Proximal tubular bicarbonate reabsorption and PCO2 in chronic metabolic alkalosis in the rat.

Studies were undertaken to define the pattern of proximal tubular bicarbonate reabsorption and its relation to tubular and capillary PCO2 in rats with chronic metabolic alkalosis (CMA). CMA was induced by administering furosemide to rats ingesting a low electrolyte diet supplemented with NaHCO3 and KHCO3. Proximal tubular bicarbonate reabsorption and PCO2 were measured in CMA rats either 4-7 or 11-14 d after furosemide injection, in order to study a wide range of filtered bicarbonate loads. A group of nine age-matched control animals, fed the same diet but not given furosemide, was studied for comparison. In a third group of controls, the filtered load of bicarbonate was varied over the same range as in the CMA rats by plasma infusion and aortic constriction. The CMA rats had significant alkalemia and hypokalemia (4-7 d: pH 7.58, HCO3 38.3 meq/liter, K+ 2.1 meq/liter; 11-14 d: pH 7.54, HCO3 38.1 meq/liter, K+ 2.5 meq/liter). Nonetheless, proximal bicarbonate reabsorption was not significantly different from that seen in control rats at any given load of filtered bicarbonate (from 250 to 1,300 pmol/min). In both control and CMA rats, 83-85% of the filtered bicarbonate was reabsorbed by the end of the accessible proximal tubule. These observations indicate that proximal bicarbonate reabsorption is determined primarily by the filtered load in chronic metabolic alkalosis. When single nephron glomerular filtration rate (SNGFR) is reduced by volume depletion in the early postfurosemide period, the filtered load and the rate of proximal bicarbonate reabsorption remain at or below control levels, maintaining metabolic alkalosis. In the late postfurosemide period, however, SNGFR returned to control levels in some instances. In these animals, both the filtered load and rate of proximal reabsorption were increased above the highest levels seen in control animals. The PCO2 gradient between the peritubular capillaries and arterial blood (Pc-Art) was significantly higher in CMA than in control, even though the rate of proximal bicarbonate reabsorption did not differ. Thus, proximal bicarbonate reabsorption did not appear to be the primary determinant of Pc-Art PCO2. PCO2 in the early proximal (EP) tubule was significantly higher than in either the late proximal (LP) tubule or peritubular capillaries in both control and CMA rats. The EP-LP PCO2 gradient correlated directly with proximal bicarbonate reabsorption (P less than 0.05). The small elevation in PCO2 in EP may be related to CO2 generated at this site in the process of bicarbonate reabsorption.

Load dependence of proximal tubular bicarbonate reabsorption in chronic metabolic alkalosis in the rat.

Studies were undertaken in Munich-Wistar rats to determine whether maintenance of chronic metabolic alkalosis (CMA) is associated with an increase in proximal HCO3- reabsorption, or whether a reduction in glomerular filtration rate (GFR) is required to sustain the elevated plasma HCO3- concentration. Superficial single nephron glomerular filtration rate (SNGFR), and absolute proximal HCO-3 (APRHCO3) and water (APRH2O) reabsorption were measured 20 +/- 3 d after the induction of CMA in eight rats and the results compared with seven age-matched control animals. Plasma [HCO3-] was 39.1 +/- 1.8 mM in CMA rats compared with 26.0 +/- 0.4 mM in controls (P less than 0.001). In the CMA rats, SNGFR was 44.8 +/- 1.1 vs. 38.2 +/- 2.1 nl/min in controls (P less than 0.025). As a result, the single nephron filtered load of HCO3- (FLHCO3) increased from 1,147 +/- 61 pmol/min in control to 2,040 +/- 108 pmol/min in CMA (P less than 0.001). APRHCO3 increased by greater than 65%, from 970 +/- 65 pmol/min in control to 1,624 +/- 86 pmol/min in CMA (P less than 0.001). APRH2O increased from 18.4 +/- 1.6 nl/min in control to 24.0 +/- 0.8 nl/min in CMA (P less than 0.005). Tubular hypertrophy resulted in an increase in the length of the proximal convoluted tubule from 5.6 +/- 0.2 to 6.5 +/- 0.2 mm (P less than 0.005). The pattern of HCO3- reabsorption along the length of the proximal convoluted tubule in CMA was indistinguishable from that found in normal rats in which FLHCO3 was varied acutely by altering SNGFR. The increase in tubular length accounted for only 30% of the increase in APRH2O and 15% of the increase in APRHCO3. We conclude that a sustained reduction in GFR is not required for maintenance of CMA in the rat. If GFR is chronically restored to normal levels, the alkalosis is maintained by an increase in APRHCO3. The increase in reabsorption is accounted for by tubular hypertrophy, a chronic adaptive response, and a load-dependent response that is indistinguishable from that seen in normal rats when FLHCO3 is increased acutely by increasing SNGFR.

Delivery dependence of early proximal bicarbonate reabsorption in the rat in respiratory acidosis and alkalosis.

In the intact rat kidney, bicarbonate reabsorption in the early proximal tubule (EP) is strongly dependent on delivery. Independent of delivery, metabolic acidosis stimulates EP bicarbonate reabsorption. In this study, we investigated whether systemic pH changes induced by acute or chronic respiratory acid-base disorders also affect EP HCO3- reabsorption, independent of delivery (FLHCO3, filtered load of bicarbonate). Hypercapnia was induced in rats acutely (1-3 h) and chronically (4-5 d) by increasing inspired PCO2. Hypocapnia was induced acutely (1-3 h) by mechanical hyperventilation, and chronically (4-5 d) using hypoxemia to stimulate ventilation. When compared with normocapneic rats with similar FLHCO3, no stimulation of EP or overall proximal HCO3 reabsorption was found with either acute hypercapnia (PaCO2 = 74 mmHg, pH = 7.23) or chronic hypercapnia (PaCO2 = 84 mmHg, pH = 7.31). Acute hypocapnia (PaCO2 = 29 mmHg, pH = 7.56) did not suppress EP or overall HCO3 reabsorption. Chronic hypocapnia (PaCO2 = 26 mmHg, pH = 7.54) reduced proximal HCO3 reabsorption, but this effect was reversed when FLHCO3 was increased to levels comparable to euvolemic normocapneic rats. Thus, when delivery is accounted for, we could find no additional stimulation of proximal bicarbonate reabsorption in respiratory acidosis and, except at low delivery rates, no reduction in bicarbonate reabsorption in respiratory alkalosis.

Control of proximal bicarbonate reabsorption in normal and acidotic rats.

This free-flow micropuncture study examined the dependence of bicarbonate reabsorption in the rat superficial proximal convoluted tubule to changes in filtered bicarbonate load, and thereby the contribution of the proximal tubule to the whole kidney's response to such changes. The independent effects of extracellular fluid (ECF) volume expansion and of acidosis on proximal bicarbonate reabsorption were also examined. When the plasma volume contraction incurred by the micropuncture preparatory surgery was corrected by isoncotic plasma infusion ( congruent with1.3% body wt), single nephron glomerular filtration rate (SNGFR), and the filtered total CO(2) load increased by 50%. Absolute proximal reabsorption of total CO(2) (measured by microcalorimetry) increased by 30%, from 808+/-47 during volume contraction to 1,081+/-57 pmol/min.g kidney wt after plasma repletion, as fractional total CO(2) reabsorption decreased from 0.90 to 0.77. Aortic constriction in these plasma-repleted rats returned the filtered load and reabsorption of total CO(2) to the previous volume contracted levels. In other animals isohydric ECF expansion with plasma (5% body wt) or Ringer's solution (10% body wt), or both, produced no further diminution in fractional proximal total CO(2) reabsorption (0.76-0.81). Metabolic acidosis was associated with very high fractional proximal total CO(2) reabsorptive rates of 0.82 to 0.91 over a wide range of SNGFR and ECF volumes. At a single level of SNGFR, end-proximal total CO(2) concentration progressively decreased from 5.6+/-0.5 to 1.6 +/-0.2 mM as arterial pH fell from 7.4 to 7.1. Expansion of ECF volume in the acidotic rats did not inhibit the ability of the proximal tubule to lower end-proximal total CO(2) concentrations to minimal levels. In conclusion, bicarbonate reabsorption in the superficial proximal convoluted tubule is highly load-dependent (75-90%) in normal and acidotic rats. No inhibitory effect of ECF volume per se on proximal bicarbonate reabsorption, independent of altering the filtered bicarbonate load, could be discerned. Acidosis enabled the end-proximal luminal bicarbonate concentration to fall below normal values and reduced distal bicarbonate delivery.

Load dependence of proximal tubular fluid and bicarbonate reabsorption in the remnant kidney of the Munich-Wistar rat.

Studies were undertaken to characterize the pattern of proximal tubular fluid (APRH2O) and bicarbonate reabsorption (APRHCO3) in the remnant kidney of euvolemic Munich-Wistar rats. The remnant kidney rats were placed on a diet containing either low or normal protein. Collections were obtained in the early, mid-, and late proximal convoluted tubule. Single nephron glomerular filtration rate (SNGFR) increased from 40.2 nl/min in controls to 58.8 nl/min in low protein remnant kidney and 78.1 nl/min in normal protein remnant kidney rats. The filtered load of bicarbonate was 1,272, 1,641, and 2,013 pmol/min, in the three groups, respectively. APRH2O and APRHCO3 increased nearly in parallel. Most of the increase in reabsorption occurred in the early proximal tubule. Tubular hypertrophy could account for at least 20-40% of the increase in reabsorption, but the majority of the increase appeared to be a delivery-dependent response similar to that observed in normal rats after an acute increase in SNGFR.

Effect of acute changes in glomerular filtration rate on Na+/H+ exchange in rat renal cortex.

Studies were undertaken in Munich-Wistar rats to assess the influence of changes in filtered bicarbonate (FLHCO3), induced by changes in GFR, on Na+/H+ exchange activity in renal brush border membrane vesicles (BBMV). Whole-kidney and micropuncture measurements of GFR, FLHCO3, and whole-kidney and proximal tubule HCO3 reabsorption (APRHCO3) were coupled with BBMV measurements of H+ gradient-driven 22Na+ uptake in each animal studied. 22Na+ uptake was measured at three Na+ concentration gradients to allow calculation of Vmax and Km for Na+/H+ exchange. GFR was varied by studying animals under conditions of hydropenia, plasma repletion, and acute plasma expansion. The increase in GFR, FLHCO3, and APRHCO3 induced by plasma administration correlated directly with an increase in the Vmax for Na+/H+ exchange in BBMV. The Km for sodium was unaffected. In the plasma-expanded rats, the Vmax for Na+/H+ exchange was 22% greater than in the hydropenic rats (P less than 0.025) whereas APRHCO3 was 86% greater (P less than 0.001). These results indicate that increases in FLHCO3, induced by acute increases in GFR, stimulate Na+/H+ exchange activity in proximal tubular epithelium. This stimulation is a mechanism which can, in part, account for the delivery dependence of proximal bicarbonate reabsorption.

Control of proximal tubule fluid reabsorption in experimental glomerulonephritis.

We have recently shown that in the early autologous phase of nephrotoxic serum nephritis (NSN) single nephron glomerular filtration rate is unchanged from values in normal hydropenic control rats, but that single nephron filtration fraction and efferent arteriolar oncotic pressure (piE) are reduced because of a marked reduction in the glomerular capillary ultrafiltration coefficient. The present study was undertaken to examine the influence of this decline in piE as well as the other known determinants of peritubular capillary fluid exchange on absolute proximal fluid reabsorption (APR) in NSN. The findings indicate that APR and proximal fractional reabsorption are reduced significantly in NSN, relative to values in a separate group of age and weight-matched normal hydropenic control rats studied concurrently. In addition to the measured decline in piE, efferent arteriolar plasma flow (Qe) and peritubular capillary hydraulic pressure (Pc) were found to increase significantly, while interstitial oncotic pressure, estimated from hilar lymph, was not significantly different from values in control rats. Using a mathematical model of peritubular capillary fluid uptake we found that, assuming that the capillary permeability-surface area product and interstitial hydraulic pressure are unchanged in NSN, the observed changes in piE and Pc are sufficient to offset the effect of the increase in QE, yielding a calculated reduction in APR of approximately 4 nl/min, in excellent agreement with the observed mean decline of 4.1 nl/min. These findings suggest that control of APR in NSN is mediated by the same factors that regulate APR under normal physiological conditions, namely, the imbalance of forces governing peritubular capillary uptake of isotonic reabsorbate.

Determinants of glomerular filtration in experimental glomerulonephritis in the rat.

Pressures and flows were measured in surface glomerular capillaries, efferent arterioles, and proximal tubules of 22 Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). Linear deposits of rabbit and rat IgG and C3 component of complement were demonstrated in glomerular capillary walls by immunofluorescence microscopy. Light microscopy revealed diffuse proliferative glomerulonephritis, and proteinuria was present. Although whole kidney and single nephron glomerular filtration rate (GFR) in NSN (0.8 plus or minus 0.04 SE2 ml/min and 2 plus or minus 2 nl/min, respectively) remained unchanged from values in 16 weight-matched NORMAL HYDROPENIC control rats (0.8 plus or minus 0.08 and 28 plus or minus 2), important alterations in glomerular dynamics were noted. Mean transcapillary hydraulic pressure difference (deltaP) averaged 41 plus or minus 1 mm Hg in NSN versus 32 plus or minus 1 in controls (P LESS THAN 0.005). Oncotic pressures at the afferent (piA) end of the glomerular capillary were similar in both groups ( 16 mm /g) but increased much less by the efferent end (piE) in NSN (to 29 plus or minus 1 mm Hg) than in controls (33 plus or minus 1, P less than 0.025). Hence, equality between deltaP and piE, denoting filtration pressure equilibrium, obtained in control but not in NSN rats. While glomerular plasma flow rate was slightly higher in NSN (88 plus or minus 8 nl/min) than in controls (76 plus or minus 6, P greater than 0.2), the failure to achieve filtration equilibrium in NSN rats was primarily the consequence of a marked fall in the glomerular capillary ultrafiltration coefficient, Kf, to a mean value of 0.03 nl/(s times mm Hg), considerably lower than that found recently for the normal rat, 0.08 nl/(s times mm Hg). Thus, despite extensive glomerular injury, evidenced morphologically and by the low Kf, GFR remained normal. This maintenance of GFR resulted primarily from increases in deltaP, which tended to increase the net driving force for filtration, and thereby compensate for the reduction in Kf.

Load dependence of HCO3 and H2O reabsorption in the early proximal tubule of the Munich-Wistar rat.

Studies were undertaken in Munich-Wistar rats to evaluate the influence of variations in the filtered load of bicarbonate (FLHCO3) and water [single nephron glomerular filtration rate (SNGFR)] on the pattern of reabsorption along the accessible proximal tubule. SNGFR and FLHCO3 were varied by examining animals under conditions of hydropenia, plasma and extracellular volume expansion (VE), and VE plus aortic constriction. Water and HCO-3 reabsorption rates were measured at intervals along the proximal tubule, from very early segments to late segments, and these values compared with previous measurements in euvolemic rats. The earliest accessible portion of the proximal tubule reabsorbed HCO3 and water avidly; 40-55% of FLHCO3 and 18-20% of SNGFR were reabsorbed within the first millimeter. Moreover, when FLHCO3 was increased to as high as 2,400 pmol/min, HCO3 reabsorption rate in the first millimeter of the tubule increased concomitantly, reaching values as high as 1,000 pmol X mm-1 X min-1. In a similar fashion, water reabsorption in the first millimeter increased in direct relation to increases in SNGFR, reaching values as high as 13 nl X mm-1 X min-1 at SNGFR values of 70 nl/min. These results indicate that the early proximal tubule has much higher HCO3 and water reabsorptive rates and a stronger load dependence than has been found in later segments of the proximal tubule. The early proximal tubule thus appears to play a critical role in the maintenance of glomerulotubular balance.

The early proximal tubule: a high-capacity delivery-responsive reabsorptive site.

The proximal convoluted tubule is responsible for reclaiming almost all of the filtered bicarbonate, glucose, and amino acids, as well as 40% or more of the filtered sodium, fluid, chloride, and phosphate. Walker and co-workers demonstrated the importance of this nephron segment as a high-capacity transport site in the first mammalian micropuncture studies, and they suggested that the first portion of the proximal tubule played a particularly important role in the ability of the nephron to adapt to variations in filtered load. Since then, many studies using micropuncture and in vivo and in vitro microperfusion techniques have confirmed that the early proximal tubule has a higher transport capacity than the late proximal tubule for a number of solutes. Moreover, at least for bicarbonate, fluid, and chloride, the transport capacity is not static, but is in a dynamic state, adapting in response to changes in filtration. In this review we have focused on the high capacity and load dependence of early proximal bicarbonate and fluid reabsorption. In addition, we summarize the evidence for axial heterogeneity along the proximal convoluted tubule for transport of a variety of other solutes.

Bradykinin B2-receptors mediate the pressor and renal hemodynamic effects of intravenous bradykinin in conscious rats.

Bradykinin (BK) is a peptide which evokes remarkably different changes in cardiovascular function. Systemic bolus injection of BK results in a rapid drop in blood pressure via an endothelium-dependent mechanism. On the other hand, local administration of BK can activate a powerful pressor reflex by stimulating afferent nerves located in the abdominal viscera, the heart, and the kidney. In the present study, the cardiovascular and renal hemodynamic effects during sustained (intravenous infusion) and transient (intravenous bolus injection) elevations in circulating BK were characterized and the receptor mechanism eliciting these effects was investigated. Mean arterial pressure (MAP), heart rate (HR), and renal blood flow (RBF) were recorded from conscious unrestrained rats while five-point cumulative dose-response curves were constructed during infusion or bolus injection of BK (5-80 microg kg(-1)). Infusion of BK produced dose-dependent increases in MAP (maximum response = 27 +/- 3 mmHg) accompanied by a significant tachycardia (maximum response = 159 +/- 20 bpm), a 28 +/- 6% increase in RBF, and no changes in renal vascular resistance (RVR). The BK-induced increases in MAP, HR, and RBF were abolished after treatment with a ganglion blocker (maximum responses: MAP = 2 +/- 3 mmHg, HR = 13 +/- 4 bpm, RBF = 4 +/- 2%) or with an agent which blocks B2-receptors (maximum responses: MAP = 1 +/- 1 mmHg, HR = 6 +/- 5 bpm, RBF = 6 +/- 2%). In marked contrast, bolus administration of BK resulted in hypotensive responses (maximum decline in MAP = -37 +/- 4 mmHg), reflex tachycardia (maximum increase in HR = 45 +/- 9 bpm), increases in RBF (maximum response = 13 +/- 4%), and significant reductions in RVR (maximum response = 38 +/- 5%). These responses were also prevented when B2-receptors were blocked (maximum responses: MAP = 3 +/- 2 mmHg, HR = 17 +/- 6 bpm, RBF = 3 +/- 3%, RVR = 9 +/- 4%). In summary, BK infusions activated a cardiopressor reflex while BK injections caused hypotension. These opposite effects were both mediated via B2-receptors. These findings suggest that BK can have complex effects on the cardiovascular system that may be dependent on the sites, magnitude, and duration of elevated BK concentrations.

Intrarenal infusion of bradykinin elicits a pressor response in conscious rats via a B2-receptor mechanism.

Bradykinin (BK) is a peptide known to activate afferent nerve fibers from the kidney and elicit reflex changes in the cardiovascular system. The present study was specifically designed to test the hypothesis that bradykinin B2 receptors mediated the pressor responses elicited during intrarenal bradykinin administration. Pulsed Doppler flow probes were positioned around the left renal artery to measure renal blood flow (RBF). A catheter, to permit selective intrarenal administration of BK, was advanced into the proximal left renal artery. The femoral artery was cannulated to measure mean arterial pressure (MAP). MAP, heart rate (HR), and RBF were recorded from conscious unrestrained rats while five-point cumulative dose-response curves during an intrarenal infusion of BK (5-80 microg x kg(-1) x min(-1)) were constructed. Intrarenal infusion of BK elicited dose-dependent increases in MAP (maximum pressor response, 26+/-3 mmHg), accompanied by a significant tachycardia (130+/-18 beats/min) and a 28% increase in RBF. Ganglionic blockade abolished the BK-induced increases in MAP (maximum response, -6+/-5 mmHg), HR (maximum response 31+/-14 beats/min), and RBF (maximum response, 7+/-2%). Selective intrarenal B2-receptor blockade with HOE-140 (50 microg/kg intrarenal bolus) abolished the increases in MAP and HR observed during intrarenal infusion of BK (maximum MAP response, -2+/-3 mmHg; maximum HR response, 15+/-11 beats/min). Similarly, the increases in RBF were prevented after HOE-140 treatment. In fact, after HOE-140, intrarenal BK produced a significant decrease in RBF (22%) at the highest dose of BK. Results from this study show that the cardiovascular responses elicited by intrarenal BK are mediated predominantly via a B2-receptor mechanism.

Maturational development of glomerular ultrafiltration in the rat.

To ascertain the cause of low glomerular filtration rate in newborn and immature mammals, we measured glomerular pressures and flows directly in immature (30- to 45-day-old) euvolemic Munich-Wistar rats with surface glomeruli. As with total kidney GFR, single nephron (SN)GFR was found to be significantly lower than in adult rats, on average by 40% when corrected for kidney weight. Equality between efferent oncotic pressure and transglomeruler hydraulic pressure difference (deltaP) was usually achieved in immature rats, indicating that the glomerular capillary ultrafiltration coefficient is not a factor limiting SNGFR and GFR in immature rats. Although the average values for deltaP in immature rats were slightly, albeit significantly, lower than in adults, markedly lower values (79 +/- 5 vs. 136 +/- 10 nl/min per g kidney wt) for glomerular plasma flow rate (QA) proved to be the primary factor responsible for the lower SNGFR and GFR values in immature rats. Considerably higher values for afferent and efferent arteriolar resistances contributed to this low QA state in immature rats.

Effects of surgery on plasma volume and salt and water excretion in rats.

Surgical preparation of rats for micropuncture resulted in a marked decrease in sodium excretion (UNaV) compared to awake animals. Associated with surgery, hematocrit (Hct) rose. Studies were performed to determine whether the rise in Hct resulted from reduced plasma volume (PV) or increased red cell volume (RCV) and to explore the relation of such alterations to the fall in UNaV. PV and RCV were determined in the calm awake rat using 125I-albumin and 51Cr-labeled red blood cells. Micropuncture surgery was performed and RCV, PV, and Hct again measured. After anesthesia and femoral artery catheterization, Hct was not different from Hct in awake animals (42.9 +/- 2.8%). The Hct increased following surgery to 48.2 +/- 2.8% (P less than 0.001), accompanied by a large fall in PV (-18.9 +/- 2.3%, P less than 0.001) with no change in RCV. Plasma volume repletion to awake values restored UNaV toward levels appropriate for dietary intake in animals on a high salt diet. Althouth plasma repletion slightly increased UNaV above awake values in low salt diet rats, they continued to avidly retain salt with respect to total salt load.

Current concepts of sodium chloride and water transport by the mammalian nephron.

The decision of the editors to solicit a review for the Medical Progress series of this journal devoted to current concepts of the renal handling of salt and water is sound in that this important topic in kidney physiology has recently been the object of a number of new, exciting and, in some instances, quite unexpected insights into the mechanisms governing sodium excretion. These developments have come about largely as a consequence of the fact that segments of nephrons previously inaccessible to direct study are now readily accessible. Many of the findings to be discussed argue for extensive revision of a number of our current widely held views concerning the renal handling of sodium chloride and water. In the opinion of the authors, the strength of this argument rests in the fact that many of these new findings were obtained under circumstances that enabled workers to gain more direct access to the nephron than has been possible heretofore. This is not to say that areas of controversy and disagreement no longer exist. Wherever possible, these have been identified. In attempting to provide a comprehensive review of this topic, it has been necessary at times to overgeneralize and to disregard minor deficiencies in some of the studies cited. Finally, we wish to emphasize that a considerable portion of the information contained herein derives from work still under active investigation. Much of this contemporary work will undoubtedly withstand the rigors of future experimental scrutiny. It is inevitable, however, as William James so aptly noted in the quotation cited below, that some of our present ideas will need to be abandoned or revised in favor of newer, more convincing evidence. Seen in this light, the present effort is intended as nothing more than a timely survey of this active and fertile topic in renal physiology.

Effect of acute increases in filtered HCO3- on renal hydrogen transporters: II. H(+)-ATPase.

Adaptive increases in renal bicarbonate reabsorption occur in response to acute increases in filtered bicarbonate (FLHCO3). In a previous study, we showed that an increase in FLHCO3 induced by plasma volume expansion increased the Vmax for Na+/H+ exchange activity in renal cortical brush border membrane vesicles (BBMV), providing a potential mechanism for the adaptive increase in HCO3- reabsorption. The present studies were undertaken to determine whether the increase in FLHCO3 induced by plasma expansion also stimulates the other major H+ transporter in cortical BBMV, the H(+)-ATPase. H(+)-ATPase activity was assessed in BBMV obtained from hydropenic and plasma expanded Munich-Wistar rats, using a NADH-linked ATPase assay. H(+)-ATPase activity was measured as the ouabain and oligomycin-insensitive, bafilomycin A1-sensitive component of total ATPase activity. Acute plasma expansion doubled single nephron FLHCO3, and this change was associated with a 64% increase in the Vmax for H(+)-ATPase activity, with no change in apparent Km. The Vmax for H(+)-ATPase activity correlated directly with whole kidney GFR and FLHCO3 (r = 0.68 and 0.72, respectively), and with single nephron GFR and FLHCO3 (r = 0.76 and 0.80, respectively). Thus, the mechanism for the adaptive increase in proximal tubular HCO3- reabsorption that occurs in response to acute increases in FLHCO3 appears to be related to increased activity of both H(+)-ATPase and Na+/H+ exchange in the apical membrane of the proximal tubule epithelium.

Metabolic acidosis stimulates bicarbonate reabsorption in the early proximal tubule.

The early proximal tubule is the major site for renal bicarbonate reabsorption but little is known about the influence of acidosis on transport in this segment. This study examined early proximal bicarbonate reabsorption in rats with chronic metabolic acidosis (MA) (induced by NH4Cl administration). Rats were studied by free-flow micropuncture techniques, after varying degrees of plasma volume expansion to vary the filtered load of bicarbonate (FLHCO3). At FLHCO3 less than 700 pmol/min, both control and acidotic animals reabsorbed greater than 80% of the filtered load by 2 mm from Bowman's space. At higher FLHCO3 (700-1,100 pmol/min), reabsorption in the early proximal tubule was significantly greater in MA rats vs. control (633 +/- 26 vs. 449 +/- 24 pmol/min, between 1 and 2 mm from Bowman's space, P less than 0.001). This MA-induced stimulation of early proximal bicarbonate reabsorption was completely reversed by restoring systemic pH to normal either by acute hypocapnia or alkali infusion. Thus bicarbonate reabsorption in the early proximal tubule correlated closely with changes in systemic pH in rats with MA when bicarbonate delivery was increased by plasma expansion. The mechanism of this effect remains to be determined.

Renal disease in rats with type 2 diabetes is associated with decreased renal nitric oxide production.

AIMS/HYPOTHESIS: In several other models of chronic renal disease, decreases in renal nitric oxide activity and nitric oxide synthase (NOS) protein abundance have been demonstrated. Here, we studied diabetic obese Zucker (ZDF Gmi fa/fa) rats that develop severe hyperglycaemia and renal disease, together with their lean control animals, to determine if renal nitric oxide deficiency also occurs in this model. METHODS: Obese Zucker rats aged 10 to 12 weeks were maintained on Purina 5008 diet until 4, 8, or 11 months of age and compared with similarly maintained, 4- and 11-month-old lean Zucker rats. NOS activity and abundance of endothelial NOS (eNOS) and neuronal NOS (nNOS) were measured on homogenates of kidney cortex. Blood was analysed for glucose, lipids, creatinine, and blood urea nitrogen and kidney tissue was obtained for histology. RESULTS: Obese rats exhibited severe hyperglycaemia from 4 months of age and developed increasing hyperlipidaemia, proteinuria, and decreasing renal function with age compared to lean counterparts. At 4 months cortical NOS activity and nNOS abundance were lower in obese rats than in lean ones. At 11 months NOS activity remained depressed and nNOS abundance had declined further in obese rats. Glomerulosclerosis in the obese rats was mild at 4 months, becoming severe by 11 months. Lean rats had only mild age-dependent increases in glomerular injury. CONCLUSIONS/INTERPRETATION: The chronic renal disease that occurs in hyperglycaemic, obese Zucker rats is associated with decreased renal cortical nitric oxide production and increasing renal injury, although the changes do not resemble those of diabetic nephropathy in man.

Cryofixation, cryosubstitution, and immunoelectron microscopy: potential role in diagnostic pathology.

In immunoelectron microscopic investigations, retention of antigenic sites is crucial. Methods for preparing samples for conventional electron microscopy involve chemical fixatives followed by dehydration in organic solvents and embedding in plastic resins, all procedures potentially detrimental to antigenicity. Cryomethods provide a physical fixation alternative for the preparation of biological samples for ultrastructural, immunocytochemical, and microanalysis studies without the use of any chemicals. This can be particularly useful in diagnostic pathology, providing an alternative to conventional fixation methods which sometimes destroy the antigen in question. The recent development of a portable cryofixation device, the PS1000 Portable Metal Mirror Ultra-Rapid Cryofixation Unit (Delaware Diamond Knives, Inc., Wilmington, DE, USA), provides an opportunity to freeze tissue immediately after procurement for use in diagnostic immunocytochemistry studies. This feasibility study examined the quality of tissue preservation with this device, in terms of both preservation of cellular ultrastructure and immunolabeling. Human tonsil and thymus tissue was slam frozen and, after cryosubstitution in Lowicryl K11M, was examined by immunoelectron microscopy. Good ultrastructural preservation was obtained and reasonable immunolabeling with antibodies to AE1/AE3 keratin filaments was also observed.