding the nephrogenesis and neurogenesis in mice and provided a practically useful knowledge to the clinical and related research.

Erythropoietin (EPO), known for its role in erythroid differentiation, has been shown to be an important growth factor for brain and heart. EPO is synthesized by fibroblast-like cells in the renal cortex. Prompted by this anatomical relationship and its significant impact on the maturation process of brain and heart, we asked whether EPO could play a role during the development of renal cortex. The relationship between the development of renal cortex and the change of EPO receptor (EPOR), through which EPO could act as a renotropic cytokine, became interesting to us. In this study, the day of birth was recorded as postnatal day 0(P0). P7, P14, P21, P28, P35, P42 and mature mice (postnatal days>56) were used as the animal model of different developmental stages. Immunohistochemistry and Western blotting were used to detect the expression of EPOR in mouse renal cortex. Results showed that expression of EPOR decreased with the development of renal cortex and became stable when kidney became mature. The expression of EPOR was detected at the renal tubule of all developmental stages and a relatively higher expression was observed at P14. However, at the renal corpuscle the expression was only observed at P7 and quickly became undetectable after that. All these suggested that a translocation of EPOR from renal corpuscle to renal tubule may take place during the developmental process of renal cortex. Also, EPO may be an essential element for the maturation of renal cortex, and the requirement for EPO was changed during postnatal development process.

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; β(1)-expression was higher in newborns than the other ages, and β(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 β(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, β(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.

Background: A mouse model of impaired renal development was developed and the effect of retinoic acid (RA) was investigated in this animal model., Methods: An angiogenesis inhibitor (SU1498) was injected s.c. into day 3 C57BL/6 newborn mice to create a model of arrested renal development. RA (2 mg/kg) was injected i.p. for 10 days. Morphometry and immunohistochemistry were done., Results: Mice injected with SU1498 demonstrated deranged renal development in tubular structure and glomerular tuft area. Cortical thickness and area of glomerular tuft were significantly decreased after vascular endothelial growth factor (VEGF) inhibitor, and were significantly restored by RA. The length of capillary loops/glomerulus, the number of podocytes/glomerulus, and density of peritubular capillaries on CD31 immunostaining were significantly decreased by VEGF blocking and recovered by RA., Conclusions: VEGF plays a major role in renal development, and RA reverses the inhibited development caused by an angiogenesis inhibitor.

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-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.

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.

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.

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.

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., ((c) 2004 Wiley-Liss, Inc.)

Renal nephropathy present in male Wistar rats more than 13 months of age was reported as an indication that the rats were in renal failure. In this study, the renal tissue damage at 14 months of age in male Munich Wistar rats was similar to that reported for Wistar rats, indicating that Munich Wistar rats could be another model for study of kidney function in the aging rat. The usual renal response to injury involves increased cell division and/or reparative processes that involve tyrosine kinase activity (TyrK) and/or guanosine triphosphate-binding (G) protein signal trans-duction pathways. This study reveals the presence of renal tissue damage coinciding with significantly reduced activity of Ras, Akt, and p34cdc2 kinase, the signaling proteins that regulate cell division and/or growth, in renal cortical tissues of aging rats compared to young rats (P < 0.005, P < 0.005, and P< 0.001, respectively). These results suggest that proteins involved in signal transduction pathways associated with cell replication are downregulated in the aging kidney cortex at a time when renal cellular damage is also present.

In rats, the secretion of p-aminohippurate (PAH) by the kidney is higher in males (M) than in females (F). The role of the major renal PAH transporters, OAT1 and OAT3, in the generation of these gender differences, as well as the responsible hormones and mechanisms, has not been clarified. Here we used various immunocytochemical methods to study effects of gender, gonadectomy, and treatment with sex hormones on localization and abundance of OAT1 and OAT3 along the rat nephron. Both transporters were localized to the basolateral membrane: OAT1 was strong in proximal tubule S2 and weak in the S3 segments, whereas OAT3 was stained in proximal tubule S1 and S2 segments, thick ascending limb, distal tubule, and in principal cells along the collecting duct. Gender differences in the expression of both transporters in adult rats (M > F) were observed only in the cortical tubules. OAT1 in the cortex was strongly reduced by castration in adult M, whereas the treatment of castrated M with testosterone, estradiol, or progesterone resulted in its complete restitution, further depression, or partial restitution, respectively. In adult F, ovariectomy weakly increased, whereas estradiol treatment of ovariectomized F strongly decreased, the expression of OAT1. The expression of OAT3 in the M and F cortex largely followed a similar pattern, except that ovariectomy and progesterone treatment showed no effect, whereas in other tissue zones gender differences were not observed. In prepubertal rats, the expression of OAT1 and OAT3 in the kidney cortex was low and showed no gender differences. Our data indicate that gender differences in the rat renal cortical OAT1 and OAT3 (M > F) appear after puberty and are determined by both a stimulatory effect of androgens (and progesterone in the case of OAT1) and an inhibitory effect of estrogens.

In postnatal weeks 2-4, cyclooxygenase-2 (COX-2) is induced in the rat kidney cortex where it is critically involved in final stages of kidney development. We examined whether changes in circulating gluco- or mineralocorticosteroids or in their renal receptors regulate postnatal COX-2 induction. Plasma corticosterone concentration peaked at birth, decreased to low levels at days 3-13, and increased to adult levels from day 22. Aldosterone peaked at birth and then stabilized at adult levels. Gluco- and mineralocorticoid receptor (GR and MR) mRNAs were expressed stably in kidney before, during, and after COX-2 induction. 11 beta-hydroxysteroid dehydrogenase 2 was induced shortly after birth and was widely distributed in the whole collecting duct system in the suckling period and then returned to an adult pattern. Supplementation with corticosterone (20 mg.kg-1.day-1) or GR-specific dexamethasone (1 mg.kg-1.day-1) during low endogenous corticosterone suppressed renal COX-2 mRNA and protein and led to a restricted distribution of COX-2 immunolabeling. The ability of glucocorticoids to affect COX-2 was reflected in colocalization of GR-alpha and COX-2 immunoreactivity and mRNAs in thick ascending limb of Henle's loop. The MR antagonist potassium canrenoate (20 mg.kg-1.day-1) enhanced COX-2 expression from days 5 to 10, but low MR-specific concentrations of DOCA (1 mg.kg-1.day-1) had no effect on COX-2. Renomedullary interstitial cells expressed GR-alpha and COX-2. Dexamethasone suppressed COX-2 in these cells. Thus low plasma concentrations of corticosterone allowed for cortical and medullary COX-2 induction during postnatal kidney development. Increased circulating glucocorticoid in the postnatal period may damage late renal development through inhibition of COX-2.

We have investigated the temporal maturation of the rat kidney during the postnatal developmental period. As a result, we observed the following: an active process of cortical cell proliferation and differentiation occurs as late as day 20. The medulla is the most immature zone at birth and displays the greatest morphological changes during this period. At birth, no distinction exists between inner and outer medulla, and the outer and inner strip of the outer medulla can be distinguished as late as day 30. Remodeling of the ECM surrounding collecting ducts occurs in the medulla twice, stopping at day 11 and it occurs in the papilla three times, stopping at day 20. The increase of kidney size is temporally different for each kidney zone. The cortex and the papilla acquire the morphological appearance of the adult kidney before the medulla does. Consequently, the medulla remains at the highest degree of immaturation among the kidney zones for a relatively long postnatal period.

The present study was designed to test the hypothesis that growth from puberty to adulthood in the rat is associated with an increase in renal cortical nitric oxide synthase (NOS) activity that results in an augmented impact of nitric oxide (NO) on hemodynamic function. Two groups of male Sprague-Dawley rats were studied: juvenile rats (approximately 2 months old) and mature rats (approximately 5 months old). NOS activity, measured as -nitro-L-arginine (NNA)-sensitive (3)H-L-citrulline production from (3)H-L-arginine, was significantly higher in the renal cortex of mature rats (57+/-2 pmol/h per mg protein) than in juveniles (42+/-3 pmol/h per mg protein). Additional animals from each group were anesthetized to determine the acute impact of NOS inhibition on arterial pressure and renal cortical blood flow, measured by single-fiber Doppler flowmetry. Cortical blood flow was higher in mature rats than in juveniles, averaging 22+/-2 and 16+/-1 perfusion units, respectively. NOS inhibition (10 mg/kg NNA i.v.) decreased renal cortical blood flow in mature rats by 35+/-7%, but only by 9+/-4% in juvenile animals. These data support the hypothesis that maturational growth in the rat is associated with augmented NOS activity coupled with an increased tonic influence of NO on renal cortical blood flow.

Carbonic anhydrase (CA) IV facilitates renal acidification by catalyzing the dehydration of luminal H(2)CO(3). CA IV is expressed in proximal tubules, medullary collecting ducts, and A-intercalated cells of the mature rabbit kidney (Schwartz GJ, Kittelberger AM, Barnhart DA, and Vijayakumar S. Am J Physiol 278: F894-F904, 2000). In view of the maturation of HCO transport in the proximal tubule and collecting duct, the ontogeny of CA IV expression was examined. During the first 2 wk, CA IV mRNA was expressed in maturing cortex and medulla at ~20% of adult levels. The maturational increase was gradual in cortex over 3-5 wk of age but surged in the medulla, so that mRNA levels appeared higher than those in the adult medulla. In situ hybridization showed very little CA IV mRNA at 5 days, with increases in deep cortex and medullary collecting ducts by 21 days. Expression of CA IV protein in the cortex and medulla was minimal at 3 days of age but then apparent in the juxtamedullary region, A-intercalated cells and medullary collecting ducts by 18 days; there was little labeling of the proximal straight tubules of the medullary rays. Thus CA IV expression may be regulated to accommodate the maturational increase in HCO absorption in the proximal tubule. In the medullary collecting duct, there is a more robust maturation of CA IV mRNA and protein, commensurate with the high rate of HCO absorption in the neonatal segment.

The postnatal pattern of renal endothelial nitric oxide synthase (eNOS) is unknown. The purpose of this study was to characterize eNOS expression during maturation and compare this to neuronal NOS (nNOS). The experiments measured whole kidney eNOS mRNA expression by RT-PCR and protein content by Western blot, as well as cortical and medullary protein content in piglets at selected postnatal ages and in adult pigs. Whole kidney eNOS mRNA was compared with nNOS. Whole kidney eNOS expression decreased from the newborn to its lowest at 7 days, returning by 14 days to adult levels. This eNOS mRNA pattern contrasted with nNOS, which was highest at birth, and progressively decreased to its lowest level in the adult. At birth, cortical eNOS protein was greater than medullary, contrasting with the adult pattern of equivalent levels. In conclusion eNOS is developmentally regulated during early renal maturation and may critically participate in renal function during this period. The eNOS developmental pattern differs from nNOS, suggesting that these isoforms may have different regulatory factors and functional contributions in the postnatal kidney.

The expression of the biogenic amine degrading enzyme monoamine oxidases-A and -B depends on several factors including regional distribution, development and hormonal environment. In the present study, we investigated the expression of monoamine oxidases in developing kidney and their regulation by dexamethasone treatment. Immunoblots and enzyme assays, performed using [14C]5-hydroxytriptamine and [14C]beta-phenylethylamine as substrates for monoamine oxidases-A and -B, respectively, showed that monoamine oxidase-A is the isoenzyme largely predominant in 9-day-old rats renal cortex. Experiments performed in 5-week-old rats showed an increase in monoamine oxidase-B activity and a decrease in monoamine oxidase-A activity and substrate affinity. The changes of monoamine oxidase-A activity and affinity were mimicked by dexamethasone treatment (0.60 mg/kg body weight injected subcutaneously three times at intervals of 24 h) of 9-day-old rats. In contrast, dexamethasone administration induced a modification of monoamine oxidase-B activity opposite to that found between 9-day- and 5-week-old rats. Dexamethasone treatment did not modify immunoreactivity and mRNA corresponding to monoamine oxidases-A and -B indicating that changes of enzyme activities were unrelated to regulation of protein synthesis and mRNA turnover. These results show that monoamine oxidases-A and -B are differently expressed in developing renal cortex and are regulated by dexamethasone treatment.

The shape and arrangement of the developing nephrons were studied three-dimensionally by scanning electron microscopy (SEM) of the neonatal mouse kidney. The specimens were treated with the KOH digestion method in order to remove extracellular connective tissue components, thus enabling the direct observation of the developing nephrons at various stages. At the subcapsular region of the renal cortex, the ureteric ducts were observed as branched tubules with terminal swellings or ampullae. Newly formed blood vessels were often associated with terminals of these ureteric ducts. The cup-shaped renal corpuscles had aggregations of mesangial cells with blood vessels in the groove. At the vascular pole of mature nephrons, extraglomerular mesangial cells were observed as a cellular sheet, which was continuous with the smooth muscle layer of afferent and efferent blood vessels. The present study also demonstrated the shape of the immature podocytes in relation to the endothelial morphology of glomerular capillaries.

To determine if the developing kidney differs from the adult in the expression of the neuronal nitric oxide synthase, NOS I, these experiments measured mRNA gene expression by RNase protection assay and protein content by Western blot of NOS I in piglets at ages newborn and 3, 7, 10, 14, and 21 days and adult pigs. Whole kidney NOS I mRNA was greatest at birth and decreased progressively during renal maturation to adult levels. NOS I protein content paralleled this developmental pattern. Cortical NOS I protein was equivalent in newborn and 14-day-old piglets and was greater at both ages than the adult. Medullary NOS I protein was relatively greater than cortical in both immature ages and decreased from a peak at birth to adult levels. We conclude the following. 1) During postnatal maturation, renal NOS I mRNA and protein content show a pattern that is developmentally regulated. 2) This developmental pattern of NOS I after birth may, in part, contribute to the enhanced functional role of NO during renal maturation.

Renal Na,K-ATPase drives active reabsorption of sodium and cotransported solutes along the nephron. There is a large increase in net Na(+) reabsorption in the postnatal rat kidney. It has previously been established that the postnatal increase in expression of sodium pump isoforms in the brain, but not the heart, is blunted in the hypothyroid neonate. The aims of this study were to establish whether the developmental increase in renal sodium transport is associated with coordinate increases in the abundance of the sodium pump alpha(1) catalytic and beta(1) glycoprotein subunits and Na,K-ATPase activity, and to determine whether thyroid status influences the postnatal increase in renal Na,K-ATPase expression. Pregnant rats were made hypothyroid with low iodine diet, propylthiouracil and perchlorate. Offspring were hypothyroid assessed by triiodothyronine/thyroxine RIA. Renal cortical membranes were prepared from euthyroid and hypothyroid rats from 6 to 24 days of age. There was no change in Na,K-ATPase activity or expression between 6 and 15 days. Between 15 and 24 days, Na,K-ATPase activity increased 1.35-fold while sodium pump alpha(1) and beta(1) subunit abundance increased coordinately to 1.7- and 2-fold over the previous period, respectively. In hypothyroid neonates, kidney weight was less than in euthyroids, and Na,K-ATPase activity, alpha(1) and beta(1) subunit pool sizes did not significantly increase as a function of age between 6 and 24 days. We conclude that the postnatal increase in sodium pump activity can be accounted for by coordinate increases in the pool sizes of alpha(1) and beta(1) subunits and that, like in brain, this increased Na,K-ATPase expression is dependent on normal thyroid status., (Copyright 2000 S. Karger AG, Basel)

We have recently demonstrated that while the osmotic water permeability (Pf) of neonatal proximal tubules is higher than that of adult tubules, the Pf of brushborder membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal tubule basolateral membranes by determining aquaporin 1 (AQP1) protein abundance and the Pf in neonatal (10-14 days old) and adult rabbit renal basolateral membrane vesicles (BLMV). At 25 degrees C the Pf of neonatal BLMV was significantly lower than the adult BLMV at osmotic gradients ranging from 40 to 160 mOsm/kg water. The activation energies for osmotic water movement were identical in the neonatal and adult BLMV (8.65 +/- 0.47 vs. 8.86 +/- 1.35 kcal x deg(-1) x mol(-1). Reflection coefficients for sodium chloride and sodium bicarbonate were identical in both the neonatal and adult BLMV and were not different from one. Mercury chloride (0.5 mM) reduced osmotic water movement by 31.3 +/- 5.5% in the adult BLMV, but by only 4.0 +/- 4.0% in neonatal vesicles (P < 0.01). Adult BLMV AQP1 abundance was higher than that in the neonate. These data demonstrate that neonatal BLMV have a lower Pf and AQP1 protein abundance than adults and that a significantly greater fraction of water traverses the basolateral membrane lipid bilayer and not water channels in neonates compared to adults. The lower Pf of the neonatal BLMV indicates that the basolateral membrane is not responsible for the higher transepithelial Pf in the neonatal proximal tubule.

The cortical collecting duct (CCD) is a major site of regulation of K+ homeostasis in the fully differentiated mammalian kidney. CCDs isolated from adult rabbits and microperfused in vitro secrete K+ into the tubular fluid at high rates. However, CCDs dissected from newborn animals show no significant net K+ secretion until the 3rd wk of life, at least in part because of a paucity of conducting apical secretory K+ (SK) channels. To determine whether the abundance of genes encoding the SK channel is developmentally regulated, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis to test for the presence of mRNA encoding rat outer medullary K+ channel (ROMK), considered to be a major subunit of the SK channel, in kidney and single CCDs isolated from maturing rabbits. Using rat ROMK-specific primers, RT-PCR of rabbit kidney yielded an amplification product of expected size and sequence. Northern blot analysis identified a single band at approximately 2.9 kb in kidney at all ages. Densitometric analysis revealed a progressive increase in steady state expression of ROMK message in kidney after birth. RT-PCR of individual CCDs yielded a single band of predicted size for ROMK in all segments isolated from animals > or =3 wk old. In contrast, transcripts were not detected in any CCD samples obtained from 1-wk-old animals and were identified in only 30% of CCD samples isolated from 2-wk-old rabbits. In all of the latter tubular samples, a specific PCR product of correct size for beta-actin mRNA was detected. These results suggest that an increase in steady state expression of ROMK mRNA contributes to the developmental appearance of conducting secretory K+ channels in the CCD.

Carbonic anhydrase (CA) IV activity facilitates renal acidification by catalyzing the dehydration of luminal carbonic acid. CA IV has been localized to the proximal tubules and medullary collecting ducts. Maturation of CA IV expression has been considered to be important in the development of renal acid excretion. The purpose of the present study was to determine the maturational expression of CA IV in rabbit kidney. A guinea pig polyclonal antibody to purified rabbit lung microsomal membrane CA IV was generated. Immunoblotting of membrane proteins after peptide-N-glycosidase F treatment revealed two N-glycosylation sites and reduction in size from approximately 52 to 35 kDa; there appeared to be heavier glycosylation in the medulla. In membrane and total proteins from the kidney cortex, CA IV was 15-30% of the adult level during the first 2 wk of life but increased to mature levels by 5 wk of age. The maturational pattern in the cortex was confirmed by measuring SDS-resistant CA hydratase activity. In the medulla, both membrane and total proteins were generally less than one-fourth of the adult level of CA IV during the first 2 wk of life before reaching mature levels by 5 wk of age. Immunohistochemistry showed staining in proximal tubules (apical > basolateral), with maximal label in the S2 segment. CA IV also appeared on the apical membranes of a minority cell type of the cortical collecting duct, presumably the alpha-intercalated cell. Several labeled cells also appeared to be the process of being extruded from medullary collecting ducts of 1- to 2-wk rabbits. The antibody did not reliably detect medullary CA IV expression in sections from mature rabbits. These studies indicate that there is a substantial postnatal increase in expression of CA IV in the maturing kidney in both the cortex and medulla. The disappearance of intercalated cells in the maturing rabbit medullary collecting duct may be part of a normal renal developmental program as previously reported [J. Kim, J.-H. Cha, C. C. Tisher, and K. M. Madsen. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F575-F592, 1996]. It is likely that the maturation of CA IV expression contributes to the increase in renal acidification observed early in postnatal life.

This study examines the ability of dietary lipids to regulate gene expression of mitochondrial and peroxisomal fatty acid beta-oxidation enzymes in the kidney cortex and medulla of 3-wk-old rats and evaluates the role of glucagon or of the alpha-isoform of peroxisome proliferator-activated receptor (PPARalpha) in mediating beta-oxidation enzyme gene regulation in the immature kidney. The long-chain (LCAD) and medium-chain acyl-CoA dehydrogenases (MCAD) and acyl-CoA oxidase (ACO) mRNA levels were found coordinately upregulated in renal cortex, but not in medulla, of pups weaned on a high-fat diet from day 16 to 21. Further results establish that switching pups from a low- to a high-fat diet for only 1 day was sufficient to induce large increases in cortical LCAD, MCAD, and ACO mRNA levels, and gavage experiments show that this upregulation of beta-oxidation gene expression is initiated within 6 h following lipid ingestion. Treatment of pups with clofibrate, a PPARalpha agonist, demonstrated that PPARalpha can mediate regulation of cortical beta-oxidation enzyme gene expression, whereas glucagon was found ineffective. Thus dietary lipids physiologically regulate gene expression of mitochondrial and peroxisomal beta-oxidation enzymes in the renal cortex of suckling pups, and this might involve PPARalpha-mediated mechanisms.

The neonatal proximal tubule has a lower rate of bicarbonate absorption than that of adults. This is due, in part, to a lower rate of apical membrane Na+/H+ antiporter activity. The purpose of these studies was to examine if thyroid hormone could be a factor in the maturational increase in Na+/H+ antiporter activity. Hypothyroid (0.01% propylthiouracil in drinking water starting at day 14 gestation and throughout the postnatal period), euthyroid, and hyperthyroid (intraperitoneal triiodothyronine, 10 micrograms/100 g body wt, once daily on days 17 to 20 of postnatal life) rats were all studied at 21 days of life. Renal cortical brush border Na+/H+ antiporter activity was 453 +/- 24, 527 +/- 30 and 608 +/- 25 pmol/mg protein in the hypothyroid, euthyroid and hyperthyroid groups, respectively (P < 0.001). Hyperthyroid neonates had approximately twofold greater renal cortical NHE-3 mRNA abundance than euthyroid and hypothyroid neonates (P < 0.05). Brush border membrane NHE-3 protein abundance in hypothyroid and hyperthyroid neonates was one-third and twofold that of euthyroid 21-day-old rats, respectively (P < 0.001). These data are consistent with a potential role of thyroid hormone in the postnatal increase in Na+/H+ antiporter activity.

The stromal cells in the renal cortex and medulla of adult rats reveal different phenotypes. Cortical peritubular fibroblasts are ecto-5'nucleotidase (5'NT)-positive and lack alpha-smooth muscle actin (alphaSMA) and vimentin immunoreactivity, whereas medullary fibroblasts are 5'NT-negative and vimentin-positive. We have studied by immunohistochemistry the postnatal (neonatal up to 8 weeks) development of renal cortical stromal cells with respect to 5'NT and to the cytoskeletal proteins alphaSMA and vimentin. Both alphaSMA and vimentin are characteristic for the renal myofibroblasts that replace stromal fibroblasts in interstitial nephritis. In new-born and 1-week-old rats, stromal cells in the cortex and medulla display alphaSMA and vimentin, but lack 5'NT. During the second postnatal week, alphaSMA and vimentin immunoreactivity in cortical interstitial cells gradually declines, whereas 5'NT reactivity becomes progressively apparent between the convoluted tubules in the juxtamedullary labyrinth. For a short time, all three proteins are found to be coexpressed in the same cells. At the end of the third week, interstitial 5'NT-immunoreactivity becomes evident also in the superficial cortical labyrinth, and alphaSMA and vimentin are no longer detectable in cortical peritubular cells. From the fourth week on, the distribution pattern and phenotype of 5'NT-positive cortical fibroblasts correspond to that in adult rats. The temporal pattern of maturation of cortical peritubular fibroblasts seems to parallel the functional maturation of cortical tubules. It is suggested that the local phenotype of peritubular fibroblasts in healthy and possibly also in injured kidneys may be controlled, at least in part, by the local tubular environment, conditioned by tubular metabolism and function.

The inducible second isoform of cyclooxygenase (COX-2) that mediates inflammation also is expressed at low levels in normal adult rat kidneys and is upregulated in response to noninflammatory stimuli (R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer, J. Clin. Invest. 94: 2504-2510, 1994). Roles in morphogenesis are indicated by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2 knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B. D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M. Czerniak, A. G. Stewart, and J. M. Trzaskos, Nature 378: 406-409, 1995; S. G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O. Smithies. Cell 83: 473-482, 1995). Blots from developing rat kidneys demonstrated that COX-2 mRNA and immunoreactive protein were present in neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult levels by the 3rd month. Immunolocalization and in situ hybridization detected intense COX-2 immunoreactivity and mRNA in a subset of thick ascending limb epithelial cells near the macula densa in each developing nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that COX-2 expression is subject to normal developmental regulation and can be sustained over extended periods; they also support the conclusion that metabolites of COX-2 play important roles in the differentiation and early functions of mammalian nephrons.

The mRNA that encodes for NaPi-2, the renal Na(+)-Pi cotransporter that is upregulated by Pi depletion in the adult rat, is low in the young animal. Yet, renal Na-Pi cotransport rates are higher in rapidly growing than in fully grown rats. The aim of this study was to unravel the molecular basis of this apparent discrepancy. Poly(A) RNA obtained from the renal cortex of young animals induced higher rates of Na(+)-Pi cotransport in oocytes than equal amounts of poly(A) mRNA obtained from the renal cortex of mature rats. Moreover, poly(A) RNA obtained from renal cortex of rapidly growing animals treated with antisense NaPi-2 oligomers or depleted of NaPi-2 transcripts by subtractive hybridization with cDNA generated from the renal cortex of adult rats retained its ability to induce Na(+)-Pi cotransport in oocytes. In addition, renal poly(A) RNA of the young subjected to subtraction hybridization generated a 379-base pair reverse transcriptase-polymerase chain reaction product common to all known type II Na(+)-Pi cotransporters. These observations permit us to surmise that the high rates of Na(+)-Pi cotransport prevailing during growth are due, at least in part, to the expression of a specific mRNA that is only partially homologous to that of NaPi-2.

The activity and expression of adenylyl cyclases (AC) were examined in the adult rat renal cortex and medulla. Northern blot analysis and in situ hybridization demonstrated that AC-6 was the predominant isoform in the adult rat kidney, whereas AC-4, -5, and -9 had a lower expression. AC-4 expression was higher in the cortex, and AC-5 and AC-6 were higher in the medulla. AC-9 expression was at the same level in both regions. AC activity was high in the fetus and declined in the adult. At all stages, AC activity was sensitive to parathyroid hormone, whereas no stimulation by vasopressin and isoproterenol was found in the fetus and the neonate. AC-5 and AC-6 mRNAs increased at day 1 and then markedly decreased, paralleling the decline in AC activity. The mRNA of AC-4 did not change and that of AC-9 increased markedly until adult. In the homozygous Brattleboro rat kidney, the expression of all these isoforms was decreased.

Phosphates are necessary for proper skeletal growth and function, as well as for growth and development of cells. Phosphate repletion depends partly on the function of the renal sodium-phosphate (Na+/Pi) transport system that functions to recover filtered urinary phosphate. It has been suggested that in order to meet the higher phosphate requirement of the developing animal, the weanling rat would have a greater adaptive response to chronic phosphate deprivation than the adolescent rat. The current study sought to characterize the adaptive response to dietary phosphate deprivation in terms of Na+/Pi transporter activity, and mRNA and immunoreactive protein levels. Weanling and adolescent rats were pair fed either a low-phosphate diet (LPD) or a control-phosphate diet (CPD) for 1 week. Maximal rates of transport (Vmax) were not different in weanling or adolescent rats on CPD (weanling 2.13 +/- 0.29 nmol/mg protein/10 sec, and adolescent 1.41 +/- 0.036 nmol/mg protein/10 sec, n = 3). K(m) values were not different in either group on CPD (weanling 0.15 +/- 0.08 mM Pi, and adolescent 0.22 +/- 0.13 mM Pi). There were no difference in mRNA abundance (Na+/Pi transporter/1B15 = 0.194 +/- 0.12 for weanling and 0.230 +/- 0.03 for adolescents, n = 3) or immunoreactive protein levels (Na+/Pi transporter/beta-actin = 0.232 +/- 0.01 for weanlings and 0.300 +/- 0.05 for adolescents, n = 3) in the two groups when fed CPD. After chronic Pi deprivation, the weanling rat showed a greater adaptive response than the adolescent as measured by Vmax values (weanling LPD/CPD = 2.01, P < 0.01; adolescent LPD/CPD not different; n = 3), mRNA signal intensity (weanling LPD/CPD = 1.86, P < 0.05; adolescent LPD/CPD not different; n = 3), and protein signal intensity (weanling LPD/CPD = 3.63, P < 0.01, and adolescent LPD/CPD 1.91, P < 0.05; n = 3). K(m) values were not affected by LPD. Immunohistochemical analysis of kidney cortex showed greater apical staining in both groups on LPD, with the increase being noticeably greater in the weanlings. Furthermore, two-way analysis of variance demonstrates a significant adaptive response in the weanling period in regard to maximum transport capacity (Vmax) and immunoreactive protein (Western), suggesting a synergistic effect between the developmental stage and low-phosphate diet. Therefore, it appears that the adaptive response is greater in the more rapidly developing animal (the weanling), and these results suggest a compensatory mechanism to conserve phosphate during periods of rapid growth.

Childhood pyelonephritis is a common cause of renal cortical scarring and hypoplastic kidneys. To understand the mechanisms underlying the cortical lesions, urinary tract infection was induced in three-week-old rats by an intravesical infusion of E. coli, type 06 K13 HL a rat nephropathogenic strain. Four days after infection, histopathological examination showed marked infiltration of leukocytes in the medullary tissue adjoining the calyces and pelvis. In the cortex, signs of inflammation were found only in the cortical zone adjacent to the pelvis. No cells indicative of inflammation were observed in other parts of the cortex. Immunohistochemistry for endogenous proliferating cell nuclear antigen (PCNA) demonstrated a marked decrease in immunoreactivity in proximal tubular (PT) cells. The mitotic response of PT cells, assessed by 3H-thymidine autoradiography, showed a highly significant decrease during the first four days after induction of the infection. Four days after infection, a transient increase in apoptotic cells was observed in cortical cells outside the inflammatory areas. No increase in apoptotic cells was detected in the cortex 10 days after infection. Only a few apoptotic cells were detected in the control kidneys. In conclusion, the data indicate that inhibition of cell proliferation and enhancement of apoptosis may contribute to the renal parenchymal loss after childhood pyelonephritis.

The effects of long-term administration of l-desamino-D-arginine vasopressin (dDAVP) on the macromorphology of the kidneys were studied in young growing lambs. Subcutaneous injections of dDAVP were started two weeks after birth and lasted for 13 weeks. Morphometric measurements showed that the volume of the cortex was reduced and both the height and volume of medulla were significantly increased in the dDAVP-treated lambs. The increase in the size of the medulla was mainly due to changes to the dimensions of the outer medulla. The surface area of the renal pelvis was enlarged by 29 per cent in the dDAVP-treated group. The mean (SEM) surface area of the renal pelvis of 15-week-old lambs was 5957 (365) mm2. These changes induced by an increased supply of vasopressin could have a beneficial effect on the recycling of urea and the concentrating ability of the kidneys.

Net K+ secretion is not detected in cortical collecting ducts (CCDs) isolated from newborn rabbits and perfused in vitro. To establish whether a low apical K+ permeability of the neonatal principal cell limits K+ secretion early in life, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to study the properties and density of conducting K+ channels in principal cells. With KCl in the pipette and a NaCl solution warmed to 37 degrees C in the bath, inward currents with a conductance of approximately 42 pS were observed in 0% (0 out of 13 or 0/13), 10% (2/21), 18% (5/28), 29% (4/14), and 56% (10/18) of cell-attached patches obtained in 1-, 2-, 3-, 4-, and 5-wk-old animals, respectively. The conductance and reversal potential of this channel led us to suspect that it represented the low-conductance K+ channel previously described in the rat CCD by L. G. Palmer, L. Antonian, and G. Frindt (J. Gen. Physiol. 104: 693-710, 1994). The mean number of open channels per patch (NPo) increased progressively (P < 0.05) after birth, from 0 at 1 wk, to 0.06 +/- 0.04 at 2 wk, to 0.40 +/- 0.18 at 3 wk, to 0.74 +/- 0.41 at 4 wk, and to 1.06 +/- 0.28 at 5 wk. The increase in NPo appeared to be due primarily to a developmental increase in N, which is the number of channels; open probability, Po, remained constant at approximately 0.5 for all channels identified after the 2nd wk of life. The increase in number of conducting K+ channels during postnatal life is likely to contribute to the maturational increase in net K+ secretion in the CCDs.

1. Renin gene expression was investigated in kidneys from 13 foetal and four adult sheep. 2. The levels of renal renin mRNA in foetuses were greater than those in adult sheep (P < 0.001). 3. There was no significant difference in renin mRNA levels between foetuses aged 91 and 134 days (term 150 days). 4. The levels of renin gene expression were higher (178 +/- 5 units; P < 0.001) in foetuses at 142 days than levels in 91 (144 +/- 4 units) and 134 days old foetuses (146 +/- 6 units). 5. It is concluded that the high level of renin gene expression in the foetal kidney is responsible for the high levels of renin in the foetal circulation.

1. Chronic angiotensin converting enzyme (ACE) inhibition or AT1 antagonism during postnatal development in the rat has been shown to cause renal tubular and vascular damage, particularly in the outer medulla. 2. The effects of ACE inhibition were investigated at a stage of development before the renal outer medulla is fully established. 3. Sprague-Dawley rat pups were given daily i.p. injections of either enalapril or saline from days 3-10. At day 11, kidneys were perfusion-fixed for either electron microscopy or immunocytochemistry. Sections were incubated in proliferating cell nuclear antigen (PCNA) antisera and the avidin-biotin immunoperoxidase method was used to detect an immunoreactive product, indicative of proliferating cells. 4. Following enalapril treatment, the normal structural arrangement of the outer medulla was disrupted compared with controls. Cell proliferation (PCNA-positive cells) in the medullary rays was reduced in enalapril-treated kidneys compared with control kidneys. 5. Thus, angiotensin II appears to be essential for normal tubular and vascular growth in postnatal renal development in the rat.

Mitochondrial fatty acid beta-oxidation plays a major role in providing the ATP required for reabsorptive processes in the adult rat kidney. However, the molecular mechanisms and signals involved in induction of the enzymes of fatty acid oxidation during development in this and other organs are unknown. We therefore studied the changes in the steady-state levels of mRNA encoding medium-chain acyl-CoA dehydrogenase (MCAD), which catalyses the initial step in mitochondrial fatty acid beta-oxidation, in the rat kidney cortex and medulla between postnatal days 10 and 30. Furthermore, we investigated whether the expression of MCAD and of mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid cycle, might be co-ordinately regulated by circulating glucocorticoids in the immature kidney during development. In the cortex, the levels of MCAD mRNA rose 4-fold between day 10 and day 21, and then decreased from day 21 to day 30. In the medulla a postnatal increase in the concentration of MCAD mRNA (8-fold) was observed during the same period. Adrenalectomy prevented the 16-21-day developmental increases in MCAD and mMDH mRNA levels in the cortex and medulla; these could be restored by dexamethasone treatment. A single injection of dexamethasone into 10-day-old rats led to a rise in MCAD and mMDH mRNA levels in the renal cortex due to stimulation of gene transcription, as shown by nuclear run-on assays. Therefore MCAD and mMDH gene expression is tightly regulated at the transcriptional level by developmental changes in circulating glucocorticoid levels. These hormones might thus represent a good candidate as a co-ordinating factor in the expression of nuclear genes encoding mitochondrial enzymes in the kidney during postnatal development.

We have previously demonstrated that the retention of phosphate required for growth is due to a a high Vmax of the Na(+)-Pi cotransport system located in the brush border membrane of the proximal tubule. Because of this and other similarities between adaptation of the kidney to a high Pi demand (growth) and that to low Pi supply, we measured the levels of NaPi-2 mRNA and cDNA present in kidney cortex of 3- and > 12-week-old rats. Like in Pi depletion, Western blots revealed that a 80 to 85 kDa protein recognized by a polyclonal antibody directed against the N-terminal region of the NaPi-2 protein was 2.3-fold more abundant in renal microvilli of the young than of adult animals. However, unlike in Pi depletion, Northern blot analysis failed to reveal a significant difference between mRNA levels at the two ages. Furthermore, suppression of NaPi-2 mRNA activity by annealing with antisense oligomers, or removal of the NaPi-2 transcripts by subtractive hybridization did not affect the rate of Na(+)-Pi cotransport induced in oocytes by polyA RNA of rapidly growing animals, while abolishing the ability of the renal cortical polyA RNA of adult rats to encode for Na(+)-Pi cotransport. RT-PCR of subtracted polyA RNA using primers specific for a region conserved in NaPi type II (Pi modulated) cotransporters yielded a product that was 98% homologous with that region, despite the absence of NaPi-2 cDNA. The results of these experiments demonstrate that the polyA RNA from kidneys of young animals contains unique mRNA transcripts able to encode for a NaPi protein homologous to, but distinct from NaPi-2.

Elevated levels of endogenous angiotensin can cause hypertensive nephrosclerosis as a result of the potent vasopressor action of the peptide. We have produced by gene targeting mice homozygous for a null mutation in the angiotensinogen gene (Atg-1-). Postnatally, Atg-1- animals show a modest delay in glomerular maturation. Although Atg-1- animals are hypotensive by 7 wk of age, they develop, by 3 wk of age, pronounced lesions in the renal cortex, similar to those of hypertensive nephrosclerosis. In addition, the papillae of homozygous mutant kidneys are reduced in size. These lesions are accompanied by local up-regulation of PDGF-B and TGF-beta1 mRNA in the cortex and down-regulation of PDGF-A mRNA in the papilla. The study demonstrates an important requirement for angiotensin in achieving and maintaining the normal morphology of the kidney. The mechanism through which angiotensin maintains the volume homeostasis in mammals includes promotion of the maturational growth of the papilla.

Immunolocalization studies of hamster kidney development were performed using polyclonal antibodies to antioxidant enzymes, including antibodies to copper, zinc and manganese superoxide dismutases, catalase, glutathione peroxidase and glutathione S-transferases and their subunits. Antibodies to extracellular matrix proteins were also studied to determine the temporal sequence between expression of immunoreactive protein for basement membrane proteins, which serve as markers of embryonic induction of nephron development, and antioxidant enzyme expression in kidney development. Immunoreactive proteins for antioxidant enzymes were not detectable in the developing kidney until after extracellular matrix proteins had been deposited. However, immunoreactive proteins for the antioxidant enzymes copper, zinc and manganese superoxide dismutases, catalase, and alpha class glutathione S-transferase Ya subunit were detected in renal tubules before birth. mu class glutathione S-transferase subunits Yb1 and Yb2 stained transitional epithelium at high levels before birth. Our results indicate: (1) each type of kidney cell has a unique antioxidant enzyme profile, (2) antioxidant enzymes are expressed in different types of cell at different times during development, but antioxidant enzyme immunoreactive protein was not present until after immunoreactive proteins for extracellular matrix molecules were detected, and (3) certain antioxidant enzymes are present before birth, indicating that high oxygen tension present at birth is not crucial for induction of immunoreactive protein.

The development of the renal vascular system requires the coordinated action of soluble morphogenic factors and specific extracellular matrix components. Despite intensive research it remains unknown whether the humoral or the environmental component is more important in the development of renal microvessels. The prolonged serum-free culture of embryonic kidney cortex explants was achieved by means of a newly developed perfusion culture system. This system made the investigation of renal vascular development under defined organotypic conditions possible. Thus, growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hormones (aldosterone, vitamin D3) could be applied without the interference with serum components. Medium supplementation with VEGF or aldosterone in combination with vitamin D3 resulted in the coordinated proliferation of endothelial cells in the explant. A well-developed collecting duct epithelium and numerous tubular structures were always observed. In contrast, only a uniform cell layer was found between fibrous organ capsule and the collecting duct epithelium after bFGF application, but neither tubular structures nor endothelial cells. Thus, the experiments indicate that bFGF alone has no stimulating effect on the growth of the renal microvasculature under perfusion culture conditions.

A major proportion of urinary dopamine derives from the renal decarboxylation of circulating dopa. This study evaluates the effects of aging on renal production of dopamine using 3- and 12-mo-old male Wistar rats. Urinary excretion of Na+, norepinephrine (NE), 3,4-dihydroxyphenylglycol, and dopa were similar in the two groups. Urinary dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were lower in older animals (dopamine, 20 +/- 6 vs. 47 +/- 7 nmol/24 h, P < 0.001; DOPAC, 142 +/- 36 vs. 304 +/- 56 nmol/24 h, P < 0.03). Urinary 3-O-methyldopa (OM-dopa) was higher in 12-mo-old rats (6.2 +/- 2.0 vs. 3.3 +/- 0.20 nmol/24 h, P < 0.03). Levels of dopa and NE in renal cortex from 12-mo-old rats were higher (P < 0.001) than in younger animals. Dopamine content in renal cortex from 3-mo-old rats was 295 +/- 64 pmol/g, whereas it was undetectable in 12-mo-old animals. Aromatic-L-amino-acid decarboxylase and monoamine oxidase activities were higher (P < 0.001) in renal cortex from 12-mo-old animals. Catechol-O-methyltransferase activity was similar in both groups. The uptake of dopa by the luminal membrane was explored using brush-border membrane vesicles. The Na(+)-gradient-driven (100 mM) uptake of dopa into vesicles from 3-mo-old animals showed at 10 s an overshoot threefold greater than the equilibrium uptake. The overshoot was blunted in 12-mo-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of NO synthase (NOS) in cells of the macula densa (MD) suggests a role for arginine-derived NO in tubulovascular information transfer. To investigate the postnatal development of the neuronal isoform of NOS and of renin in the kidney, the cellular distribution of these enzymes was examined in perfusion-fixed kidneys of 2-, 6-, and 15-day-old rats at both the protein and mRNA level (n = 4 rats/group). NOS and renin and their mRNAs were localized by immunohistochemical and in situ hybridization methods. In addition, NOS levels were assessed by using NADPH diaphorase (NADPH-d) histochemistry. For quantification, the fraction of NOS- and renin-positive glomeruli as well as the number of NOS-positive MD cells was evaluated at all stages. Presence of NOS in single cells of the developing distal tubule was encountered already in the S-shaped body. Full expression of a NOS signal in MD cells was seen as soon as a glomerular urinary space was developed. Double labeling with NADPH-d and antibody to Tamm-Horsfall protein (THP) indicated mutual exclusiveness of NADPH-d-positive MD cells and neighboring THP-positive distal tubule cells at all levels of development. The relative intensity of renin status was 2 day > 6 day > 15 day, whereas NOS expression was maximal on postnatal day 6. Our data are consistent with an involvement of MD NO synthesis in the early organization of the juxtaglomerular apparatus during nephrogenesis and suggest an interdependent relation with renin-producing cells.

The neonatal proximal tubule has a lower rate of bicarbonate absorption and Na+/H+ antiporter activity than the proximal tubule of adult animals. Two isoforms of the Na+/H+ antiporter have been localized to the proximal tubule. NHE3 is located on the apical membrane, whereas NHE1, the isoform found on most mammalian cells, is present on the basolateral membrane. The Na+/H+ antiporter isoforms that increase with renal maturation are unknown. The purpose of the present study was to examine the maturation of rabbit renal cortical NHE3 and NHE1 mRNA and protein abundance and to determine whether the rate of maturation of these isoforms was affected by glucocorticoids. Renal cortex from neonatal rabbits (1 wk) had approximately one-fourth the NHE3 mRNA and protein abundance as that from adult animals. Renal cortical NHE1 mRNA and protein abundance did not change significantly during maturation. Glucocorticoids have been shown to accelerate the maturation of neonatal bicarbonate absorption and apical membrane Na+/H+ antiporter activity. Daily subcutaneous administration of dexamethasone starting at 4 days of age (10 micrograms/100 g body wt) for 3 days and 2 h before being killed resulted in a twofold increase in NHE3 mRNA abundance and a threefold increase in NHE3 protein abundance. NHE1 mRNA and protein abundance were unaffected. These data show that there is selective maturation of NHE3 during renal cortical development, which can be accelerated by administration of glucocorticoids.