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

Department of Physiology and Pharmacology, University of Southern Denmark, Odense, Denmark

Submitted 23 April 2007 ; accepted in final form 13 June 2007

	ABSTRACT

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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.

aldosterone; mineralocorticoid; kidney; prostaglandin

In rats, plasma glucocorticoid exhibits a developmental nadir in the second postnatal week (9). Exposure to elevated glucocorticoid in this period leads to hypertension in the adult rat (21) and inhibits proliferation of the loop of Henle and the kidney outer medulla (27). Little is known about factors that initiate and maintain proliferation of loop cells at the outer medulla-cortex junction. Pharmacological and genetic approaches employed to inhibit or delete components of the renin-angiotensin-aldosterone system in the postnatal period have consistently shown a range of abnormalities with diminished kidney growth, decreased ability to concentrate urine, and pathological changes of the kidney medulla (6, 7, 14, 18, 20, 29, 30). In some of these experimental models, plasma aldosterone is suppressed (29) and mice deficient in aldosterone synthase (CYP11B2^–/–) share several phenotypic characteristics with kidneys from animals with interrupted ANG II signaling (14). These observations imply a point of convergence at the aldosterone-mineralocorticoid receptor (MR) pathway for renal phenotypic alterations. It is not known whether aldosterone is necessary for proliferation or apoptotic remodeling within the renal medulla and at which time during postnatal development such sensitivity is present. Mineralocorticoid might exert direct effects on the loop epithelium or influence proliferation and growth indirectly through effects on Na⁺ homeostasis. In this context, renal prostanoids appear particularly relevant. Mineralocorticoid suppresses cortical cyclooxygenase-2 (COX-2) expression in developing and adult kidney, whereas administration of AT₁-receptor blockers and MR antagonists stimulate renal COX-2 in the postnatal period (26, 31, 32). Intrarenal COX-2 is elevated and promotes NaCl wasting in NaCl-loosing tubulopathies (11, 19, 28). Whether enhanced COX-2 activity contributes to renal dysfunction in conditions with impaired postnatal mineralocorticoid signaling has not previously been analyzed. We hypothesized that 1) aldosterone promotes renal medullary development in the late postnatal phase after nephrogenesis by stimulating tubular cell proliferation and remodeling and that 2) aldosterone promotes development of urinary-concentrating ability by indirect effects on COX-2 activity.

These hypotheses were tested in rat pups subjected to adrenalectomy in the postnatal developmental "window," during which medullary proliferation peaks and nephron formation is complete [between postnatal days 10 and 20 (P10–P20)]. Pups were given differential replacement therapy by chronic implantation of pellets that contained gluco- and mineralocorticoid. In addition, short-term intervention with a COX-2 blocker was used to elucidate the role of COX-2 for impaired concentrating ability in adrenalectomized rats.

	MATERIALS AND METHODS

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Animal Surgery and Protocols

All procedures conformed to the Danish national guidelines for the care and handling of animals and the published guidelines from the National Institutes of Health. The Danish Animal Experiments Inspectorate under the Department of Justice approved all experimental procedures that involved the use of animals (no. 2003/561-761).

Female Sprague-Dawley rats had free access to standard pathogen-free rat chow (Altromin-1310, Lage, Germany) (2 g/kg Na⁺, 5 g/kg Cl^–) and tap water. In the first 24 h after birth, litters were reduced to 10 to ensure equal feeding with five males and females in each litter, if possible. The dams and pups were kept at a 12:12-h light-dark cycle. At P10, pups were anesthetized with ketamine (25 mg/kg) and xylazine (5 mg/kg), and bilateral adrenalectomy (ADX) was performed through two lumbodorsal incisions. After surgery, rats were divided into four groups: 1) sham group, which underwent a surgical protocol without removal of adrenals [a pellet (50 mg cholesterol) was inserted in the subcutis]; 2) ADX group, which did not have corticosteroid substitution and where a 50-mg pure cholesterol pellet was inserted. In pilot experiments, most pups in this group died before P20. Therefore, we adopted a protocol shown to sustain survival in MR^–/– mice where pups were injected subcutaneously with isotonic NaCl in an amount corresponding to urinary Na⁺ losses (2). The final two groups were as follows: 3) ADX with corticosterone replacement only (ADX-C), which had a pellet (50 mg) with 12.5% corticosterone inserted in the subcutis; and 4) ADX with corticosterone and mineralocorticoid (DOCA) substitution (ADX-CD), which had a pellet (50 mg) with 12.5% corticosterone and 12.5% DOCA inserted. Pellets were manufactured as described by Meyer et al. (15) for adult rats and reduced from a 100-mg to a 50-mg size to adjust for different body sizes and to maintain corticosterone at a level characteristic for the second postnatal week in the rat (9). Pellets were "sterilized" by heating for 2 h at 105°C. Pellets were stored at 4°C for a maximum of 2 wk.

Experimental Series

Series 1. Spot urine was collected at day 20 from the four experimental groups, and mixed trunk blood was sampled in EDTA-coated or heparin-coated vials after decapitation. Kidneys were dissected in outer medulla-cortex and inner medulla, snap frozen in liquid nitrogen, and stored at –80°C.

Series 2. To determine urinary-concentrating ability, the dam was removed from the pups between P19 and P20 for 20 h. Spot urine was collected at decapitation, and mixed trunk blood was sampled. Kidneys were dissected in outer medulla-cortex and inner medulla and then frozen in liquid nitrogen and stored at –80°C.

Series 3. Pups from the four groups were anesthetized at P20 as described above. Kidneys were fixed by systemic perfusion through the left cardiac ventricle with 4% phosphate-buffered paraformaldehyde for 4 min. Coronal tissue blocks were processed for immunohistochemistry as described (27).

Series 4. Four litters were adrenalectomized at P10 and only supplemented with isotonic NaCl injections. Pups were given subcutaneous injections of a COX-2-selective antagonist (parecoxib; Dynastat; Merck), at 5 mg·kg^–1·day^–1 divided in two daily doses (2.5 µg/g in 5 µl/g) from P17 to P20. The pups were then fluid deprived for 20 h. Spot urine was collected, and pups were decapitated. Plasma, urine, and kidneys were sampled. Inner medulla and cortex-outer medulla were dissected out and rapidly frozen.

Plasma and Tissue Electrolyte Concentrations

In initial experiments, papillas were homogenized in pure water and then centrifuged 10 min at 13.000 g, and supernatants were used for measurements. In subsequent experiments, osmolytes were quantitatively extracted as described (27). Osmolality was analyzed by freeze-point depression (Osmomat 030-D, Gonotec, Bie & Berntsen). Na⁺ and K⁺ concentrations were determined in plasma and renal medullary interstitial fluid by flame photometry (model IL 943; Instrumentation Laboratory, Lexington, MA). Urea was determined kinetically as the amount of NADH consumed over time measured spectrometrically (UV) after hydrolysis of urea by urase and the formation of L-glutamate by glutamate dehydrogenase (ABX Pentra Urea CP, ABX Diagnostics).

Hormone Analyses

Plasma aldosterone was measured in all pups from series 1, 2, and 4 as a control of complete removal of all adrenal tissue with a commercial kit (COAT-A-COUNT; Diagnostic Products, Los Angeles, CA). The detection limit was 13.0 pg/ml, and the intra-assay coefficient of variation was <4%. Plasma total corticosterone was measured with the radioimmunoassay kit from Amersham's Biotrak series, using ¹²⁵I-labeled corticosterone as a tracer. Plasma renin concentration was analyzed as described (22). For tissue PGE₂, samples were homogenized as described for Western blotting, and 250 µl of homogenate were acidified with 18 µl of 50% formic acid. Samples were extracted with 1 ml ethylacetate-hexane (70/30) by vortexing and subsequent centrifugation. The supernatants were vacuum dried and dissolved in 250 µl of assay buffer. PGE₂ was determined by ELISA (Assay Designs).

Messenger RNA Analyses

Isolation of RNA and quantitative RT-PCR were performed as described previously (27). A standard curve was constructed by plotting threshold cycle against serial dilutions of linearized plasmids. A subset of quantitative RT-PCR data sets was validated by subsequent ribonuclease protection assay as previously described (27).

Western Blotting

Tissue samples were homogenized in buffer as previously described in detail (27). Protein concentration determination, gel electrophoresis, electroblotting, and chemiluminescence detection were done as previously described (27). Densitometry was evaluated by QuantityOne software (BioRad). Primary antibodies used were as follows: COX-2 (1:2,000, M-19, Santa Cruz); Na⁺-K⁺-ATPase ₁ (1:2,000, Upstate); proliferating cell nuclear antigen (PCNA; 1:2,000, Santa Cruz); and sodium-potassium-2-chloride cotransporter (NKCC2; 1:2,000, Chemicon).

Morphometry and Immunohistochemistry

Fixation and paraffin embedding for immunochemistry was as described previously (27). Primary antibodies for immunohistochemistry were as follows: COX-2 (1:1,000, M-19, Santa Cruz); NKCC2 (1:1,000, Chemicon); the potassium channel ROMK (1:500, Alomone Labs); Na⁺-K⁺-ATPase ₁ (1:100, Upstate); PCNA (1:100, Santa Cruz); and Tamm-Horsfall glycoprotein (1:200, Biotrend). For morphometric analysis, coronal sections through the tip of papilla were labeled for Tamm-Horsfall glycoprotein to allow precise delineation of kidney outer medulla and inner medulla. Juxtamedullary glomeruli defined the cortical-outer medulla junction in morphometric analysis. Measurement of kidney dimensions was done with Image Tool version 3.0 (National Institutes of Health) as described (27).

Statistics

If data were not normally distributed, they were log-transformed and tested for normality. Statistical significance between several groups was evaluated by one-way ANOVA followed by the Tukey-Kramer multiple comparisons test. Unpaired Student's t-test was used when two groups were evaluated. If data were not normally distributed or variance was not homogenous, a nonparametric Mann-Whitney test was used to test for significant differences between two groups, and differences between more than two groups were estimated by the Kruskal-Wallis test followed by Dunn's multiple comparisons test. P values <0.05 were considered significant. Arithmetic means are shown as ± SE; for log-transformed data, geometric means are shown. All calculations were performed with GraphPad Prism InStat software (version 3.0; GraphPad, San Diego, CA).

	RESULTS

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Effect of Adrenalectomy on Body and Kidney Growth and Plasma Hormone and Electrolyte Concentrations

Plasma aldosterone concentration was 87 ± 22 pg/ml in sham-operated rats at P20 (n = 13) and was below the detection limit after adrenalectomy in all included rats (not shown; assay limit of 11 pg/ml, n = 7–9 animals in each group). In control pups not subjected to surgery, plasma corticosterone increased 10 times between P10 and weaning at P20 (13.4 ± 1.3 ng/ml vs. 128.7 ± 28 ng/ml; n = 5 in both groups), which was not different from plasma corticosterone in sham-operated pups at P20 (137 ± 30 ng/ml; n = 13). In the ADX group that received no steroid replacement, corticosterone was detectable in plasma at P20, but the developmental surge was prevented (Table 1). In ADX-C pups, plasma corticosterone was not significantly different from sham animals at P20 (Table 1). In ADX-CD pups, plasma corticosterone was significantly lower than in sham animals (Table 1).

In ADX pups supplemented with NaCl and in ADX-C pups, plasma renin concentration was significantly elevated compared with sham and ADX-CD groups (Table 1). Kidney renin mRNA level was significantly elevated in the ADX group compared with sham and ADX-CD, whereas, in ADX-C pups, renin mRNA was not different from sham (Table 3).

View larger version (89K): [in this window] [in a new window]   Fig. 1. A: kidneys from the 4 experimental groups of pups were sectioned in the frontal plane. Pups (n = 5 in each group) were substituted with cholesterol pellets with no steroid [with sham operation (sham) or with adrenalectomy (ADX)], with 12.5% corticosterone (ADX-C), and with 12.5% corticosterone and 12.5% DOCA (ADX-CD). B: compound pictures show low-power magnification micrographs of histological sections of pup kidneys from the 4 experimental groups at postnatal day 20 (P20). Sections are stained with hematoxylin. Bars = 500 µm. C: micrographs show histological sections of kidneys from the 4 experimental groups at P20. Sections are reacted with a proliferating cell nuclear antigen (PCNA)-specific antibody by histochemical reaction. D, left: result of Western blotting experiment using whole kidney homogenate from sham and nonsubstituted ADX rat pups at P20 reacted with a specific antibody for PCNA. D, right: result of densitometric evaluation of the film. OD, optical density.

In the ADX group, COX-2 mRNA and protein abundances in kidney cortex-outer medulla fraction were significantly higher than those shown in sham animals (Fig. 2A). Corticosterone replacement did not decrease kidney COX-2 mRNA or protein levels compared with ADX animals. For the ADX-CDgroup, renal COX-2 mRNA and protein levels were similar to those for sham animals (Fig. 2A). Tissue concentration of PGE₂ in cortex-outer medulla tissue from ADX-CD rats was about four times lower than that shown in ADX pups [20.7 ± 8.5 pg/mg protein (n = 6) vs. 84.7 ± 9.1 pg/mg protein (P < 0.05) (n = 6)]. Immunohistochemical labeling of kidney tissue showed that COX-2 signal was localized predominantly in cortex in all four experimental groups (Fig. 2B). COX-2 was not readily detectable in the medulla, and visualization required amplification. COX-2 was associated with inner medullary interstitial cells (not shown). Labeling of kidney tissue from ADX rats for COX-2 yielded more intense immunoreactivity and more widely distributed signal than shown for sham and ADX-CD kidneys (Fig. 2B). Kidney sections from ADX-C rats also displayed more widely distributed COX-2-immunoreactive protein than shown in sham and ADX-CD animals (Fig. 2B). At high-power magnification, COX-2-positive signals were associated with the cytoplasm in the majority of loop of Henle cells in the kidney cortex of ADX rats (Fig. 2B, bottom). In contrast, COX-2-immunoreactive signal from ADX-CD rat kidney was more faint and detectable only in a minority of loop of Henle cells (Fig. 2B, bottom).

View larger version (90K): [in this window] [in a new window]   Fig. 2. A: cyclooxygenase-2 (COX-2) abundance in rat pup kidneys at P20 at the level of mRNA (left) and protein (right). Pups were sham operated (sham) or adrenalectomized at postnatal day 10 (P10) and substituted with cholesterol pellets with no steroid (sham and ADX), with 12.5% corticosterone (ADX-C), and with 12.5% corticosterone and 12.5% DOCA (ADX-CD); n = 5 or 6 in each group in the quantitative RT-PCR experiments and n = 4 in the Western blotting experiments. *P < 0.05. B, top: micrographs of histological sections of kidneys at P20 in sham or ADX groups at P10 that are reacted with a COX-2-specific antibody by histochemical reaction. Bars = 200 µm. B, bottom: high-power micrographs of kidney sections labeled for COX-2 from an ADX rat (left) and ADX-CD rat (right). Bars = 50 µm.

In normohydrated pups, there were no significant differences in urine osmolality between the four experimental groups at P20 (Table 1). There was a tendency for lower plasma Na⁺ concentration ([Na⁺]) in ADX and ADX-C groups, but this was not statistically significant; however, plasma [Na⁺] in ADX-CD rats was significantly elevated compared with that shown for ADX-C rats (Table 1). In response to fluid deprivation, plasma [Na⁺] was significantly lower in ADX-C and ADX pups than in sham rats (Table 1). In the ADX-CD group, plasma [Na⁺] was significantly elevated compared with that shown for all other groups (Table 1). Fluid deprivation increased urine osmolality in sham-operated rats compared with normohydrated sham-operated pups (Table 1). In ADX and ADX-C groups, urine-concentrating ability was significantly lower than that shown for sham rats (Table 1). In response to fluid deprivation, urine osmolality in ADX and ADX-C rats did not increase significantly compared with normohydrated counterparts (Table 1). In the ADX-CD group, urinary-concentrating ability was not different from that shown in sham animals and significantly higher than that shown in ADX and ADX-C groups (Table 1). Osmolality and [Na⁺] in papillary tissue interstitial fluid changed similarly to urine-concentrating ability; these levels were significantly reduced in ADX and ADX-C groups compared with sham, whereas levels in ADX-CD rats were not significantly different from those in sham animals (Table 1). NaCl transport molecule expression was determined in cortex-outer medulla tissue fraction. Adrenalectomy with or without gluco- and mineralocorticosteroid substitution did not significantly alter mRNA abundances of the investigated NaCl transport molecules [NKCC2, ROMK, Na⁺-K⁺-ATPase ₁-subunit, renal Na⁺/H⁺ exchanger (NHE3), and -epithelial Na⁺ channel] and 11-hydroxysteroid dehydrogenase-2 as determined by quantitative RT-PCR (Table 3). Ribonuclease protection assays for NKCC2 and Na⁺-K⁺-ATPase ₁-subunit mRNAs corroborated the results obtained by quantitative RT-PCR (Fig. 3A). Western blotting experiments showed a lower abundance of Na⁺-K⁺-ATPase ₁-subunit protein in kidney tissue from ADX group (Fig. 3B), whereas NKCC2 was not changed (not shown). Immunohistochemical staining for NKCC2 (Fig. 3C) and ROMK (not shown) confirmed the pathological change with a thinner outer medulla but did not reveal any appreciable differences in tissue distribution or labeling intensity of immunoreactive proteins. NKCC2 was associated with the loops of Henle in outer medulla and in cortical medullary rays (Fig. 3C). Labeling intensity was reduced for Na⁺-K⁺-ATPase ₁-subunit in ADX rats compared with the other groups (Fig. 3C). Immunoreactive signal for Na⁺-K⁺-ATPase was associated with the basolateral aspect of epithelial cells in the distal convoluted and connecting tubules in cortex and loop of Henle cells in the outer medulla (Fig. 3C). There were no obvious differences in the distribution pattern and staining intensity between control, ADX-C, and ADX-CD groups (Fig. 3C).

View larger version (76K): [in this window] [in a new window]   Fig. 3. A, top: result of ribonuclease protection assays for NKCC2, Na+-K+-ATPase 1-subunit (NaK-1), and -actin. Total RNA from kidneys of sham and nonsubstituted ADX pups at P20 was used for hybridization with specific radiolabeled probes. A, bottom: quantitative evaluation of the assays; n = 5 in each group. B, top: Western blotting experiment of whole kidney homogenate for Na+-K+-ATPase 1-subunit from sham, ADX, ADX-C, and ADX-CD rat pups. B, bottom: densitometric values (OD); n = 4 in each group. *P < 0.05. C: micrographs of histological sections of kidneys at P20 in sham and ADX groups (ADX, ADX-C, and ADX-CD) at P10 that are reacted with a NKCC2-specific antibody (top) and an Na+-K+-ATPase 1-subunit antibody (bottom) by histochemical reaction. Scale bars = 500 µm (top) and 50 µm (bottom).

At P17, ADX rats were divided into two groups: vehicle (weight: 33.5 ± 0.7 g, mean ± SE, n = 13) and parecoxib (weight: 34.1 ± 1 g, n = 14). Subcutaneous injection of the COX-2-selective antagonist parecoxib (5 mg·kg^–1·day^–1, P17-P20) enhanced weight gain significantly (Fig. 4A). The COX-2 inhibitor reduced weight loss after fluid deprivation for 20 h (Fig. 4A). In response to fluid deprivation, plasma osmolality and hematocrit increased significantly less in parecoxib-treated rats than in vehicle-treated ADX pups (Table 4). Parecoxib treatment significantly improved urinary-concentrating ability and augmented osmolality of papillary interstitial fluid (Fig. 4, B and C). The increase in osmolality of papillary interstitial fluid after COX-2 inhibition was achieved through significantly increased accumulation of Na⁺ and urea but not K⁺ (Table 4). The COX-2 inhibitor lowered plasma renin concentration significantly to a level 1/30 of that in vehicle-injected fluid-deprived pups (Fig. 4D).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Effect of a COX-2 inhibitor (parecoxib, 5 mg·kg–1·day–1) from P17 to P20 on weight change, urinary concentration ability, and plasma renin concentration in ADX rat pups at P10. No. of animals is shown in each column. A: relative body weight (BW) gain of ADX rats with and without COX-2 inhibition from P17 to P20 (left) and relative body weight loss after fluid deprivation for 20 h between P20 and P21 (right). *P < 0.05. B: effect of a COX-2 inhibitor on urine osmolality after fluid deprivation of ADX rats for 20 h between P20 and P21. *P < 0.05. C: effect of a COX-2 inhibitor on inner medullary tissue interstitial osmolality (geometric means) in ADX rats subjected to fluid deprivation for 20 h between P20 and P21. *P < 0.05. D: effect of a COX-2 inhibitor on plasma renin concentration (geometric means) in ADX, fluid-deprived pups (20 h, P20–P21). *P < 0.05.

	DISCUSSION

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The observations in the ADX and ADX-C groups that plasma [Na⁺] is not elevated and does not increase significantly after fluid deprivation indicate that rats suffer primarily from NaCl losses and subsequent water losses. The level of Na⁺-K⁺-ATPase ₁-subunit was lower in the ADX group, whereas levels of other NaCl transport proteins were not changed (ROMK, NHE3, NKCC2, and epithelial Na⁺ channel) in outer medulla-cortex tissue, consistent with findings in aldosterone synthase- and MR-deficient mice (1, 14). The data imply that the effect of corticosteroids on Na⁺ transport are not mediated predominantly at the level of transport protein abundance.

COX-2 activity contributed significantly to the NaCl-wasting phenotype elicited by absence of aldosterone in the postnatal period. Administration of a COX-2 inhibitor, parecoxib, increased body weight gain abruptly, which implies an effect through renal Na⁺ handling with subsequent expansion of extracellular volume. Moreover, in response to fluid deprivation, parecoxib attenuated the increases in hematocrit, plasma osmolality, plasma [Na⁺], and renin and improved the accumulation of Na⁺ and urea in the medullary interstitium. The improved interstitial hypertonicity achieved by COX-2 inhibition in the renal medulla is the most likely explanation for the improved urine-concentrating ability. This effect could be accomplished by inhibition of prostaglandin effects on the tubular epithelium and, indirectly, by an improved action of vasopressin and/or by decreased washout of solutes through a decrease in medullary perfusion. The primary deficiency in mineralocorticoid and the negative Na⁺ balance augments renal COX-2 activity, which further enhances renal Na⁺ loss. A similar mechanism is likely to be involved in renal dysfunction in aldosterone synthase-deficient mice, where intrarenal COX-2 expression is increased fivefold (14). Concentrating ability was improved significantly by short-term COX-2 inhibition (Fig. 4) but not reestablished to the level normally observed at weaning in adrenal-intact rats (2,000 mosmol/kgH₂O). Whether intervention with COX inhibitors beyond 3 days can rescue the medullary phenotype induced by absence of mineralocorticoid is not resolved by the present experiments. We chose a short period of intervention because COX-2 inhibition or gene deletion from birth by itself causes renal injury in rodents, primarily in the renal cortex (5, 12, 17).

As noted by Makhanova et al. (14), the medullary injury after mineralocorticoid deficiency bears a striking resemblance to the effect of ureteral obstruction during postnatal development. In accord, administration of angiotensin-converting enzyme inhibitors from birth leads to dilatation of tubules in the rat kidney medulla (13), and deletion of AT₁ receptors leads to increased intrapelvic pressure and impaired peristaltic propagation of urine in mice (16). In the present study, ANG II signaling was not impaired, but medullary damage was evident, suggesting a distinct role for aldosterone. The ADX pups are likely to exhibit polyuria similar to other situations with impaired renin-angiotensin signaling. This may directly lead to increased pelvic pressure as suggested to be the case also in Ren1C-deficient mice, where aldosterone concentration is strongly decreased (29). Extreme polyuria in the neonatal period as induced by furosemide or NKCC2 gene deletion causes more pronounced but basically similar pathological changes in the medulla (28). Supplementation with isotonic NaCl after adrenalectomy was necessary for survival of pups in the present study, but it further augments urine flow rate. The difference in fluid load presented to the kidney and urinary tract may explain why hydronephrosis was observed less consistently in kidneys from aldosterone synthase- and Ren1C-deficient mice (14, 29) compared with the present study and to NKCC2-deficient mice (28). Thus, to survive mineralocorticoid deficiency with a defect in urine-concentrating mechanism, pups must drink a volume that is likely to damage the kidney.

We conclude that mineralocorticoid is required for normal postnatal development of the renal medulla. Renal COX-2 activity contributes significantly to NaCl loss, extracellular volume depletion, and decreased urine-concentrating ability in postnatal mineralocorticoid deficiency. COX inhibitor administration seems an attractive approach to reduce renal Na⁺ and water losses in such conditions.

	GRANTS

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The present work received grant support from the Danish Research Council for Health and Disease (22040305), the NOVO Nordisk Foundation, The Danish Kidney Association Research Foundation, Helen and Ejnar Bjørnows foundation, A. J. Andersen og Hustrus Fond, Jens C. Christoffersens mindefond, and Else Poulsens mindefond.

	ACKNOWLEDGMENTS

 
The technical assistance of Mette Svendsen, Dina Dræby, Mette Fredenslund, Inge Andersen, and Lis Teusch is gratefully acknowledged.

	FOOTNOTES

 

Address for reprint requests and other correspondence: B. L. Jensen, Dept. of Physiology and Pharmacology, Univ. of Southern Denmark, Winsløwparken 21, 3, DK-5000 Odense C, Denmark (e-mail: bljensen{at}health.sdu.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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