Phylogenetic, ontogenetic, and pathological aspects of the urine-concentrating mechanism

The urine-concentrating mechanism is one of the most fundamental functions of avian and mammalian kidneys. This particular function of the kidneys developed as a system to accumulate NaCl in birds and as a system to accumulate NaCl and urea in mammals. Based on phylogenetic evidence, the mammalian urine-concentrating mechanism may have evolved as a modification of the renal medulla's NaCl accumulating system that is observed in birds. This qualitative conversion of the urine-concentrating mechanism in the mammalian inner medulla of the kidneys may occur during the neonatal period. Human kidneys have several suboptimal features caused by the neonatal conversion of the urine-concentrating mechanism. The urine-concentrating mechanism is composed of various functional molecules, including water channels, solute transporters, and vasopressin receptors. Abnormalities in water channels aquaporin (AQP)1 and AQP2, as well as in the vasopressin receptor V2R, are known to cause nephrogenic diabetes insipidus. An analysis of the pathological mechanism involved in nephrogenic diabetes insipidus suggests that molecular chaperones may improve the intracellular trafficking of AQP2 and V2R, and, in the near future, such chaperones may become a new clinical tool for treating nephrogenic diabetes insipidus.     

Regulation of renal urea transporters

Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.     

Molecular approaches to urea transporters

Urea plays a critical role in the urine-concentrating mechanism in the inner medulla. Physiologic data provided evidence that urea transport in red blood cells and kidney inner medulla was mediated by specific urea transporter proteins. Molecular approaches during the past decade resulted in the cloning of two gene families for facilitated urea transporters, UT-A and UT-B, encoding several urea transporter cDNA isoforms in humans, rodents, and several nonmammalian species. Polyclonal antibodies have been generated to the cloned urea transporter proteins, and the use of these antibodies in integrative animal studies has resulted in several novel findings, including: (1) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; (2) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (3) UT-A protein abundance is upregulated in uremia in both liver and heart; and (4) UT-B is expressed in many nonrenal tissues and endothelial cells. This review will summarize the knowledge gained from using molecular approaches to perform integrative studies into urea transporter protein regulation, both in normal animals and in animal models of human diseases, including studies of uremic rats in which urea transporter protein is upregulated in liver and heart.     

Characterization of aldose reductase from the thick ascending limb of Henle's loop of rabbit kidney

BACKGROUND: The organic osmolyte sorbitol plays an important role in the osmoregulation of immortalized epithelial cells of the thick ascending limb of Henle's loop (TALH) of rabbit. The intracellular sorbitol content seems to depend strongly on the extracellular osmolarity. To investigate the nature of the osmotic regulation we characterized the aldose reductase. METHODS: We determined aldose reductase activity enzymatically and the content of organic osmolytes by HPLC. RESULTS: The aldose reductase activity correlates with the extracellular tonicity. Elevating the osmolarity of the medium from 300 to 600 mosm/l by addition of NaCl or sucrose resulted in a significant increase of maximal velocity (V(max)) of the adapted cells from 8 +/- 1 micromol/g x min (300 mosm/l) to 322 +/- 28 micromol/g x min (600 mosm/l, NaCl) or 54 +/- 9 micromol/g x min (600 mosm/l, sucrose), respectively, while affinity (K(m)) remained unchanged. But we found no rise of aldose reductase activity when extracellular urea concentration was elevated. Similar alterations in V(max) were observed when the activity of the highly enriched enzyme was determined with glucose as substrate. Elevation of the extracellular osmolarity by NaCl and sucrose strongly induced the expression of aldose reductase protein with an apparent molecular weight of 39 kD. The affinity of glucose is characteristically low with a K(m) above 300 mmol/l. Aldose reductase utilizes both NADPH and with lower affinity NADH as coenzymes. In vitro sulfate ions (0.4 mol/l) results in a two-fold activation of the aldose reductase activity whereas sodium (200-400 mmol/l) decreased the activity significantly (22-33%). Potassium and chloride up to 400 mmol/l did not alter the aldose reductase activity in vitro. CONCLUSIONS: These results indicate that the aldose reductase of TALH cells of the outer medulla is osmotically regulated and has many similarities with aldose reductase in renal inner medulla. Therefore, intracellular sorbitol synthesis seems to be of similar importance in the osmoregulation of TALH cells as in the inner medulla.     

Mechanisms to concentrate the urine: an opinion

PURPOSE OF REVIEW: Our goal is to suggest how the renal concentrating mechanism is regulated in vivo. RECENT FINDINGS: The majority of descending thin limbs of the loop of Henle lack aquaporin-1 water channels, and loops of Henle in the inner medulla lack urea transporters. SUMMARY: Lack of water permeability in the descending thin limbs of the loop of Henle offers several advantages. First, since much less water is added to the outer medullary interstitial compartment, inhibitory control mechanisms on sodium and chloride reabsorption from the medullary thick ascending of loop of Henle initiated by water addition from the medullary collecting duct can be effective. Second, recycling of urea is efficient, as little urea will be washed out of the medulla. Third, delivery of a larger volume of filtrate to the medullary thick ascending limb of the loop of Henle permits both an appreciable reabsorption of sodium along with only a small fall in the luminal concentration of sodium in each of these liters. Hence there need be only a small lumen positive voltage in the medullary thick ascending limb of the loop of Henle. The absence of urea transporters in the loop of Henle in the inner medulla is required for a passive mechanism of sodium and chloride reabsorption in the inner medulla. Control of urea reabsorption from the medullary collecting duct is needed to prevent excessive oliguria in electrolyte-poor urine.     

Alloreactive T suppressor cells in the rat. I. Evidence of three distinct subsets of splenic suppressor T cells resistant to cyclosporine

In this study we examined the effect of cyclosporine on three distinct subsets of T suppressor (Ts) cells identified in a rat renal allograft model. Ts inducer (Ts1) cells having the CD4 marker are found in the spleens of DA rats undergoing acute rejection of LEW kidneys. Transducer (Ts2) and effector (Ts3) cells both carry the CD8 marker and are found in the spleens of long-term surviving DA rats bearing LEW kidney allografts made tolerant by donor-specific blood transfusions or by cyclosporine (in most cases). These latter cells are distinguished by their susceptibility to cyclophosphamide (CY), Ts2 cells being resistant while Ts3 cells are sensitive to CY. When Ts cells from DA rats undergoing acute graft rejection of LEW kidneys or bearing long-term-surviving LEW kidneys that had been treated with cyclosporine (10mg/kg/day) for 2 or 10 days, respectively, were adoptively transferred into lightly irradiated DA recipients, these cells were still able to specifically induce long-term survival of LEW kidneys. LEW kidney survival was not prolonged in DA rats given no cells or cells from rats treated with cyclosporine for 10 days. Thus it would appear that the three functional subsets of Ts cells demonstrated in this renal allograft model by adoptive transfer of spleen lymphocytes are not inhibited by cyclosporine, suggesting that this resistance of Ts cells to cyclosporine may be partly responsible for the immunosuppressive effect of this agent.     

Polynocturia in chronic kidney disease is related to natriuresis rather than to water diuresis.

BACKGROUND: Nocturnal polyuria has been well known in renal insufficiency. Recently, we found that as renal function deteriorated in chronic kidney disease (CKD), natriuresis was enhanced during the night with nocturnal blood pressure elevation. In the present study, we investigated whether nocturnal polyuria in CKD was due to the inability to concentrate urine, as previously proposed, or based on osmotic diuresis mainly by natriuresis. METHODS: In 27 CKD patients, circadian rhythms of urinary sodium, potassium, urea and osmolar excretion rates (U(Na)V, U(K)V, U(urea)V, U(osm)V) as well as of urinary volume (V) and free-water clearance (C(H(2)O)) were estimated during both daytime (6:00 to 21:00) and nighttime (21:00 to 6:00). Then, the night/day ratios of these parameters were analysed in relation to creatinine clearance (C(cr)) as a marker of glomerular filtration rate. RESULTS: C(cr) had significantly negative relationships with night/day ratios of V (R = -0.69; P < 0.0001), U(osm)V (R = -0.54; P = 0.004) and U(Na)V (R = -0.63; P = 0.0005), but no correlation with night/day ratios of C(H(2)O) (R = -0.33; P = 0.1), U(K)V (R = -0.29; P = 0.1) or U(urea)V (R = -0.31; P = 0.1). Linear and multiple regression analysis identified nocturnal natriuresis rather than urea excretion as an independent determinant of nocturia. CONCLUSION: As renal function deteriorated, nocturnal polyuria was seen, being consistent with classical recognition. Furthermore, this increase in nocturnal urine volume seemed related to osmotic diuresis mainly by natriuresis rather than to water diuresis or urea excretion.     

Historadioautographic localization, pharmacology and ontogeny of V(1a) vasopressin binding sites in the rat kidney.

The localization and pharmacological characteristics of vasopressin (VP) binding sites of the V(1a) subtype in developing and adult rat kidney were investigated by radioautography on kidney sections incubated in the presence of a radioiodinated selective V(1a) antagonist. Their localization after in vivo systemic infusion of the radioligand was also investigated. V(1a) binding sites first appear at embryonic day 16 on vascular elements. In the adult, they were localized in the cortex (vascular and tubular structures, juxtaglomerular apparatus), the outer medulla outer stripe (vasa recta) and inner stripe (thin descending limbs of short looped nephrons) and the inner medulla (collecting ducts). Data obtained in vitro were confirmed by in vivo binding at postnatal day 30 (PN30). Whatever their localizations, the V(1a) binding sites exhibited full V(1a) pharmacological profile in postnatal stages rats and in adult rats: a high affinity (nM range) for VP and for the V(1a) agonist, a lower affinity (microM range) for oxytocin and no affinity for the oxytocin agonist. The presence of V(1a) binding sites in these different structures raises the question of the putative roles of VP in modulating renal functions. A striking finding is the presence of V(1a) binding sites in the outer medullary thin descending limbs of short looped nephrons suggesting their colocalization with urea transporters.     

Ionic dialysance allows an adequate estimate of urea distribution volume in hemodialysis patients

BACKGROUND: An adequate estimation of urea distribution volume (V) in hemodialysis patients is useful to monitor protein nutrition. Direct dialysis quantification (DDQ) is the gold standard for determining V, but it is impractical for routine use because it requires equilibrated postdialysis plasma water urea concentration. The single pool variable volume urea kinetic model (SPVV-UKM), recommended as a standard by Kidney Disease Outcomes Quality Initiative (K/DOQI), does not need a delayed postdialysis blood sample but it requires a correct estimate of dialyser urea clearance. METHODS: Ionic dialysance (ID) may accurately estimate dialyzer urea clearance corrected for total recirculation. Using ID as input to SPVV-UKM, correct V values are expected when end-dialysis plasma water urea concentrations are determined in the end-of-session blood sample taken with the blood pump speed reduced to 50 mL/min for two minutes (U(pwt2')). The aim of this study was to determine whether the V values determined by means of SPVV-UKM, ID, and U(pwt2') (V(ID)) are similar to those determined by the "gold standard" DDQ method (V(DDQ)). Eighty-two anuric hemodialysis patients were studied. RESULTS: V(DDQ) was 26.3 +/- 5.2 L; V(ID) was 26.5 +/- 4.8 L. The (V(ID)-V(DDQ)) difference was 0.2 +/- 1.6 L, which is not statistically significant (P= 0.242). Anthropometric volume (V(A)) calculated using Watson equations was 33.6 +/- 6.0 L. The (V(A)-V(DDQ)) difference was 7.3 +/- 3.3 L, which is statistically significant (P < 0.001). CONCLUSION: Anthropometric-based V values overestimate urea distribution volume calculated by DDQ and SPVV-UKM. ID allows adequate V values to be determined, and circumvents the problem of delayed postdialysis blood samples.     

Short-Term Functional Adaptation of Aquaporin-1 Surface Expression in the Proximal Tubule,a Component of Glomerulotubular Balance

Transepithelial water flow across the renal proximal tubule is mediated predominantly by aquaporin-1 (AQP1). Along this nephron segment, luminal delivery and transepithelial reabsorption are directly coupled, a phenomenon called glomerulotubular balance. We hypothesized that the surface expression of AQP1 is regulated by fluid shear stress, contributing to this effect. Consistent with this finding, we found that the abundance of AQP1 in brush border apical and basolateral membranes was augmented >2-fold by increasing luminal perfusion rates in isolated, microperfused proximal tubules for 15 minutes. Mouse kidneys with diminished endocytosis caused by a conditional deletion of megalin or the chloride channel ClC-5 had constitutively enhanced AQP1 abundance in the proximal tubule brush border membrane. In AQP1-transfected, cultured proximal tubule cells, fluid shear stress or the addition of cyclic nucleotides enhanced AQP1 surface expression and concomitantly diminished its ubiquitination. These effects were also associated with an elevated osmotic water permeability. In sum, we have shown that luminal surface expression of AQP1 in the proximal tubule brush border membrane is regulated in response to flow. Cellular trafficking, endocytosis, an intact endosomal compartment, and controlled protein stability are the likely prerequisites for AQP1 activation by enhanced tubular fluid shear stress, serving to maintain glomerulotubular balance.     

Renal phenotype of UT-A urea transporter knockout mice

The urea transporters UT-A1 and UT-A3 mediate rapid transepithelial urea transport across the inner medullary collecting duct (IMCD). In a previous study, using a new mouse model in which both UT-A1 and UT-A3 were genetically deleted from the IMCD (UT-A1/3(-/-) mice), we investigated the role of these transporters in the function of the renal inner medulla. Here the authors report a new series of studies investigating more generally the renal phenotype of UT-A1/3(-/-) mice. Pathologic screening of 33 tissues revealed abnormalities in both the testis (increased size) and kidney (decreased size and vascular congestion) of UT-A1/3(-/-) mice. Total urinary nitrate and nitrite (NOx) excretion rates in UT-A1/3(-/-) mice were more than double those in wild-type mice. Total renal blood flow was not different between UT-A1/3(-/-) and wild-type mice but underwent a greater percentage decrease in response to NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME) infusion. Whole kidney GFR (FITC-inulin clearance) was not different in UT-A1/3(-/-) mice compared with controls and underwent a similar increase in response to a greater dietary protein intake. Fractional urea excretion was markedly elevated in UT-A1/3(-/-) mice on a 40% protein diet, reaching 102.4 +/- 8.8% of the filtered load, suggesting that there may be active urea secretion somewhere along the renal tubule. Although there was a marked urinary concentrating defect in UT-A1/3(-/-) mice, there was no decrease in aquaporin 2 or aquaporin 3 expression. Furthermore, although urea accumulation in the inner medulla was markedly attenuated, there was no decrease in sodium ion concentration in tissue from outer medulla or two levels of the inner medulla. These results support our conclusion that the urinary concentrating defect in UT-A1/3(-/-) mice is caused by a failure of urea transport from the IMCD lumen to the inner medullary interstitium, resulting in osmotic diuresis.     

The medullary microcirculation

Like other regional circulations, the medullary circulation supplies oxygen and other primary substrates to the medulla and removes carbon dioxide and other waste metabolites. It also acts as a countercurrent exchanger and simultaneously removes water reabsorbed from the renal tubule to preserve mass balance. Our present understanding of how the medulla serves both these functions at the same time is illustrated in Figure 3. Blood leaves the efferent arteriole with an elevated plasma protein concentration as a consequence of glomerular filtration, and flows down descending vasa recta within a vascular bundle. The increased interstitial osmotic-concentration coupled with a finite capillary reflection coefficient for small solutes causes additional water to be extracted so that at the termination of descending vasa recta, the plasma protein concentration exceeds that in the systemic circulation by approximately twofold. Solute, urea more than sodium chloride, also enters descending vasa recta. As blood flows through the interconnecting capillary plexus and up ascending vasa recta, transcapillary oncotic and osmotic pressure differences combine to cause capillary uptake of fluid. There is also simultaneous loss of urea such that the medullary trapping of urea is very effective. Countercurrent exchange of sodium chloride, however, appears to be less efficient and as a consequence, not only water but sodium chloride is removed from the medulla. Antidiuretic hormone reduces medullary blood flow, both directly by its vasoconstrictor (V1-receptor mediated) effect and indirectly by its antidiuretic (V2-receptor mediated) effects. Prostaglandins are able to enhance medullary blood flow by counteracting vasoconstrictive influences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of (CO2 + HCO3-) on electrical conductance in cortical thick ascending limbs

These experiments in isolated mouse cortical thick ascending limbs (cTALH) provide information about: the relative contributions of cellular and paracellular pathways to the transepithelial electrical conductance Ge (mS cm-2); the effects of (CO2 + HCO3-) on Ge; the ratio of net K+ secretion (JKnet) to net Cl- absorption (JClnet); and the K+ requirement for apical membrane furosemide-sensitive NaCl entry. The combination of luminal Ba++, zero K+ reduced Ge; at 5 mM luminal Ba++, the residual conductance was about 75% of the control Ge. The Ba++-insensitive Ge of 73.1 +/- 6.4 mS cm-2 was only slightly greater than the shunt conductance of 57 mS cm-2 computed from the sum of the dissipative bath to lumen fluxes of 22Na+ and 36Cl-. Moreover, luminal 5 mM Ba++, zero K+ had no effect on the Na+/Cl- permselectivity ratio of the paracellular pathway. Thus, Ba++ blockaded transcellular conductance by blocking apical membrane K+ channels. The combination of (CO2 + HCO3-) in external solutions increased Ge solely by augmenting the Ba++-sensitive, that is, transcellular, component of Ge. Finally, in paired experiments, the ratio JKnet/JClnet was 0.27 +/- 0.05; and both Ve, the spontaneous transepithelial voltage (mV), and the equivalent short circuit current Je (pEq sec-1 cm-2) were reduced dramatically by luminal K+ omission. Thus, apical membranes of the cTALH appear to contain a pathway for net K+ secretion, and apical membrane NaCl entry may involve co-transport with K+. Transcellular conductance contributes at least 25% to the total Ge, and the combination (CO2 + HCO3-) augments transcellular conductance.     

Time-dependent up-regulation of endothelin-A receptors and down-regulation of endothelin-B receptors and nitric oxide synthase binding sites in the renal medulla of a rabbit model of partial bladder outlet obstruction: potential clinical relevance

OBJECTIVE: To assess the density of endothelin (ET) receptors (ET-1 is a potent vasoconstrictor peptide acting on two known receptors, ETA and ETB ) and nitric oxide synthase (NOS) binding sites in the kidney of a rabbit model of bladder outlet obstruction (BOO). MATERIALS AND METHODS: Partial BOO was created in adult New Zealand White rabbits; after 1, 3, 4 and 6 weeks of BOO, kidney sections were incubated with radioligands for ET-1, ETA, ETB receptors and with [3H]-NOARG (a ligand for NOS). Autoradiographs were generated and analysed densitometrically. Sections were also assessed by NADPH histochemistry. Plasma creatinine, urea and electrolyte levels were regularly monitored. The control and 6-week BOO kidneys were also evaluated ultrastructurally by electron microscopy. RESULTS: There was no significant change in plasma creatinine, urea and electrolyte levels. ETA and ETB receptor density was significantly greater in the medulla than in the cortex (P<0.001) in all animals. There was an up-regulation of ETA receptors (P=0.03) and down-regulation of ETB receptors (P=0.03) and NOS binding sites (P<0.001), as well as decreased NADPH staining in the medulla of 6-week partial BOO kidneys. Electron microscopy detected glomerular disruption of the obstructed kidneys. CONCLUSION: The time-dependent changes in ETA and ETB receptors, NOS binding sites and NADPH staining in the renal medulla, as well as ultrastructural changes, occur despite normal renal function. These changes appear to be an early event and may play a role in the development of renal failure. Hence, the use of ETA receptor antagonists at this early stage may prevent the development of renal failure/impairment in BOO.     

Aquaporin-1 is not expressed in descending thin limbs of short-loop nephrons

In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1-positive proximal tubules to aquaporin-1-negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.     

Kidney vacuolar H+ -ATPase: physiology and regulation

The vacuolar H(+)-ATPase is a multisubunit protein consisting of a peripheral catalytic domain (V(1)) that binds and hydrolyzes adenosine triphosphate (ATP) and provides energy to pump H(+) through the transmembrane domain (V(0)) against a large gradient. This proton-translocating vacuolar H(+)-ATPase is present in both intracellular compartments and the plasma membrane of eukaryotic cells. Mutations in genes encoding kidney intercalated cell-specific V(0) a4 and V(1) B1 subunits of the vacuolar H(+)-ATPase cause the syndrome of distal tubular renal acidosis. This review focuses on the function, regulation, and the role of vacuolar H(+)-ATPases in renal physiology. The localization of vacuolar H(+)-ATPases in the kidney, and their role in intracellular pH (pHi) regulation, transepithelial proton transport, and acid-base homeostasis are discussed.