Immunomorphometric study of rat renal inner medulla.
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We utilized immunofluorescent immunolabeling of renal tissue sections to identify and count tubules at specified depths of the rat renal inner medulla. We used primary antibodies to aquaporin-1 (AQP1; labeling thin descending limbs), aquaporin-2 (AQP2; labeling inner medullary collecting ducts), the rat kidney-specific chloride channel (ClC-K1; labeling thin ascending limbs), and von Willebrand factor (labeling descending vasa recta). Secondary antibodies conjugated to different fluorophores were used, giving up to a three-color display. Labeled structures were then identified and counted. At each level sampled in the inner medulla, many more thin limbs were labeled by ClC-K1 than AQP1. In addition, thin limbs were found to label with antibodies to ClC-K1 on both sides of their hairpin turns. We conclude that the descending thin limbs shift from expressing AQP1 to expressing ClC-K1 some distance before the point where they turn and begin to ascend. Mathematical models can use our quantitative data to explore implications for the urine-concentrating mechanism. (+info)
Posttranscriptional compensation for heterozygous disruption of the kidney-specific NaK2Cl cotransporter gene.
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Mice homozygous for a loss of function mutation of the kidney-specific NaK2Cl cotransporter, BSC1/NKCC2, do not survive. Here the effects of loss of one copy of the gene are studied. NKCC2 mRNA of NKCC2 +/- kidney was 55 +/- 6% of +/+, yet no differences were found between NKCC2 +/+ and +/- mice in BP, blood gas, electrolytes, creatinine, plasma renin concentration, urine volume and osmolality, ability to concentrate and dilute urine, and response to furosemide. When mice were challenged with 180 mM NH(4)Cl, plasma ammonia and urinary ammonia excretion were increased twofold and fivefold, respectively, but there was still no difference between the two genotypes. NKCC2 +/- mice had a near-normal level of NKCC2 protein and no clear change in the distribution of NKCC2 in the thick ascending limb (TAL) cells. In vitro microperfusion of isolated TAL showed no significant difference between the two genotypes in the basal and vasopressin-stimulated capacity to reabsorb NaCl. There was no difference in the mRNA expressions of thiazide-sensitive NaCl cotransporter, epithelial Na channel (ENaC), aquaporin-2, ROMK, and NaKATPase. Halving the mRNA expression of NKCC2 does not affect BP or fluid balance because of compensatory factors that restore the protein level to near normal. One possible factor is a regulated increase in the movement of cytoplasmic protein to the luminal membrane leading to a restoration of functional transporter to an essentially wild type level. (+info)
Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes.
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We reported increased water permeability and a low urea reflection coefficient in Xenopus oocytes expressing urea transporter UT-B (former name UT3), suggesting that water and urea share a common aqueous pathway (Yang, B., and Verkman, A. S. (1998) J. Biol. Chem. 273, 9369-9372). Although increased water permeability was confirmed in the Xenopus oocyte expression system, it has been argued (Sidoux-Walter, F., Lucien, N., Olives, B., Gobin, R., Rousselet, G., Kamsteeg, E. J., Ripoche, P., Deen, P. M., Cartron, J. P., and Bailly, P. (1999) J. Biol. Chem. 274, 30228-30235) that UT-B does not transport water when expressed at normal levels in mammalian cells such as erythrocytes. To quantify UT-B-mediated water transport, we generated double knockout mice lacking UT-B and the major erythrocyte water channel, aquaporin-1 (AQP1). The mice had reduced survival, retarded growth, and defective urinary concentrating ability. However, erythrocyte size and morphology were not affected. Stopped-flow light scattering measurements indicated erythrocyte osmotic water permeabilities (in cm/s x 0.01, 10 degrees C): 2.1 +/- 0.2 (wild-type mice), 2.1 +/- 0.05 (UT-B null), 0.19 +/- 0.02 (AQP1 null), and 0.045 +/- 0.009 (AQP1/UT-B null). The low water permeability found in AQP1/UT-B null erythrocytes was also seen after HgCl(2) treatment of UT-B null erythrocytes or phloretin treatment of AQP1 null erythrocytes. The apparent activation energy for UT-B-mediated water transport was low, <2 kcal/mol. Estimating 14,000 UT-B molecules per mouse erythrocyte, the UT-B-dependent P(f) of 0.15 x 10(-4) cm/s indicated a substantial single channel water permeability of UT-B of 7.5 x 10(-14) cm(3)/s, similar to that of AQP1. These results provide direct functional evidence for UT-B-facilitated water transport in erythrocytes and suggest that urea traverses an aqueous pore in the UT-B protein. (+info)
Structural-functional correlations in the kidneys and observations of colon and cloacal morphology in certain Australian birds.
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1. Variations in renal microstructure between the zebra finch and Senegal dove were consistent with their relative renal concentrating abilities (urine/plasma ratios of 2-8 and 1-7, respectively). Compared with dove kidneys, those of the finch contained a higher fraction of mammalian-type nephrons (with Henle's loops), and a lower fraction of reptilian-type nephrons (without loops). 2. Singing honeyeaters concentrated their urine almost as well as zebra finches, although honeyeater kidneys were less specialized (fewer mammalian-type nephrons). Such findings emphasize the need to clarify other osmoregulatory parameters. 3. No significant microstructural differences were found in the kidneys of domesticated as compared with those of wild zebra finches. Hence, osmoregulatory differences between tame and wild birds must be related to physiological factors rather than morphological. 4. Thickness of the renal medulla seemed to be directly correlated with urine concentrating ability. However, certain inconsistencies obscure this relationship such that its resolution will require further research. 5. Histological features of the mucosae of the colon and cloaca are described. The galah and kookaburra displayed a mammalian (non-villous) pattern of mucosal organization. Zebra finches, singing honeyeaters, and particularly emus, possessed colonic and cloacal villi and hence an increased surface area per volume in this region of the gut. This raises the possibility that the colon and cloaca are involved in uring concentration and osmoregulatory activities in these species. (+info)
Impaired urine concentration and absence of tissue ACE: involvement of medullary transport proteins.
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ACE.2 mice lack all tissue angiotensin-converting enzyme (ACE) but have 33% of normal plasma ACE activity. They exhibit the urine-concentrating defect and hyperkalemia present in mice that lack all ACE, but in contrast to the complete knockout, ACE.2 mice have normal medullary histology and creatinine clearance. To explore the urine-concentrating defect in ACE.2 mice, renal medullary transport proteins were analyzed using Western blot analysis. In the inner medulla, UT-A1, ClC-K1, and aquaporin-1 (AQP1) were significantly reduced to 28 +/- 5, 6 +/- 6, and 39 +/- 5% of the level in wild-type mice, respectively, whereas AQP2 and UT-B were unchanged. In the outer medulla, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2/BSC1) and AQP1 were significantly reduced to 56 +/- 11 and 29 +/- 6%, respectively, whereas Na(+)-K(+)-ATPase, UT-A2, UT-B, and AQP2 were unchanged, and renal outer medullary potassium channel was significantly increased to 711 +/- 187% of the level in wild-type mice. The abnormal expression of these transporters was similar in ACE.2 mice backcrossed onto a C57BL/6 or a Swiss background and was not rescued by ANG II infusion. We conclude that the urine-concentrating defect in ACE.2 mice is associated with, and may result from, downregulation of some or all of these key urea, salt, and water transport proteins. (+info)
A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 null mutant mice develop renal lesions mimicking obstructive nephropathy.
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BACKGROUND: A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 is distinguished from other a disintegrin and metalloproteinase molecules by the presence of thrombospondin type 1 motifs at its C-terminus and anchors to the extracellular matrix. We studied the biological role of ADAMTS-1 in the kidney. METHODS: We developed ADAMTS-1 null mice by replacing exons 2-4, which encode most of the metalloproteinase domain, with the neomycin resistance gene. RESULTS: In normal mice, ADAMTS-1 was detected in the epithelial cells of collecting ducts, and more intensely in the urinary epithelium at the ureteropelvic junction in kidney. We found that targeted disruption of the mouse ADAMTS-1 gene resulted in enlarged renal calices accompanied by bilateral hydronephrosis and papillary atrophy approximately 4 weeks after birth. Electron microscopic examination revealed the fibrotic changes and hypervascularity of capillaries between the urinary epithelial cell layer and smooth muscle cell layer at the ureteropelvic junction. CONCLUSION: ADAMTS-1 appears necessary for normal kidney morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease. (+info)
Altered expression of major renal Na transporters in rats with unilateral ureteral obstruction.
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It has been demonstrated previously that ureteral obstruction was associated with downregulation of renal AQP2 expression and an impaired urinary concentrating capacity (Li C, Wang W, Kwon TH, Isikay L, Wen JG, Marples D, Djurhuus JC, Stockwell A, Knepper MA, Nielsen S, and Frokiaer J. Am J Physiol Renal Physiol 281: F163-F171, 2001). In the present study, changes in the expression of major renal Na transporters were examined in a rat model with 24 h of unilateral ureteral obstruction (UUO) to clarify the molecular mechanisms of the marked natriuresis seen after release of UUO. Urine collection for 2 h after release of UUO revealed a significant reduction in urinary osmolality, solute-free water reabsorption, and a marked natriuresis (0.29 +/- 0.03 vs. 0.17 +/- 0.03 micromol/min, P < 0.05). Consistent with this, immunoblotting revealed significant reductions in the abundance of major renal Na transporters: type 3 Na(+)/H(+) exchanger (NHE3; 24 +/- 4% of sham-operated control levels), type 2 Na-P(i) cotransporter (NaPi-2; 21 +/- 4%), Na-K-ATPase (37 +/- 4%), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1; 15 +/- 3%), and thiazide-sensitive Na-Cl cotransporter (TSC; 15 +/- 4%). Immunocytochemistry confirmed the downregulation of NHE3, BSC-1, and TSC in response to obstruction. In nonobstructed contralateral kidneys, a significant reduction in the abundance of inner medullary Na-K-ATPase and cortical NaPi-2 was found. This may contribute to the compensatory increase in urinary production (23 +/- 2 vs. 13 +/- 1 microl x min(-1). kg(-1)) and increased fractional excretion of urinary Na (0.62 +/- 0.03 vs. 0.44 +/- 0.03%, P < 0.05). In conclusion, downregulation of major renal Na transporters in rats with UUO may contribute to the impairment in urinary concentrating capacity and natriuresis after release of obstruction, and reduced levels of Na-K-ATPase and NaPi-2 in the contralateral nonobstructed kidney may contribute to the compensatory increase in water and Na excretion from that kidney during UUO and after release of obstruction. (+info)
Inner medullary lactate production and urine-concentrating mechanism: a flat medullary model.
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We used a mathematical model to explore the possibility that metabolic production of net osmoles in the renal inner medulla (IM) may participate in the urine-concentrating mechanism. Anaerobic glycolysis (AG) is an important source of energy for cells of the IM, because this region of the kidney is hypoxic. AG is also a source of net osmoles, because it splits each glucose into two lactate molecules, which are not metabolized within the IM. Furthermore, these sugars exert their full osmotic effect across the epithelia of the thin descending limb of Henle's loop and the collecting duct, so they are apt to fulfill the external osmole role previously attributed to interstitial urea (whose role is compromised by the high urea permeability of long descending limbs). The present simulations show that physiological levels of IM glycolytic lactate production could suffice to significantly amplify the IM accumulation of NaCl. The model predicts that for this to be effective, IM lactate recycling must be efficient, which requires high lactate permeability of descending vasa recta and reduced IM blood flow during antidiuresis, two conditions that are probably fulfilled under normal circumstances. The simulations also suggest that the resulting IM osmotic gradient is virtually insensitive to the urea permeability of long descending limbs, thus lifting a longstanding paradox, and that this high urea permeability may serve for independent regulation of urea balance. (+info)