An independent effect of osmolality on urea transport in rat terminal inner medullary collecting ducts.

We have shown that urea transport across the terminal inner medullary collecting duct (terminal IMCD) is mediated by a vasopressin-stimulated, facilitated diffusion process exhibiting properties consistent with a transporter. To investigate whether hypertonic NaCl, as exists in vivo in the inner medulla, affects urea permeability, we studied isolated perfused rat terminal IMCD segments. Perfusate and bath osmolality were varied symmetrically by adding or removing NaCl or mannitol. Urea permeability rose progressively when osmolality was increased with NaCl or mannitol from 290 to 690 mOsm/kg H2O in the absence of vasopressin; there was no further increase at 890 mOsm/kg H2O. In the presence of 10(-8) M arginine vasopressin, urea permeability increased when NaCl was added to raise osmolality from 290 to 490 mOsm/kg H2O but there was no further increase at 690 mOsm/kg H2O. When 1 mM 8-bromo cyclic AMP was added to the bath, raising NaCl still increased urea permeability. These results suggest that urea transport across the rat terminal IMCD is regulated both by vasopressin and by osmolality at values present in the renal inner medulla. Osmolality seems to activate urea transport across the rat terminal IMCD by mechanisms distinct from those of vasopressin or cyclic AMP.

Evidence for sodium-dependent active urea secretion in the deepest subsegment of the rat inner medullary collecting duct.

Active reabsorption of urea appears in the initial IMCD (IMCD1) of rats fed a low-protein diet. To determine whether active urea transport also occurs in the deepest IMCD subsegment, the IMCD3, we isolated IMCDs from the base (IMCD1), middle (IMCD2), and tip (IMCD3) regions of the inner medulla from rats fed a normal protein diet and water ad libitum. IMCDs were perfused with identical perfusate and bath solutions. A significant rate of net urea secretion was present only in IMCD3s. Replacing perfusate Na+ with NMDG+ reversibly inhibited net urea secretion but replacing bath Na+ with NMDG+ or perfusate Cl- with gluconate- had no effect. Net urea secretion was significantly inhibited by: (a) 250 microM phloretin (perfusate); (b) 100 nM triamterene (perfusate); (c) 1 mM ouabain (bath); and (d) cooling the tubule to 23 degrees C. Net urea secretion was significantly stimulated by 10 nM vasopressin (bath). Next, we perfused IMCD3s from water diuretic rats (given food ad libitum) and found a significant, fivefold increase in net urea secretion. In summary, we identified a secondary active, secretory urea transport process in IMCD3s of normal rats which is upregulated in water diuretic rats. This new urea transporter may be a sodium- urea antiporter.

Active sodium-urea counter-transport is inducible in the basolateral membrane of rat renal initial inner medullary collecting ducts.

Rat inner medullary collecting ducts (IMCD3s) possess a luminal Na+-dependent, active urea secretory transport process, which is upregulated by water diuresis. In this study of perfused IMCDs microdissected from base (IMCD1), middle (IMCD2), or tip (IMCD3) of the inner medulla, we tested whether furosemide diuresis alters active urea transport. Rats received furosemide (10 mg/d s.c. for 3-4 d) and were compared with pair-fed control rats. Furosemide significantly decreased urine osmolality and urea clearance, and increased blood urea nitrogen. IMCD3s from furosemide-treated rats had significantly lower rates of active urea secretion than IMCD3s from control rats. IMCD2s showed no active urea transport in control or furosemide-treated rats. IMCD1s from control rats had no active urea transport, but IMCD1s from furosemide-treated rats expressed significant rates of active urea reabsorption. In IMCD1s, this active urea reabsorptive transport process was inhibited by: (i) 0. 25 mM phloretin (bath); (ii) 1 mM ouabain (bath); and (iii) replacing bath Na+ with NMDG+; it was stimulated by 10 nM bumetanide (bath). In summary, we found that furosemide decreased active urea secretion in IMCD3s and induced active urea reabsorption in IMCD1s. The new Na+- dependent, active urea reabsorptive transport process may be a basolateral Na+-urea antiporter.

Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium.

To compare passive urea transport across the inner medullary collecting ducts (IMCDs) and the papillary surface epithelium (PSE) of the kidney, two determinants of passive transport were measured, namely permeability coefficient and surface area. Urea permeability was measured in isolated perfused IMCDs dissected from carefully localized sites along the inner medullas of rats and rabbits. Mean permeability coefficients (X 10(-5) cm/s) in rat IMCDs were: outer third of inner medulla (IMCD1), 1.6 +/- 0.5; middle third (IMCD2), 46.6 +/- 10.5; and inner third (IMCD3), 39.1 +/- 3.6. Mean permeability coefficients in rabbit IMCDs were: IMCD1, 1.2 +/- 0.1; IMCD2, 11.6 +/- 2.8; and IMCD3, 13.1 +/- 1.8. The rabbit PSE was dissected free from the underlying renal inner medulla and was mounted in a specially designed chamber to measure its permeability to urea. The mean value was 1 X 10(-5) cm/s both in the absence and presence of vasopressin (10 nM). Morphometry of renal papillary cross sections revealed that the total surface area of IMCDs exceeds the total area of the PSE by 10-fold in the rat and threefold in the rabbit. We conclude: the IMCD displays axial heterogeneity with respect to urea permeability, with a high permeability only in its distal two-thirds; and because the urea permeability and surface area of the PSE are relatively small, passive transport across it is unlikely to be a major source of urea to the inner medullary interstitium.

Low protein diet alters urea transport and cell structure in rat initial inner medullary collecting duct.

Low protein diets reverse the urea concentration gradient in the renal inner medulla. To investigate the mechanism(s) for this change, we studied urea transport and cell ultrastructure in initial and terminal inner medullary collecting ducts (IMCD) from rats fed 18% protein or an isocaloric, 8% protein diet for 4 wk. Serum urea, aldosterone, and albumin were significantly lower in rats fed 8% protein, but total protein and potassium were unchanged. Vasopressin stimulated passive urea permeability (Purea) threefold (P < 0.05) in initial IMCDs from rats fed 8% protein, but not from rats fed 18% protein. Luminal phloretin reversibly inhibited vasopressin-stimulated Purea. However, in terminal IMCDs from rats fed either diet, vasopressin stimulated Purea. Net transepithelial urea flux (measured with identical perfusate and bath solutions) was found only in initial IMCDs from rats fed 8% protein. Reducing the temperature reversibly inhibited it, but phloretin did not. Electron microscopy of initial IMCD principal cells from rats fed 8% protein showed expanded Golgi bodies and prominent autophagic vacuoles, and morphometric analysis demonstrated a marked increase in the surface density and boundary length of the basolateral plasma membrane. These ultrastructural changes were not observed in the terminal IMCD. Thus, 8% dietary protein causes two new urea transport processes to appear in initial but not terminal IMCDs. This is the first demonstration that "active" urea transport can be induced in a mammalian collecting duct segment.

Atrial natriuretic factor inhibits vasopressin-stimulated osmotic water permeability in rat inner medullary collecting duct.

The inner medullary collecting duct (IMCD) has been proposed to be a site of atrial natriuretic factor (ANF) action. We carried out experiments in isolated perfused terminal IMCDs to determine whether ANF (rat ANF 1-28) affects either osmotic water permeability (Pf) or urea permeability. In the presence of a submaximally stimulating concentration of vasopressin (10(-11) M), ANF (100 nM) significantly reduced Pf by an average of 46%. Lower concentrations of ANF also significantly inhibited vasopressin-stimulated Pf by the following percentages: 0.01 nM ANF, 18%; 0.1 nM, 46%; 1 nM, 48%. Addition of exogenous cyclic GMP (0.1 mM) mimicked the effect of ANF, decreasing Pf by an average of 48%. ANF also inhibited cyclic AMP-stimulated Pf by an average of 31%. ANF did not affect urea permeability, nor did it alter vasopressin-stimulated cyclic AMP accumulation. We conclude that ANF at physiological concentrations causes a large inhibition of vasopressin-stimulated Pf in the rat terminal IMCD, and that cyclic GMP is the second messenger mediating the effect. ANF appears to act at a site distal to cyclic AMP generation in the chain of events linking vasopressin receptor binding to an increase in osmotic water permeability.

Sodium-dependent net urea transport in rat initial inner medullary collecting ducts.

We reported that feeding rats 8% protein for 3 wk induces net urea transport and morphologic changes in initial inner medullary collecting ducts (IMCDs) which are not present in rats fed 18% protein. In this study, we measured net urea transport in microperfused initial IMCDs from rats fed 8% protein for > or = 3 wk and tested the effect of inhibiting Na+/K(+)-ATPase activity and found that adding 1 mM ouabain to the bath reversibly inhibited net urea transport from 14 +/- 3 to 6 +/- 2 pmol/mm per min (P < 0.01), and that replacing potassium (with sodium) in the bath reversibly inhibited net urea transport from 18 +/- 3 to 5 +/- 0 pmol/mm per min (P < 0.01). Replacing perfusate sodium with N-methyl-D-glucamine reversibly inhibited net urea transport from 12 +/- 2 to 0 +/- 1 pmol/mm per min (P < 0.01), whereas replacing bath sodium had no significant effect on net urea transport. Adding 10 nM vasopressin to the bath exerted no significant effect on net urea transport. Finally, we measured Na+/K(+)-ATPase activity in initial and terminal IMCDs from rats fed 18% or 8% protein and found no significant difference in either subsegment. Thus, net urea transport in initial IMCDs from rats fed 8% protein for > or = 3 wk requires sodium in the lumen, is reduced by inhibiting Na+/K(+)-ATPase, and is unchanged by vasopressin or phloretin. These results suggest that net urea transport may occur via a novel, secondary active, sodium-urea cotransporter.

Cloning and regulation of expression of the rat kidney urea transporter (rUT2).

In mammals, urea is the predominant end-product of nitrogen metabolism and plays a central role in the urinary-concentrating mechanism. Urea accumulation in the renal medulla is critical to the ability of the kidney to concentrate urine to an osmolality greater than systemic plasma. Regulation of urea excretion and accumulation in the renal medulla depends on the functional state of specialized phloretin-sensitive urea transporters. To study these transporters and their regulation of expression we isolated a cDNA which encodes the rat homologue (rUT2) of rabbit UT2 (You, G., C.P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M.A. Hediger, et al. Nature (Lond.). 1993. 365:844-847). Rat UT2 has 88% amino acid sequence identity to rabbit UT2 and 64% identity to the recently cloned human erythrocyte urea transporter, HUT11 (Olives, B., P. Neav, P. Bailly, M.A. Hediger, G. Rousselet, J.P. Cartron, and P. Ripoch J. Biol. Chem. 1994. 269:31649-31652). Analysis of rat kidney mRNA revealed two transcripts of size 2.9 and 4.0 kb which had spatially distinct distributions. Northern analysis and in situ hybridization showed that the 4.0-kb transcript was primarily responsive to changes in the protein content of the diet whereas the 2.9-kb transcript was responsive to changes in the hydration state of the animal. These studies reveal that the expression levels of the two rUT2 transcripts are modulated by different pathways to allow fluid and nitrogen balance to be regulated independently. Our data provide important insights into the regulation of the renal urea transporter UT2 and provide a basis on which to refine our understanding of the urinary concentrating mechanism and its regulation.

Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct.

During antidiuresis, increases in vasopressin (AVP)-elicited osmotic water permeability in the terminal inner medullary collecting duct (tIMCD) raise luminal calcium concentrations to levels (> or = 5 mM) above those associated with the formation of calcium-containing precipitates in the urine. Calcium/polycation receptor proteins (CaRs) enable cells in the parathyroid gland and kidney thick ascending limb of Henle to sense and respond to alterations in serum calcium. We now report the presence of an apical CaR in rat kidney tIMCD that specifically reduces AVP-elicited osmotic water permeability when luminal calcium rises. Purified tIMCD apical membrane endosomes contain both the AVP-elicited water channel, aquaporin 2, and a CaR. In addition, aquaporin 2-containing endosomes also possess stimulatory (G(alpha q)/G(alpha 11) and inhibitory (G(alpha i1, 2, and 3)) GTP binding proteins reported previously to interact with CaRs as well as two specific isoforms (delta and zeta) of protein kinase C. Immunocytochemistry using anti-CaR antiserum reveals the presence of CaR protein in both rat and human collecting ducts. Together, these data provide support for a unique tIMCD apical membrane signaling mechanism linking calcium and water metabolism. Abnormalities in this mechanism could potentially play a role in the pathogenesis of renal stone formation.

Changes in aquaporin-2 protein contribute to the urine concentrating defect in rats fed a low-protein diet.

Low-protein diets cause a urinary concentrating defect in rats and humans. Previously, we showed that feeding rats a low (8%) protein diet induces a change in urea transport in initial inner medullary collecting ducts (IMCDs) which could contribute to the concentrating defect. Now, we test whether decreased osmotic water permeability (Pf) contributes to the concentrating defect by measuring Pf in perfused initial and terminal IMCDs from rats fed 18 or 8% protein for 2 wk. In terminal IMCDs, arginine vasopressin (AVP)-stimulated osmotic water permeability was significantly reduced in rats fed 8% protein compared to rats fed 18% protein. In initial IMCDs, AVP-stimulated osmotic water permeability was unaffected by dietary protein. Thus, AVP-stimulated osmotic water permeability is significantly reduced in terminal IMCDs but not in initial IMCDs. Next, we determined if the amount of immunoreactive aquaporin-2 (AQP2, the AVP-regulated water channel) or AQP3 protein was altered. Protein was isolated from base or tip regions of rat inner medulla and Western analysis performed using polyclonal antibodies to rat AQP2 or AQP3 (courtesy of Dr. M.A. Knepper, National Institutes of Health, Bethesda, MD). In rats fed 8% protein (compared to rats fed 18% protein): (a) AQP2 decreases significantly in both membrane and vesicle fractions from the tip; (b) AQP2 is unchanged in the base; and (c) AQP3 is unchanged. Together, the results suggest that the decrease in AVP-stimulated osmotic water permeability results, at least in part, in the decrease in AQP2 protein. We conclude that water reabsorption, like urea reabsorption, responds to dietary protein restriction in a manner that would limit urine concentrating capacity.

Maturation of aldose reductase expression in the neonatal rat inner medulla.

Newborns are less able to concentrate urine than adults are. With development of the concentrating system and a hypertonic medullary interstitium, there is a need to generate intracellular osmolytes such as sorbitol, which is produced in a reaction catalyzed by the enzyme aldose reductase. We sought to discriminate between two possible mechanisms of aldose reductase induction during development: (a) a response to an osmotic stimulus generated by the concentrating mechanism; or (b) part of the genetic program for development of the kidney. We measured the change in aldose reductase mRNA and activity in terminal inner medullary collecting ducts (IMCDs) microdissected from Sprague-Dawley rats during the first month of life. Aldose reductase mRNA was assayed by Northern analysis of total RNA from inner medulla and by detection of the reverse transcription-polymerase chain reaction (RT-PCR) product obtained from single IMCDs using aldose reductase-specific primers. Aldose reductase activity was measured in IMCDs taken from the same rats using a fluorescent microassay. Newborn rat IMCDs had minimal aldose reductase mRNA or activity, however mRNA was readily detected in IMCDs from rats older than 3 d of age, with peak expression occurring at 1-3 wk of age before decreasing to adult levels. In contrast, the mRNA level for a housekeeping metabolic enzyme, malate dehydrogenase, did not change during maturation. Aldose reductase enzyme activity was readily detectable by 6 d of age, peaked at 20 d, then decreased to adult levels. Urine osmolality remained < 600 mosmol/kg until 16 d, then increased to > 1,100 mosmol/kg after 20 d. Thus, aldose reductase mRNA and activity increased before urinary osmolality reached 870 mosmol/kg. Because urine osmolality may not be indicative of inner medullary osmolality and because mother's milk may provide excessive free water to the pups under 3 wk of age, half of the animals in several litters were separated from their mothers for 1 d and inner medullary osmolality, in addition to urine osmolality, was measured by vapor pressure osmometry, while aldose reductase mRNA was assessed densitometrically in IMCDs after RT-PCR. Although fluid restriction resulted in a near doubling of urine osmolality and a tendency towards increased aldose reductase mRNA, there was no consistently significant increase in aldose reductase mRNA or inner medullary osmolality during the first 13 d of life compared to the suckling animals. On the other hand, 2-3-wk-old rats showed significant increases in aldose reductase mRNA, accompanied by increases in inner medullary osmolality, after fluid restriction. Thus, the dissociation between the increases in aldose reductase expression and inner medullary hyperosmolality indicates that the maturational induction of the aldose reductase gene is not a consequence of osmotic stimulation, but rather, part of the developmental program of the kidney.

UT-A urea transporter protein in heart: increased abundance during uremia, hypertension, and heart failure.

Urea transporters have been cloned from kidney medulla (UT-A) and erythrocytes (UT-B). We determined whether UT-A proteins could be detected in heart and whether their abundance was altered by uremia or hypertension or in human heart failure. In normal rat heart, bands were detected at 56, 51, and 39 kDa. In uremic rats, the abundance of the 56-kDa protein increased 1.9-fold compared with pair-fed, sham-operated rats, whereas the 51- and 39-kDa proteins were unchanged. We also detected UT-A2 mRNA in hearts from control and uremic rats. Because uremia is accompanied by hypertension, the effects of hypertension per se were studied in uninephrectomized deoxycorticosterone acetate salt-treated rats, where the abundance of the 56-kDa protein increased 2-fold versus controls, and in angiotensin II-infused rats, where the abundance of the 56 kDa protein increased 1.8-fold versus controls. The 51- and 39-kDa proteins were unchanged in both hypertensive models. In human left ventricle myocardium, UT-A proteins were detected at 97, 56, and 51 kDa. In failing left ventricle (taken at transplant, New York Heart Association class IV), the abundance of the 56-kDa protein increased 1.4-fold, and the 51-kDa protein increased 4.3-fold versus nonfailing left ventricle (donor hearts). We conclude that (1) multiple UT-A proteins are detected in rat and human heart; (2) the 56-kDa protein is upregulated in rat heart in uremia or models of hypertension; and (3) the rat results can be extended to human heart, where 56- and 51-kDa proteins are increased during heart failure.

Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter.

We cloned the Slc14a2 gene and determined the genomic organization of the rat urea transporter UT-A. Slc14a2, the gene encoding the rat UT-A transporter, extends for more that 300 kb. The four known rat mRNA isoforms: UT-A1, UT-A2, UT-A3, and UT-A4 are transcribed from 24 exons. The Slc14a2 genomic map also accounts for 3'-untranslated sequences expressed alternatively in UT-A1, UT-A2, and UT-A3. We previously identified a TATA-less, tonicity-responsive promoter controlling the transcription of UT-A1, UT-A3, and UT-A4 from a single initiation site in the 5'-flanking region of the gene. Here, we describe a second, internal promoter in intron 12, which controls the transcription of UT-A2 starting from exon 13. This region contains a TATA motif upstream from the UT-A2 transcription start site, and shows consensus sequences for the cAMP response element (CRE) and for the tonicity enhancer (TonE) motif. Stimulation by cAMP induces UT-A2 mRNA expression in mIMCD3 cells, and luciferase activity in mIMCD3 cells transfected with those pGL3 constructs including the CRE sequences. Although long-term exposure to hypertonicity induces UT-A2 expression in mIMCD3 cells, hypertonicity does not induce significantly the activity of the promoter in intron 12. In summary, we describe the genomic structure of the rat UT-A urea transporter, encoded by the Slc14a2 gene. Our findings suggest that two promoters regulate transcription of the four UT-A isoforms, and that stimulation of transcription by vasopressin, mediated by cAMP and CRE sequences, and controlled by an intronic promoter, may contribute to the increase in UT-A2 expression during water deprivation.

Prospective evaluation of low-dose ketoconazole plus hydrocortisone in docetaxel pre-treated castration-resistant prostate cancer patients.

BACKGROUND: Ketoconazole is a well-known CYP17-targeted systemic treatment for castration-resistant prostate cancer (CRPC). However, most of the published data has been in the pre-chemotherapy setting; its efficacy in the post-chemotherapy setting has not been as widely described. Chemotherapy-naïve patients treated with attenuated doses of ketoconazole (200-300 mg three times daily) had PSA response rate (>50% decline) of 21-62%. We hypothesized that low-dose ketoconazole would likewise possess efficacy and tolerability in the CRPC post-chemotherapy state. METHODS: Men with CRPC and performance status 0-3, adequate organ function and who had received prior docetaxel were treated with low-dose ketoconazole (200 mg orally three times daily) and hydrocortisone (20 mg PO qAM and 10 mg PO qPM) until disease progression. Primary endpoint was PSA response rate (>50% reduction from baseline) where a rate of 25% was to be considered promising for further study (versus a null rate of <5%); 25 patients were required. Secondary endpoints included PSA response >30% from baseline, progression-free survival (PFS), duration of stable disease and evaluation of adverse events (AEs). RESULTS: Thirty patients were accrued with median age of 72 years (range 55-86) and median pre-treatment PSA of 73 ng ml(-1) (range 7-11,420). Twenty-nine patients were evaluable for response and toxicity. PSA response (>50% reduction) was seen in 48% of patients; PSA response (>30% reduction) was seen in 59%. Median PFS was 138 days; median duration of stable disease was 123 days. Twelve patients experienced grade 3 or 4 AEs. Of the 17 grade 3 AEs, only 3 were attributed to treatment. None of the two grade 4 AEs were considered related to treatment. CONCLUSIONS: In docetaxel pre-treated CRPC patients, low-dose ketoconazole and hydrocortisone is a well-tolerated, relatively inexpensive and clinically active treatment option. PSA response to low-dose ketoconazole appears historically comparable to that of abiraterone in this patient context. A prospective, randomized study of available post-chemotherapy options is warranted to assess comparative efficacy.

Urea transport in the kidney.

Urea transport within the kidney is regulated and varies dramatically between different nephron segments. The terminal IMCD displays very high rates of transepithelial urea transport enabling delivery of large amounts of urea into the deepest portions of the inner medulla to maintain a high interstitial osmolality for concentrating the urine maximally. Urea in the terminal IMCD is transported by a specific urea transporter that is stimulated by vasopressin and hyperosmolarity. Although the urea transporter has not been cloned, individuals have been identified who lack the urea transporter. Individuals who lacked the Kidd antigen (a minor blood group antigen) also lacked carrier-mediated urea transport in their erythrocytes (and presumably in their kidneys). These same subjects were unable to concentrate their urine above 800 mOsm following overnight water deprivation. This experiment of nature illustrates the critical importance of the urea transporter to concentrating ability in humans.

Effect of age and testosterone on the vasopressin and aquaporin responses to dehydration in Fischer 344/Brown-Norway F1 rats.

To determine if the aging-associated decline in testosterone results in attenuated vasopressin (VP) responses to dehydration, testosterone implants were given to aged male Fischer 344Brown-Norway F1(F344BNF1) rats. Water deprivation caused comparable dehydration, increased plasma VP (pVP), and decreased posterior pituitary (PP) VP content in 4-, 15-, and 28-month-old rats. Dehydration increased VP mRNA content of supraoptic nuclei only at 4 months, whereas VP mRNA length was increased at both 4 and 15 months of age. The elevated pVP in the water-deprived aged rats indicates that even without an increase in VP mRNA content, PP VP storage was adequate to maintain elevated pVP. Dehydration increased aquaporin-2 content at 4, but not at 15 or 28 months of age, suggesting decreased renal responsiveness to VP. Testosterone replacement did not produce dehydration-induced increases in VP mRNA or aquaporin-2. Therefore, testosterone deficiency does not result in altered VP responses to dehydration in aged F344BNF1 rats.

Current concepts of the countercurrent multiplication system.

The production of a concentrated urine is achieved by countercurrent multiplication in the renal medulla. While the single effect in the outer medulla is known to be active NaCl reabsorption in the thick ascending limb, the single effect in the inner medulla is not definitively established. However, the passive model of Kokko and Rector [1] and Stephenson [2] remains the most widely accepted mechanism for the single effect in the inner medulla. Continued experimental studies of transport in perfused inner medullary nephron segments and mathematical simulations that incorporate these new experimental values and anatomic complexity will be needed to fully elucidate the process of urinary concentration. In addition, the availability of molecular reagents will permit investigation into the molecular mechanisms that regulate transport proteins which play crucial roles in the urinary concentrating mechanism.

Regulation of urea transporter proteins in kidney and liver.

Due to urea's role in producing concentrated urine, its transport is critically important to the conservation of body water. Within the renal inner medulla, urea is transported by both facilitated and active urea transport mechanisms. The vasopressin-regulated, facilitated urea transporter (UT-A1) in the terminal inner medullary collecting duct (IMCD) permits high rates of transepithelial urea transport and results in delivery of large quantities of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for maximal urine concentration. Four cDNA isoforms of the UT-A urea transporter family have been cloned. In addition, there are three secondary active, sodium-dependent, urea transport mechanisms in IMCD subsegments: (1) active urea secretion in the apical membrane of the terminal IMCD from untreated rats; (2) active urea absorption in the apical membrane of the initial IMCD from low-protein fed or hypercalcemic rats; and (3) active urea absorption in the basolateral membrane of the initial IMCD from furosemide-treated rats. This review will focus on integrative studies of the rapid and long-term regulation of urea transporters in rats with reduced urine concentrating ability. These studies led to the surprising result that the basal-facilitated urea permeability in the terminal IMCD and UT-A1 protein abundance are increased during in vivo conditions associated with an impaired urine concentrating ability. In contrast, there are two response patterns of active urea transporters: (1) hypercalcemia, a low-protein diet, and furosemide result in induction of active urea absorption in the initial IMCD, albeit by different mechanisms, and inhibition of active urea secretion in the terminal IMCD; while (2) water diuresis results in up-regulation of active urea secretion in the terminal IMCD without any active urea absorption in the initial IMCD. The first pattern contributes to the urine concentrating defect by increasing urea delivery to the base of the inner medulla, thus decreasing urea delivery distally to the inner medullary tip. The second response pattern will directly decrease urea content in the deep inner medulla. UT-A urea transporters are also expressed outside the kidney. Recent studies show that the liver has phloretin-inhibitable urea transport and that it occurs via a 49 kDa UT-A protein. When rats are made uremic, the abundance of this 49 kDa UT-A protein increases in the liver in vivo. This up-regulation of the 49 kDa UT-A protein may allow hepatocytes to increase ureagenesis to reduce the accumulation of ammonium and/or bicarbonate in uremia.

Hemolytic-uremic syndrome following "crack" cocaine inhalation.

The authors present a patient who experienced cocaine-related thrombotic microangiopathy and patchy renal cortical necrosis, associated with the clinical syndrome of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure, characteristics of the Hemolytic-Uremic syndrome. The proposed pathogenetic mechanisms include: (1) cocaine-induced vasoconstriction and endothelial damage and (2) procoagulant effects of cocaine.

Bartter's syndrome, supraventricular tachycardia, mitral valve prolapse, and asthma: a therapeutic challenge.

A 25-year-old man with acquired Bartter's syndrome, mitral valve prolapse, and supraventricular tachycardia secondary to a low atrial focus was diagnosed with asthma. The unique aspects of managing these coexisting diseases are evaluated. Calculation of free-water clearance in the diagnosis of Bartter's syndrome and the etiology and characteristics of the syndrome are discussed.