Regulation of the renal Na+-Cl- cotransporter by phosphorylation and ubiquitylation.

The activity of the renal thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule plays a key role in defining arterial blood pressure levels. Increased or decreased activity of the NCC is associated with arterial hypertension or hypotension, respectively. Thus it is of major interest to understand the activity of NCC using in vivo models. Phosphorylation of certain residues of the amino-terminal domain of NCC has been shown to be associated with its activation. The development of phospho-specific antibodies against these sites provides a powerful tool that is helping to increase our understanding of the molecular physiology of NCC. Additionally, NCC expression in the plasma membrane is modulated by ubiquitylation, which represents another major mechanism for regulating protein activity. This work presents a review of our current knowledge of the regulation of NCC activity by phosphorylation and ubiquitylation.

Aldosterone paradox: differential regulation of ion transport in distal nephron.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.

WNK3 and WNK4 amino-terminal domain defines their effect on the renal Na+-Cl- cotransporter.

Loss of physiological regulation of the renal thiazide-sensitive Na+-Cl- cotransporter (NCC) by mutant WNK1 or WNK4 results in pseudohypoaldosteronism type II (PHAII) characterized by arterial hypertension and hyperkalemia. WNK4 normally inhibits NCC, but this effect is lost by eliminating WNK4 catalytic activity or through PHAII-type mutations. In contrast, another member of the WNK family, WNK3, activates NCC. The positive effect of WNK3 on NCC also requires its catalytic activity. Because the opposite effects of WNK3 and WNK4 on NCC were observed in the same expression system, sequences within the WNKs should endow these kinases with their activating or inhibiting properties. To gain insight into the structure-function relationships between the WNKs and NCC, we used a chimera approach between WNK3 and WNK4 to elucidate the domain of the WNKs responsible for the effects on NCC. Chimeras were constructed by swapping the amino or carboxyl terminus domains, which flank the central kinase domain, between WNK3 and WNK4. Our results show that the effect of chimeras toward NCC follows the amino-terminal domain. Thus the amino terminus of the WNKs contains the sequences that are required for their activating or inhibiting properties on NCC.

Differential distribution of the vasopressin V receptor along the rat nephron during renal ontogeny and maturation.

BACKGROUND: Ontogeny and cellular distribution of vasopressin receptors in the kidney are key factors determining the role of vasopressin in renal physiology. Expression of vasopressin V(2) receptor (V(2)R) mRNA and the immunoreactive protein in rat kidney were investigated. METHODS: An antiserum directed to epitope TLD25 of the rat V(2)R sequence was characterized by Western blotting. Expression of V(2)R mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR), and on protein level by immunohistochemistry. RESULTS: Specificity of the antiserum was documented by Western blots from cells expressing a fusion protein of V(2)R and GFP. Using lysates of rat kidney and of native cell lines expressing V(2)R but not V(1)R, our antiserum to peptide TLD25 revealed a major band of 55 kD corresponding to the monomeric form of V(2)R, and a band of 110 kD most likely representing the homodimeric form of the receptor. This highly specific antiserum allowed us to localize the V(2)R in thick ascending limbs, distal convoluted and connecting tubules, and in collecting ducts. During ontogeny, immunoreactivity was first observed at the luminal membrane on prenatal day 20, emerging at the basolateral side from postnatal day 5 on. RT-PCR demonstrated V(2)R transcripts from prenatal day 18 to gradually increasing thereafter. CONCLUSION: Expression of V(2)R is first detectable in the late embryonic stage of rat ontogeny starting from day E18 and gradually increasing with kidney maturation. In the adult kidney, V(2)R is differentially distributed in the various nephron segments.

Recovery of distal nephron enzyme activity after release of unilateral ureteral obstruction.

PURPOSE: Unilateral ureteral obstruction (UUO) for 24 hours results in a severe compromise of distal tubular function. The acidification defect is believed to be localized in the collecting duct. To characterize distal tubular function recovery one month after junction release, clearance studies in whole animals and enzyme studies in microdissected segments were performed in an experimental model of unilateral ureteral obstruction. MATERIALS AND METHODS: Following release of ureteral obstruction of 24 hours duration, a significant decrease of whole kidney glomerular filtration rate was observed in the postobstructed kidney (POK) with a marked increase in urinary pH, fractional excretion of bicarbonate (FEHCO3-) and decrease in urinary osmolality. By orthograde stop flow experiment, bicarbonate excretion rate (Fr:Ff HCO3-/Fr:Ff Inutest) increased in the first and second urine fractions of 120 microl. corresponding to the collecting segment in the POK, one day after release. Decrease in U-P pCO2 (p<0.01) suggested an impaired H+ secretion on distal nephron in POK. Recovery of inulin clearance and values of urinary pH, FEHCO3- and urinary osmolality near contralateral and control kidneys were observed thirty days following ureteral release. The decline in enzyme activity in the distal nephron due to structural damage from high intratubular pressure was evaluated. Bafilomycin sensitive H+ -ATPase activity measurement in the medullary collecting duct segments of the POK showed an important decrease (68%), with lightly reduced activity (20%) in the cortical collecting duct, 24 hours after obstruction release. Localized in the connecting tubule cells and secreted into the tubular fluid in the late distal nephron, renal kallikrein has been involved in bicarbonate transport at cortical collecting duct segments. The renal kallikrein-like activity was reduced in POK (p<0.01). RESULTS: Recovery of enzyme activity was shown thirty days after unilateral ureteral obstruction. Our results show severe functional damage of the collecting duct after 24 hours of unilateral ureteral obstruction. H+ -ATPase activity was markedly decreased on medullary collecting duct segments. CONCLUSIONS: A correlation between the functional impairment of distal H+ secretion and decreased distal nephron enzyme activity has been shown. Recovery of both the functional and the enzyme activity at the distal nephron was demonstrated thirty days after obstruction release.

Micropuncture study on urea movements in the kidney cortical tubules of low protein fed sheep.

Micropuncture studies of late proximal, early and late distal cortical tubules were carried out on kidneys of normal (NP) and low (LP) protein fed sheep in order to investigate the participation of these segments in the urea sparing induced by protein restriction in the diet. A low protein diet induced significant reductions in the fractional (-54%) and total (-84%) urea excretion, revealing an enhanced capacity for urea conservation. Micropuncture data did not show any difference in the proximal tubule functions between both groups of sheep. In distal cortical tubules the fractional delivery of urea (early distal, 0.61 +/- 0.06 for NP and 0.77 +/- 0.06 for LP sheep, not significant (NS); late distal, 0.45 +/- 0.07 for NP and 0.71 +/- 0.09 for LP sheep, P < 0.05) showed a relatively larger amount of urea present in the late distal tubule of protein restricted sheep. The tubular fluid-to-plasma inulin ratio in the late distal tubule was found to be lower in LP sheep (4.33 +/- 0.23 versus 8.58 +/- 0.9 in NP sheep, P < 0.01). The tubular flow rate, reduced in the early distal tubules of LP sheep (10.87 +/- 0.99 versus 18.92 +/- 2.58 nL.min-1 in NP sheep, P < 0.05), was not different in the late distal tubules from values in normally fed animals (6.65 +/- 0.90 versus 7.73 +/- 0.94 nL.min-1 in NP sheep, NS). These findings suggest a decreased distal water reabsorption coincident with the relatively larger amounts of intraluminal urea in LP sheep. This relatively larger urea delivery to the initial collecting duct could increase the subsequent urea reabsorption in protein restricted sheep.

Mechanisms and regulation of H+ transport in distal tubule epithelial cells.

The mechanism of acidification in the cortical distal tubule of mammalian kidney was analysed by "in vivo" microperfusion and using MDCK cells in culture, by electrophysiological and by cell pH microfluorescence techniques. An electrogenic effect of the vacuolar H(+)-ATPase, which has been localized to the intercalated cells of the cortical distal tubule (connecting segment and initial collecting duct) was only observed after blocking Cl- channels by NPPB. In MDCK cells, the recovery of cell pH after an acid pulse in Na(+)-free medium was also depressed by NPPB, indicating that Cl- ions have an important role in the function of H+ ATPase. The regulation by hormonal agents of distal H+ transport due to Na+/H+ exchange and to vacuolar H+ ATPase, was also studied by microperfusion and cell pH techniques. Angiotensin and vasopressin at picomolar concentrations stimulated both transport mechanisms in late distal tubule, and only Na+/H+ exchange in the early segment. In MDCK cells, cell pH recovery in the presence of Na+ was stimulated by picomolar concentrations of angiotensin and vasopressin, and inhibited by micromolar levels, both effects being reverted by micromolar ANP. Studies with specific antagonists suggest that the luminal effect of angiotensin is mediated by AT1 receptors, and of vasopressin by V1 receptors. There is evidence that cell Ca2+ may have an important regulatory role in the action of these hormones.

Renal actions of angiotensin-(1-7)

The heptapeptide angiotensin-(1-7) is considered to be a biologically active endproduct of the renin-angiotensin system. This angiotensin, which is devoid of the most known actions of angioatensin II such as induction of drinking behavior and vasoconstriction, has several selective effects in the brain and periphery. In the present article we briefly review recent evidence for a physiological role of angiotensin-(1-7) in the control of hydroelectrolyte balance.

Role of thyroid hormones in renal tubule acidification.

Renal tubule acidification was studied in thyroparathyroidectomized rats which had the parathyroids reimplanted into cervical muscle tissue, by stopped-flow microperfusion using ion-exchange resin microelectrodes. Hypothyroid rats had decreased rates of proximal and late distal bicarbonate reabsorption. This reduction occurred in the absence of changes in pH gradients, and was due mostly to decreases in acidification half-times, that is, of the rate of bicarbonate exit from the tubule lumen. H+ back-flux from the lumen measured during luminal perfusion with solutions at pH 6 (below stationary pH) was decreased in proximal tubule of hypothyroid rats, showing that the acidification defect was not due to an increased H+ shunt across the epithelium. These data indicate that in hypothyroid rats the proximal tubule luminal density of Na+/H+ exchangers or their turnover is decreased in the absence of alterations in the driving force (H+ and Na+ gradients across the luminal membrane) for H+ secretion. The effect observed in distal tubule may be due to action on Na+/H+ exchangers that are present also on this site, or to an impairment of the action of other H+ transporters such as H(+)-ATPases, including the provision of energy for them. 9

The role of the distal nephron in the regulation of acid-base equilibrium by the kidney.

The present paper reviews mechanisms by which the kidney controls systemic acid-base balance, with emphasis on the role of the distal nephron, and particularly of the cortical distal tubule. These mechanisms are essentially based on H-ion transport along the whole nephron. In proximal tubule cells, approximately 80% of H-ion secretion is mediated by Na+/H+ exchange, and 20% by H(+)-ATPase. In the distal nephron, acid-base transport mechanisms are located mainly in intercalated cells. H-ion secretion is effected by vacuolar H(+)-ATPase in alpha-intercalated cells and, in K-depleted animals, also by the gastric type H/K ATPase. In animals in alkalosis, beta-intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger, while a basolateral H-ATPase transfers H-ions into the interstitium. In cortical distal tubule, these mechanisms have been shown to be present in the intercalated cells of the connecting segment and of the initial collecting duct (the late distal tubule of micropuncture experiments). In the convoluted distal tubule (early distal tubule), most H-ion secretion occurs by means of the Na+/H+ exchanger. These data show that the distal nephron, including the cortical distal tubule, is a nephron segment responsible for a sizeable portion of bicarbonate reabsorption and titratable acid generation, as well as for bicarbonate secretion under appropriate metabolic conditions, being therefore the site of fine regulation of renal mechanisms that maintain acid-base homeostasis.

The role of the distal nephron in the regulation of acid-base equilibrium by the kidney

The present paper reviews mechanisms by which the kidney controls systemic acid-base balance, with emphasis on the role of the distal nephron, and particularly of the cortical distal tubule. These mechanisms are essentially based on H-ion transport along the whole nephron. In proximal tubule cells, approximately 80 of H-ion secretion is mediated by Na+/H+ exchange, and 20 by H(+)-ATPase. In the distal nephron, acid-base transport mechanisms are located mainly in intercalated cells. H-ion secretion is effected by vacuolar H(+)-ATPase in alpha-intercalated cells and, in K-depleted animals, also by the gastric type H/K ATPase. In animals in alkalosis, beta-intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger, while a basolateral H-ATPase transfers H-ions into the interstitium. In cortical distal tubule, these mechanisms have been shown to be present in the intercalated cells of the connecting segment and of the initial collecting duct (the late distal tubule of micropuncture experiments). In the convoluted distal tubule (early distal tubule), most H-ion secretion occurs by means of the Na+/H+ exchanger. These data show that the distal nephron, including the cortical distal tubule, is a nephron segment responsible for a sizeable portion of bicarbonate reabsorption and titratable acid generation, as well as for bicarbonate secretion under appropriate metabolic conditions, being therefore the site of fine regulation of renal mechanisms that maintain acid-base homeostasis.

Mechanism of acidification along cortical distal tubule of the rat.

The cellular mechanism of luminal acidification (bicarbonate reabsorption) was studied in cortical distal tubules of rat kidney. The stopped-flow microperfusion technique was applied to early distal (ED) and late distal (LD) segments, perfused with bicarbonate Ringer solution to which specific inhibitors were added, to measure bicarbonate reabsorption [HCO3 flux (JHCO3)]. pH and transepithelial potential difference (Vt) were recorded by double-barreled H+ exchange resin/reference (1 M KCl) electrodes. Amiloride increased stationary pH and reduced Vt in both early and late segments. Hexamethylene-amiloride (HMA), a specific Na(+)-H+ exchange blocker, reduced JHCO3 in both segments (ED by 43.6 and LD by 40.3%) without affecting Vt. Benzamil, an Na(+)-channel blocker, reduced Vt by 75.9 in ED and 74.9% in LD but had no significant effect on acidification in both segments. The specific inhibitor of H(+)-ATPase, bafilomycin A1, inhibited LD JHCO3 at a concentration of 2 x 10(-7) M by 49%, but ED was inhibited by 24% only at 2 x 10(-6) M. Sch-28080, an inhibitor of gastric H(+)-K(+)-ATPase, reduced JHCO3 by 35% in LD of K(+)-depleted rats but not in control rats and had no effect on ED. These data indicate that, in ED, bicarbonate reabsorption is mediated mostly by Na(+)-H+ exchange. In LD, there is evidence for contribution of Na(+)-H+ exchange, vacuolar H(+)-ATPase, and H(+)-K(+)-ATPase (in K(+)-depleted rats) to bicarbonate reabsorption.

Effect of peritubular hypertonicity on water and urea transport of inner medullary collecting duct.

The effect of bath fluid hypertonicity on hydraulic conductivity (Lp) and [14C]urea permeability (Pu) of the distal inner medullary collecting duct (IMCD) was studied in the absence and in the presence of vasopressin (VP) using the in vitro microperfusion technique of rat IMCD. In the first three groups of IMCD, we observed that in the absence of VP the Lp was not different from zero when the osmotic gradient was created by hypotonic perfusate and isotonic bath fluid, but it was significantly greater than 1.0 x 10(-6) cm.atm-1.s-1 when the osmotic gradient was created by hypertonic bath and isotonic perfusion fluid. The increase in Lp was observed when the hypertonicity of the bath fluid was produced by the addition of NaCl or raffinose, but no such effect was observed with urea. The stimulated effect of bath fluid hypertonicity on Lp was also observed in the IMCD obtained from Brattleboro homozygous rats in which VP is absent. The NaCl hypertonic bath increased the Pu in the absence of VP. In another series of experiments with VP (10(-10) M) we observed that the hypertonic bath fluid increased in a reversible manner the VP-stimulated Lp of distal IMCD. However, the NaCl hypertonicity of the bath fluid was not able to increase dibutyryladenosine 3',5'-cyclic monophosphate-stimulated Lp. The Pu stimulated by VP (10(-10) M) increased twofold when the bath fluid was hypertonic. Therefore hypertonicity of the peritubular fluid produced by the addition of NaCl or raffinose increases the Lp and Pu in the absence and in the presence of VP. No such effect was noted with the addition of urea.

Sodium dependence of early distal H+ secretion in rat kidney.

In order to study the mechanism of H-ion secretion in cortical distal tubules of the rat kidney, the luminal pH and transepithelial potential difference (PD) were measured with double-barrelled, pH-sensitive, resin/reference microelectrodes. Perfusion of peritubular capillaries with low-sodium solutions increased luminal pH by 0.28 +/- 0.024 units. Perfusion of the lumen with 1 mM amiloride increased luminal pH by 0.67 +/- 0.01 units. These changes could not be attributed to modification of transepithelial PD. We conclude that early distal acidification is sodium-dependent, possibly owing to the presence of Na+/H+ exchange.

Sodium dependence of eraly distal H+ secretion in rat kidney

In order to study the mechanism of H-ion secretion in cortical distal tubules of the rat kedney, the luminal pH and transepithelial potential difference (PD) were measured with double-barrelled, pH-sensitive, resin/reference microelectrodes. perfusion of peritubular capillaries with low-sodium solutions increased luminal pH by 0.28 ñ 0.024 units. Perfusion of the lumen with 1 m§ amiloride increased luminal pH by 0.67 ñ 0.01 units. These changes could not be atributed to modification of transepithelial PD. We conclude that early distal acidification is sodium-dependent, possibly owing to the presence of Na+/H+ exchange

Distal nephron sodium handling by normal, nephrotic, pyelonephritic and partially nephrectomized rats.

The renal tubular handling of sodium was evaluated in 10 normal (N), 9 edematous nephrotoxic serum nephrotic (Ne), 9 pyelonephritic (Py) and 12 partially nephrectomized (Nx) Wistar rats during maximal free-water clearance induced by intravenous infusion of hypotonic sodium chloride solutions. Fractional deliveries of sodium to distal nephron [(CH2O + CNa)/GFR%] were similar (P greater than 0.05) in N (23.2 +/- 0.69%), Ne (24.3 +/- 1.30%) and Nx (26.9 +/- 2.6%) rats. However, the fractional distal sodium reabsorption [CH2O/(CH2O + CNa)%] was significantly higher (P less than 0.05) in Ne (76.8 +/- 1.80%) and lower (P less than 0.05) in Nx (33.3 +/- 3.1%) rats when compared to normal rats (50.3 +/- 1.80%). Py rats exhibited a higher fractional delivery of sodium to distal nephron (33.5 +/- 3.4%) and a lower fractional distal sodium reabsorption (35.9 +/- 5.9%) when compared to normal rats (P less than 0.05). These data suggest that the distal nephron, by altering sodium reabsorption, contributes to the maintenance of sodium balance in Nx rats, whereas in Ne rats the distal nephron generates a positive sodium balance and edema. At very low glomerular filtration rates (GFR), as is the case for Py rats, sodium balance is maintained both by the proximal and distal portions of the nephron.

Properties of the isolated rabbit proximal convoluted tubule and pars recta after long-term storage in a modified Collins solution.

The effect of storage of cortical slices in modified Collins solution on the viability of rabbit proximal convoluted tubules (PCT) and pars recta (PR) was evaluated by microperfusion of isolated tubules in vitro. Cortical slices of rabbit kidney were stored for 1, 24, 48, 72 and 97 h at 4 degrees C in modified Collins solution. PCT and PR were dissected from these slices and perfused with a modified Krebs-Henseleit solution in vitro. Fluid absorption (JV) was 1.21 +/- 0.3 nl min-1 mm-1 (N = 8) and the transepithelial potential difference (PD) was -2.3 +/- 0.3 mV (N = 6) for PCT from cortical slices stored 0 to 3 h. JV was 1.01 +/- 0.09 nl min-1 mm-1 (N = 8) and PD was -1.9 +/- 0.6 mV (N = 5) for PCT from tissue stored 23 to 97 h. JV of PR from tissue stored 5 to 26 h was 0.75 +/- 0.1 nl min-1 mm-1 (N = 5) and PD was -1.5 +/- 0.3 mV (N = 5). JV was 0.76 +/- 0.1 nl min-1 mm-1 (N = 3) for PR stored 72 to 74 h, and PD was not measured. There was no correlation between storage time and JV or PD. Furthermore, the JV and PD values for these tubules were similar to those obtained for freshly dissected tubules. These results show that the isolated tubule preparation is useful for the direct assessment of the efficacy of different kidney preservation solutions on nephron function.