Renal physiology in health , Renal physiology in health , کتابخانه الکترونیک و دیجیتال - آذرسا

I am deeply honored to be the 2001 corecipient of the A.N. Richards Award and I am grateful to Professor Andreoli and the members of the board of the International Society of Nephrology for choosing me to receive this honor. For over 40 years, nephrology has been the core of my professional and intellectual life. My intellectual heroes were the renal physiologists Homer Smith, Robert Pitts, and Robert Berliner. For me, nephrology was more than just an intellectual discipline—it was a worldwide community of scientists and scholars with a shared interest in the mysteries of the kidney. It was in this community where I made some of my closest friends—Ike Robinson, Tom Andreoli, and Klaus Thurau. Over the years, I have been privileged to work with a large group of very gifted colleagues. Neither time, nor memory, permits mentioning them all, but a few deserve special mention. While I was in Dallas, Norman Carter's work on pH-sensitive microelectrodes made possible our studies on the mechanism of bicarbonate reabsorption in the proximal tubule. Juha Kokko's studies on the transport properties of the thin limbs of Henle's loop provided the basis for our development of the passive model for countercurrent multiplication for the inner medulla. In San Francisco, Robert Alpern, Christine Berry, Martin Cogan, and Patricia Preisig all made important contributions to our studies in delineating the active and passive components of proximal tubule transport. The clinical studies on a very unusual patient by Morris Schambelan and Anthony Sebastian provided the basis for identifying a unique chloride ion shunt in the distal tubule as the underlying pathogenetic mechanism for a form of aldosterone-resistant hyperkalemia and hypertension (Gordon's syndrome). Early in my career, one of the most remarkable men in American medicine became my mentor, my collaborator, and my close personal friend. To have Donald Seldin present this A.N. Richards award makes this a very special and intensely moving occasion for me. Thank you.

To analyze the rate of basolateral Cl exit and the magnitude and relative contributions of KCl cotransport and Na-dependent and -independent Cl/HCO3 exchange to Cl exit across the basolateral membrane (BLM) during transcellular Cl absorption, rabbit proximal convoluted tubules (PCT) were perfused with high-Cl, low-HCO3 plus formate solutions and bathed with plasma ultrafiltrate-like plus formate solutions. The initial rates of intracellular Cl activity (AiCl) reduction following bath Cl removal were compared when bath Cl was 0, when bath Na and Cl were 0, and when bath HCO3 and Cl were 0. The initial rate of AiCl reduction following bath Cl removal was 4.4 +/- 0.4 mM/s. After bath Na and Cl removal, this rate was reduced to 25.1 +/- 5.0%. After bath HCO3 and Cl removal, it was reduced to 18.0 +/- 4.8%. The difference between bath Na and Cl removal and bath HCO3 and Cl removal was not significant. Cl efflux following bath HCO3 and Cl removal may be due to a KCl symporter. The contribution of a KCl symporter was examined by raising bath K to 20 mM, thus eliminating the chemical driving force for KCl exit. After bath K increase, the initial rate of AiCl increase was 0.057 +/- 0.005 mM/s. These data suggest that Cl efflux at BLM of rabbit PCT is 1) large enough to explain transcellular Cl transport, 2) predominately due to an Na-dependent Cl/HCO3 exchanger, and 3) negligibly due to an Na-independent Cl/HCO3 exchanger or a KCl symporter.

In the mammalian proximal tubule NaCl reabsorption occurs by both passive and active transport processes. Passive NaCl reabsorption occurs in the presence of a high luminal chloride and a low luminal bicarbonate concentration. These anion gradients provide the driving forces for diffusive Na and Cl movement. Na is driven by the lumen positive PD effected by the greater permeability of the tubular wall to Cl than to HCO3. Cl is driven by its high tubular concentration. Passive NaCl reabsorption accounts for only about 10% to 15% of total proximal NaCl transport. The remaining proximal NaCl is reabsorbed by active transport processes and occurs both in the presence or absence of anion gradients reabsorption. Two mechanisms of active NaCl reabsorption participate in active NaCl reabsorption along the proximal tubule. Firstly, active NaCl reabsorption is electrogenic. In the early proximal tubule Na enters to cell coupled to organic solute transport. This Na reabsorption generates a lumen negative PD and effects "coupled" electrogenic NaCl reabsorption. This mechanism is limited by the supply of organic solutes and is blunted by the greater Na than Cl permeability in the proximal tubule; it probably can account for no more than 10% of proximal NaCl reabsorption. In the terminal proximal tubule, the proximal straight tubule, the apical membrane appears to possess a channel for Na entry. This Na reabsorption also generates a lumen negative PD and effects "simple" electrogenic NaCl reabsorption. This mechanism is limited by the low transport capacity of this segment and probably accounts for no more than 5% to 10% of total proximal NaCl reabsorption. The great bulk of proximal NaCl reabsorption occurs along the entire proximal tubule by active, transcellular electroneutral NaCl reabsorption. The precise cellular transport mechanisms responsible for this process are only recently being defined. At the apical membrane parallel ion exchangers are responsible for NaCl entry into the cell. Na enters via the apical membrane Na-H antiporter. Cl most likely crosses the apical membrane by some combination of Cl-OH and Cl-HCO2 exchangers but not via a Cl-HCO3 exchanger. The relative contributions of Cl-OH and Cl-HCO2 exchange have not been defined. There are two important considerations in this question. First is the availbility of OH versus HCO2. Although there is an infinite supply of OH and a small equilibrium supply of HCO2, it is possible that the luminal concentration of HCO2 could be increased by an USL that raises the concentration of HCO2 to a degree sufficient to supply H2CO2 recycling for physiological transcellular Cl transport rates.(ABSTRACT TRUNCATED AT 400 WORDS)

The mammalian proximal tubule is an important mediator of the renal adaptive response to systemic acidosis. In chronic metabolic and respiratory acidosis the bicarbonate reabsorptive (or proton secretory) capacity is increased. This increase is mediated, at least in part, by an increase in Vmax of the luminal Na/H antiporter. To determine whether this adaptation involves increased mRNA expression, Na/H antiporter mRNA levels were measured by Northern analysis in renal cortex of rats with metabolic (6 mmol/kg body wt NH4Cl for 2 or 5 d) and respiratory (10% CO2/air balanced for 2 or 5 d) acidosis and of normal, pair-fed rats. Na/H antiporter mRNA levels were unchanged after 2 d of both metabolic and respiratory acidosis. After 5 d, however, Na/H antiporter mRNA expression was increased 1.76 +/- 0.12-fold in response to metabolic acidosis (P less than 0.005, n = 8), but was not different from normal in response to respiratory acidosis: 1.1 +/- 0.2 (NS, n = 8). Thus, the renal adaptive response to metabolic acidosis involves increased cortical Na/H antiporter mRNA levels. In contrast, the enhanced proximal tubule Na/H antiporter activity and bicarbonate reabsorption in respiratory acidosis seem to involve mechanisms other than increased Na/H antiporter gene expression.

AbstractThe sections in this article are:Newer TechniquesCarbonic AnhydraseMechanisms of Proximal Tubular AcidificationApical MembraneBasolateral MembraneLeak PathwaysCarbonic AnhydraseSummaryMechanisms of Loop of Henle AcidificationMechanisms of Distal Nephron AcidificationMechanism of Proton Secretion (Bicarbonate Absorption)Mechanism of Bicarbonate SecretionLeak PathwaysCells Mediating H+/TransportCarbonic AnhydraseMechanisms of Regulation of AcidificationProximal TubuleDistal Tubule

To examine the basolateral Cl transport mechanisms of proximal convoluted tubules (PCT), intracellular Cl activity (AiCl) was measured with double-barreled Cl-selective microelectrodes. When rabbit PCT were perfused in vitro with high Cl, low HCO3, and bathed with ultrafiltrate-like solutions, AiCl was 29.9 +/- 0.4 mM and basolateral membrane voltage (Vbl) was -47.7 +/- 0.4 mV (n = 247). Possible basolateral Cl transport mechanisms that we examined were as follows: Cl conductance, KCl cotransport, and Na-dependent Cl-HCO3 exchange. Cl conductance was negligible, since the voltage clamp of Vbl to 30 mV above and below the spontaneous Vbl did not change AiCl even in the absence of luminal Cl. KCl cotransport was suggested by 1) increasing bath K, increased AiCl, and 2) decreasing bath K decreased AiCl. KCl cotransport was Na independent and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), barium, and furosemide insensitive. Na-dependent Cl-HCO3 exchange was suggested by 1) bath HCO3 reduction increased AiCl, which was greatly inhibited by bath Na removal or bath SITS, and 2) bath Na removal increased AiCl, which was completely blocked by bath SITS. We conclude that 1) Cl conductance is negligibly small at the basolateral membrane and 2) SITS-insensitive KCl cotransport and SITS-sensitive Na-dependent Cl-HCO3 exchange are present at the basolateral membrane.

To determine the effect of luminal pH on cell pH and basolateral cell membrane potential difference (Vbl) of rabbit proximal convoluted tubules, Vbl was measured by conventional microelectrodes and intracellular pH was measured microfluorometrically. Lowering lumen pH acidified the cell and depolarized Vbl. Three factors contributed to depolarization of Vbl. Lowering lumen pH decreased apical cell membrane potassium permeability (PK) as indicated by the following: 1) at lumen pH 7.4 raising lumen [K] depolarized Vbl; 2) lowering lumen pH eliminated the depolarization of Vbl induced by increasing lumen [K]. An additional effect was suggested by the following: lumen Ba2+ blunted, but did not eliminate, the Vbl response to lowering lumen pH. An effect on basolateral K permeability (PK) via its effect on cell pH was suggested by the fact that lowering lumen pH dramatically reduced the depolarization induced by increasing bath [K]. Lowering lumen pH might influence Vbl by inhibiting H+-HCO3- transport. Addition of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath solution hyperpolarized Vbl and enhanced the depolarization induced by lowering luminal pH. At luminal pH 6.0 SITS had no effect, suggesting elimination of H+ secretion. Addition of 1 mM luminal amiloride had no effect on Vbl or the response of Vbl to lowering luminal pH, but in the presence of amiloride SITS still hyperpolarized Vbl, suggesting amiloride-insensitive electrogenic H+ secretion. These results suggest that lumen pH-dependent depolarization of Vbl is due to 1) a decrease in apical PK; 2) cell acidification with secondary effects on basolateral PK; and 3) a decrease in apical electrogenic H+ transport.

The proximal tubule utilizes multiple mechanisms to reabsorb filtered NaCl. In the early PCT electrogenic Na-coupled organic solute transport generates a lumen-negative PD which drives Cl- passively through the paracellular pathway. Preferential reabsorption of HCO3- and organic solutes in the early PCT elevates luminal Cl- concentration, which in the late PCT provides the driving force for passive reabsorption of both Na+ and Cl-. However, most of the NaCl reabsorbed in the PCT is mediated by an electroneutral mechanism in which equivalent amounts of Na+ and Cl- move transcellularly across apical and basolateral membranes. In the mammalian PCT the evidence overwhelmingly supports parallel Na+-H+ and Cl- -base exchangers as the mechanism by which Na+ and Cl- cross the apical membrane during electroneutral, transcellular NaCl reabsorption. OH-, HCO3-, formate and Ox- have all been suggested to be the anion exchanged for Cl-. An important physiologic contribution of formate has been shown in in vitro microperfusion studies [29]. Measurements of intracellular pH using fluorescent dyes [59, 60] support a quantitatively important role for formate and argue against a large contribution of OH- and HCO3-. The absence of a role for HCO3- is also supported by in vivo microperfusion studies using methoxazolamide [53]. The potential role of oxalate requires physiologic evaluation. To date, the experimental data suggest that Cl- -formate is probably the predominant anion exchange mechanism. One may ask why, in a process so critical as NaCl reabsorption, the tubule would choose to use a "toxin" rather than one of those ions more familiar to renal physiologists?(ABSTRACT TRUNCATED AT 250 WORDS)

The value of rapid, contrast-enhanced, diuretic magnetic resonance (MR) imaging (using ferrioxamine B and furosemide) in demonstrating partial unilateral ureteral obstruction and the potential of such MR imaging in differentiating obstructive from nonobstructive hydronephrosis was assessed in six micropigs. MR imaging (0.35 Tesla, partial-flip technique with repetition time [TR] of 125 milliseconds, echo-delay time [TE] of 20 milliseconds, and flip angle of 70 degrees) was performed before, and at 5, 12, and 19 days after partial ureteral obstruction. Additionally, MR images were acquired 5, 12, and 19 days after release of obstruction. The diuretic was injected 10 minutes after the contrast medium. MR findings were correlated with results from nuclear scintigraphy (99mTc-DMSA uptake). MR images provided good morphologic detail from which renal size, parenchymal thickness, and degree of hydronephrosis could be determined. Contrast medium allowed assessment of cortical uptake and urinary excretion. The course of cortical signal enhancement best characterized the difference between obstructive and nonobstructive hydronephrosis. Normal kidneys and kidneys with nonobstructive hydronephrosis showed progressive decrease in cortical signal enhancement (-11.7% within 40 minutes) after furosemide injection. The kidneys with obstructive hydronephrosis demonstrated a plateau of signal enhancement without decrease (-0.7% within 40 minutes). These results demonstrate the utility of rapid contrast-enhancing, diuretic MR imaging in differentiating obstructive from nonobstructive hydronephrosis.

We recently showed that, in the presence of physiological sodium concentrations, 4.3 mM luminal amiloride inhibits 90% of apical membrane Na+-H+ antiporter activity in the in vivo microperfused rat proximal convoluted tubule. In the present studies we examined the effect of 4.3 mM luminal amiloride on transepithelial NaCl absorption from a high-chloride, low-bicarbonate perfusate, simulating the tubular fluid of the late proximal tubule. Both chloride and volume absorption were inhibited approximately 44%, consistent with inhibition of most of transcellular NaCl absorption, and suggestive of parallel Na+-H+ and Cl(-)-base exchange as the mechanism of NaCl uptake across the apical membrane. Methazolamide (10(-4) M), a potent inhibitor of renal carbonic anhydrase, had no significant effect on either volume or chloride absorption, suggesting that a carbonic anhydrase-independent mechanism is at least partially involved in chloride absorption. Hydrochlorothiazide (1 mM), an inhibitor of electroneutral NaCl cotransport in tight epithelia, did not significantly affect either volume or chloride absorption. Thus these studies suggest that, in the rat, the mechanism of apical membrane electroneutral NaCl uptake is Na+-H+ and Cl- -base exchange.

To assess the presence and nature of steady-state anion current across the basolateral membrane in in vitro rabbit proximal convoluted tubules bathed and perfused with a high-chloride, low-bicarbonate solution simulating late proximal tubular fluid, steady-state basolateral cell membrane potential difference (Vb1) was measured by conventional microelectrodes. The mean value of Vb1 was -52 mV. Addition of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath solution hyperpolarized Vb1 by 30 mV, suggesting the presence of basolateral anion current. Total chloride removal did not change Vb1 significantly, and formate, a presumptive stimulant of electroneutral sodium chloride transport, depolarized Vb1 both in the presence and absence of chloride, suggesting that the formate-stimulated change in Vb1 was chloride independent. In the total absence of chloride and bicarbonate, 1 mM bath SITS and 0.1 mM lumen and bath acetazolamide hyperpolarized Vb1 by 27-35 and 23 mV, respectively. These results suggest that the SITS-sensitive change in Vb1 is independent of chloride and associated with a basolateral anion current that is predominantly due to bicarbonate exit. In the absence of exogenous CO2, cell-to-bath HCO3-dependent anion current can be derived from metabolic CO2.

The mechanism of basolateral membrane base transport was examined in the in vitro microperfused rabbit proximal convoluted tubule (PCT) in the absence and presence of ambient CO2/HCO3- by means of the microfluorometric measurement of cell pH. The buffer capacity of the cells measured using rapid NH3 washout was 42.8 +/- 5.6 mmol.liter-1.pH unit-1 in the absence and 84.6 +/- 7.3 mmol.liter-1.pH unit-1 in the presence of CO2/HCO3-. In the presence of CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.30 pH units and lowering peritubular Na from 147 to 0 mM acidified the cell by 0.25 pH units. Both effects were inhibited by peritubular 4-acetamido-4'- isothiocyanostilbene-2,2'-disulfonate (SITS). In the absence of exogenous CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.25 pH units and lowering peritubular Na from 147 to 0 mM decreased cell pH by 0.20 pH units. Lowering bath pH from 7.4 to 6.8 induced a proton flux of 643 +/- 51 pmol.mm-1.min-1 in the presence of exogenous CO2/HCO3- and 223 +/- 27 pmol.mm-1.min-1 in its absence. Lowering bath Na from 147 to 0 mM induced proton fluxes of 596 +/- 77 pmol.mm-1.min-1 in its absence. The cell acidification induced by lowering bath pH or bath Na in the absence of CO2/HCO3- was inhibited by peritubular SITS or by acetazolamide, whereas peritubular amiloride had no effect. In the absence of exogenous CO2/HCO3-, cyanide blocked the cell acidification induced by bath Na removal, but was without effect in the presence of exogenous CO2/HCO3-. We reached the following conclusions. (a) The basolateral Na/base n greater than 1 cotransporter in the rabbit PCT has an absolute requirement for CO2/HCO3-. (b) In spite of this CO2 dependence, in the absence of exogenous CO2/HCO3-, metabolically produced CO2/HCO3- is sufficient to keep the transporter running at 30% of its control rate in the presence of ambient CO2/HCO3- . (c) There is no apparent amiloride-sensitive Na/H antiporter on the basolateral membrane of the rabbit PCT.