Tubule Fluid Research Articles

Determination of disequilibrium pH in the rat kidney in vivo: evidence of hydrogen secretion.

The recent demonstration of elevated PCO2 in structures of the rat renal cortex indicated that previous determinations of disequilibrium pH (pHDq), and thus the differentiation of H+ secretion from bicarbonate reabsorption per se, required further evaluation. A new aspiration pH electrode was developed to allow tubule fluid to achieve chemical equilibrium at the PCO2 prevailing in vivo. In control and bicarbonate-loaded rats a pHDq was not observed in either proximal or distal tubules. After intravenous benzolamide a significant acid pHDq was observed in the proximal (but not the distal) nephron, and increased further during metabolic alkalosis. During combined metabolic alkalosis and respiratory acidosis a significant pHDq was present in the distal but not in the proximal tubule. Aldosterone administration to bicarbonate-loaded, hypercapnic rats did not alter the distal pHDq further. When present, the pHDq in the distal tubule was obliterated by carbonic anhydrase infusion. We conclude that proximal but not distal tubule fluid is in functional contact with carbonic anhydrase; the enzyme is in excess in the proximal lumen and H2CO3 did not accumulate even during conditions associated with increased H+ secretion; the basal rate of H+ secretion in the distal nephron accessible to cortical micropuncture is less than previously assumed. The data support the view that H+ secretion is the major mechanism of renal bicarbonate reabsorption.

The permeability characteristics of the isolated malpighian tubules of the housefly Musca domestica

1. 1. The permeability characteristics of the Malpighian tubules of Musca domestica have been determined by measuring tubule fluid: bathing medium ratios for d-, l- and CH 3-glueose, inulin. dextrans and polyethylene glycols. 2. 2. Molecules cross the epithelium in inverse relation to their molecular size, although the permeability to polyethylene glycols is independent of molecular chain length. 3. 3. No correlation between tubule fluid: bathing medium ratio and rates of fluid secretion is observed with CH 3-glueose and polyethylene glycols, but an inverse relationship does exist for inulin and dextrans indicating a molecular sieving effect for these molecules. 4. 4. The results are interpreted in terms of fluid flow occurring via the intercellular spaces with filtration across the tubule epithelium being linked with potassium secretion.

Terminal papillary collecting duct reabsorption of water, sodium, and potassium in Psammomys obesus.

Micropuncture studies were performed to assess the contribution of the terminal collecting duct to reabsorption of water, sodium, and potassium in the desert rodent, Psammomys obesus. Tubule fluid was collected at base and tip of the exposed papilla before (period I) and after (period II) administration of furosemide. In period I, a significant rise occurred in tubule fluid-to-plasma (TF/P) ratios of inulin, osmolality, and sodium and potassium from base to tip; 2.3 +/- 0.8% of filtered water and 5.1 +/- 1.7% of filtered sodium were reabsorbed by the collecting duct. In period II water, sodium, and potassium delivery to the collecting duct was significantly increased. Although TF/P ratios for inulin, osmolality, and sodium and potassium were lower at base and tip in period II than corresponding values in period I, all ratios increased between base and tip. Approximately 3 times as much water (7.4 +/- 1.8%) and twice as much sodium (10.1 +/- 3.0%) were reabsorbed by the exposed collecting duct in period II. These results reveal a remarkable reabsorptive capacity and suggest that the terminal collecting duct plays an important role in regulation of water and sodium excretion in Psammomys obesus.

3H]3-O-methyl-D-glucose transport from blood into the lumina of the seminiferous and epididymal tubules in intact and vasectomized hamsters.

The investigation demonstrated a restriction of [3H]3-O-methyl-D-glucose ([3H]3-OMG) net transport into seminiferous tubule fluid. Net transport of [3H]3-OMG into the cauda epididymidal tubule was even more limited. By 4 months after vasectomy, there was no statistical difference in the net transport of the isotope into the seminiferous tubule fluid, but there was a significant increase in net transport into cauda epididymidal fluid. These results demonstrate that the blood-epididymal barrier can be even more restrictive to molecular transport than the blood-testis barrier and that vasectomy can have subtle effects on characteristics of molecular transport in the cauda epididymidis.

Failure of tubule fluid osmolarity to affect feedback regulation of glomerular filtration.

Experiments were performed in Sprague-Dawley rats in order to distinguish between sodium chloride and total solute concentration as possible luminal signals capable of eliciting tubuloglomerular feedback responses. Early proximal flow rate (VEP), an index of nephron filtration rate, was measured without perfusion of the loop of Henle and during retrograde perfusion with solutions containing 20, 35, 60 to 100 mM NaCl and varying amounts of either urea or mannitol to achieve total solute concentrations of 130, 280, or 400 mosM. Perfusion flow rate was kept constant at 20 nl/min. Perfusion with a solution containing 20 mM NaCl and made hypo-, iso-, or hypertonic with urea or mannitol caused little or no change in VEP. Perfusion with a 35 mM NaCl solution made hypo-, iso-, or hypertonic with mannitol resulted in a fall of VEP of 6-7 nl/min. When NaCl concentration was 60 mM, VEP fell by 10-14 nl/min with solutions made hypo-, iso-, or hypertonic with urea or mannitol. With 100 mM NaCl solutions made hypo-, iso-, or hypertonic with mannitol, VEP fell approximately 12 nl/min. These results indicate that feedback responses are determined by the NaCl concentration of the perfusate and that this NaCl dependency is not modified by varying perfusate osmolarity between 130 and 400 mosM with urea or mannitol as osmotic agents.

Effects of sodium orthovanadate on whole kidney and single nephron function

The renal effects of sodium vanadate (Na3VO4), an inhibitor of sodium-potassium-ATPase recently shown to be a potent diuretic, were studied by using clearance and micropuncture techniques in nondiuretic anesthetized rats. Administration of 1.0 mumole of sodium vanadate (high dose) increased urine flow rate (V) from 9.8 +/- 1.4 to 17.5 +/- 4.0 microliter/min (mean +/- SEM, P < 0.025), UNaF from 1.73 +/- 0.36 to 3.05 +/- 0.65 microEq/min (P < 0.025), and FENa from 0.67 +/- 0.15 to 1.24 +/- 0.28% (P < 0.025)., No significant changes in GFR or RPF were observed. Late proximal tubular-fluid-to-plasma (F/P) inulin decreased from 2.28 +/- 0.19 to a minimum value of 1.38 +/- 0.06 (P < 0.025). Absolute water reabsorption decreased from 15.8 +/- 3.5 to 6.5 +/- 1.7 nl/min (P < 0.025) and fractional water reabsorption from 52.0 +/- 4.4 to 26.5 +/- 4.1% (P < 0.025). The injection of 0.5 mumole of sodium vanadate (low dose) resulted in no significant changes in V. Late proximal F/P inulin decreased, however, from 2.37 +/- 0.14 to a minimum value of 1.59 +/- 0.12 (P < 0.025). SNGFR remained unchanged, as did GFR and RPF. UNaV increased from 1.41 +/- 0.35 to 2.25 +/- 0.35 microEq/min (P < 0.025), and FENa rose from 0.64 +/- 0.16 to 0.91 +/- 0.15% (P < 0.025). The decrease in F/P inulin was observed in all but one animal, even in the absence of a diuretic response. The amount of fluid remaining in the lumen of the late proximal tubule was virtually the same in both low- and high-dose animals (18.9 +/- 3.0 and 19.5 +/- 3.4 nl/min, respectively). We conclude that sodium vanadate causes a decrease in superficial proximal tubule fluid and salt reabsorption. Inasmuch as the low dose does not result necessarily in a diuretic response, an increase in fluid reabsorption distal to the late proximal tubule must take place.

Isolated nephron segments in a rabbit model of ischemic acute renal failure.

Renal artery occlusion has been extensively used in animal models to cause acute renal failure. The present isolated tubule microperfusion studies were designed to examine the transport characteristics of multiple nephron segments of the rabbit after 60 min of total renal ischemia. Preliminary studies showed that this maneuver produced significant and persistent elevations of serum creatinine. The tubules were perfused and bathed with artificial solutions simulating ultrafiltrate and studied at 37 degrees C. Four nephron segments were examined. Ischemia reduced proximal convoluted tubule fluid reabsorption 77% (0.72 +/- 0.11 vs. 0.14 +/- 0.06 nl . mm-1 . min-1, P less than 0.01) and cortical proximal straight tubule fluid reabsorption 88% (0.54 +/- 0.10 vs. 0.06 +/- 0.03 nl . mm-1 . min-1, P less than 0.005). Ischemia reduced the ability of the thick ascending limb of Henle's loop to lower perfusate chloride ion concentration 60% (-47 +/- 9 vs. -19 +/- 3 meq/liter, P less than 0.02) and its diluting ability 49% (-87 +/- 15 vs. -44 +/- 7 mosmol/kg H2O, P less than 0.01). Ischemia reduced the antidiuretic hormone-dependent osmotic water permeability of the cortical collecting tubule 59% (0.0203 +/- vs. 0.0083 +/- 0.0020 cm/s, P less than 0.01). Morphologic alterations were noted in the proximal segments but not in the distal segments of the nephron. The current studies demonstrate that 60 min of renal ischemia impairs the transport capability of all proximal and distal nephron segments studied.

Importance of efferent arteriolar vascular tone in regulation of proximal tubule fluid reabsorption and glomerulotubular balance in the rat.

Micropuncture study was performed in 21 mildly volume-expanded Munich-Wistar rats before and during partial aortic constriction to examine the effects of endogenous prostaglandins (PG) and angiotensin II (AII) on single nephron glomerular filtration rate (SNGFR) and absolute proximal reabsorption rate (APR). Animals received either vehicle (group 1), indomethacin (group 2), or indomethacin plus saralasin (group 3). Before aortic constriction, these inhibitors were without effect on values of SNGFR and APR. In group 1 rats, reduction in mean renal arterial perfusion pressure (RAP) to approximately 65 mm Hg resulted in marked and proportional declines in SNGFR and APR. With equivalent reduction in RAP in group 2 rats, however, SNGFR fell to a lesser extent and APR tended to increase slightly above preconstriction values. Indomethacin administration was therefore associated with disruption of glomerulotubular balance. In view of the roughly equivalent declines in afferent arteriolar resistance measured in groups 1 and 2, the magnitude of increase in efferent arteriolar resistance (R(E)) appeared to be of major importance in determining the observed presence or absence of glomerulotubular balance. Thus, the lesser fall in SNGFR in group 2 than in group 1 was a result of the higher value for glomerular capillary hydraulic pressure in group 2, a consequence of the higher value of R(E). The higher average value for APR during reduced RAP in group 2 than in group 1 is also attributable to this pronounced rise in R(E), the effect of which was to augment the net reabsorptive pressure both by favoring higher postglomerular oncotic pressure and lower downstream (peritubular capillary) hydraulic pressure. Since intrarenal release of AII is enhanced when RAP declines, and because AII is known to raise R(E) selectively, it is likely that endogenous AII brought about the marked increase in R(E) in group 2, which was readily demonstrable only in indomethacin-treated rats, presumably because endogenous PG synthesis was suppressed. In keeping with this conclusion, when the action of endogenous AII was inhibited by saralasin in group 3 rats, reduction in RAP failed to induce a rise in R(E), so that net filtration and reabsorption pressures again declined proportionally, as did SNGFR and APR. The present evidence therefore suggests that glomerulotubular balance is influenced to an important extent by the prevailing vasomotor tone of the efferent arteriole.

Tubular transport processes in proximal tubules of hypothyroid rats. Micropuncture studies on isotonic fluid, amino acid and buffer reabsorption.

In three groups of rats: control animals (n=18), hypothyroid (TX) rats (n=21) 8–10 weeks after i. p. injection of Na131I (0.4–1 mCi), and hypothyroid rats (n=16) substituted with thyroxine (TX+T3, 50 μg/kg B.W. for 7 days) proximal tubule fluid reabsorption, and transtubular concentration difference of3H-l-histidine and3H-glycodiazine at zero net flux were measured. The effectiveness of TX was confirmed by delayed growth (200±12 as compared to 400±42 g B. W.), diminished plasma concentration of thyroxine (8.0 as compared to 53 μg/l), and increased plasma concentration of TSH (0.75 as compared to 0.15 mg/l).

Some factors affecting Malpighian tubule fluid secretion and transepithelial potential in Locusta migratoria L.

Both fluid secretion and transepithelial potential were stimulated by cAMP. Fluid secretion was unaffected by 5-HT over the concentration range 10(-8)-10(-4) M. The presence of ouabain in the bathing medium effected a decrease in transepithelial potential.

Volume expansion-induced alterations in proximal tubule chloride gradient in the rat.

To elucidate the mechanisms by which acute volume expansion (AVE) induces a decrease in proximal tubule transepithelial chloride gradient, male Sprague-Dawley rats were studied before and after AVE with Ringer lactate. In group 1, after AVE equivalent to 10% body wt, there were decreases in both tubule fluid to plasma inulin ratio ((TF/P)In) (from 2.28 +/- 0.10 to 1.57 +/- 0.05) and tubule fluid to ultrafiltrate chloride ratio ((TF/UF)Cl) (from 1.25 +/- 0.02 to 1.18 +/- 0.02). Group 2 was studied during carbonic anhydrase inhibition (CAI) produced by benzolamide before and during superimposed AVE (20% body wr). Both (TF/P)In (from 1.91 +/- 0.10 to 1.41 +/- 0.08) and (TF/UF)Cl (from 1.07 +/- 0.02 to 1.01 +/- 0.01) decreased. Group 3 was studied during maintained AVE (15% body wt) as a control for group 4, in which CAI was superimposed on maintained AVE. In group 3, (TF/P)In and (TF/UF)Cl did not change, but in group 4 CAI was associated with a decrease in (TF/P)In (from 1.55 +/- 0.05 to 1.21 +/- 0.05) and in (TF/UF)Cl (from 1.16 +/- 0.01 to 1.04 +/- 0.07). These data suggest that in the superficial proximal convoluted tubule of the rat, AVE-induced alterations in transepithelial chloride gradient are dependent on a mechanism(s) other than changes in carbonic anhydrase-mediated bicarbonate reabsorption.

Suppression of potassium-recycling in the renal medulla by short-term potassium deprivation

Recently we proposed that potassium, like urea, normally undergoes medullary recycling from collecting tubule to the pars recta or descending limb of the juxtamedullary nephron and suggested that the extent of recycling is a function of the concentration of potassium in collecting tubule fluid. To test this hypothesis further, we fed young rats a potassium-free diet for 3 days and then prepared them for micropuncture of the left renal papilla. Compared to findings in normally fed animals, potassium deprivation caused a significant fall in plasma potassium and urinary excretion of potassium. There was a striking decrease in the fraction of filtered potassium remaining at the end of the justamedullary descending limb for 94 +/- 11% to 38 +/- 3% (P less than 0.001). The latter value is not significantly different from the fraction of filtered sodium remaining (36 +/- 4%) and suggests that net addition of potassium to the pars recta or descending limb was completely abolished. A correlation was observed between the fraction of filtered potassium remaining at the end of the descending limb and either urinary potassium excretion (P less than 0.001) or urinary potassium concentration(P less than 0.001) in the contralateral unexposed kidney. These results lend further support to the hypothesis of medullary recycling of potassium.

Luminal fluid proteins of the male rat reproductive tract.

Micropuncture samples from rat testis and epididymis were subjected to protein analysis, microelectrophoresis and ABP determinations. Rete testis fluid contained only 5% of the total protein concentration of blood serum. Other reproductive tract fluids contained one-third to one-half the total protein concentrations of blood. Electrophoresis of intratubular fluids from the various testicular and epididymal zones allowed separation of reproductive tract-specific protein bands in each fluid studied. Luminal fluids from the epididymal tubule contained several proteins not appearing in blood. Analysis for androgen binding activity revealed higher concentrations in rete testis fluid than in seminiferous tubule fluid. Caput epididymal fluid contained the highest ABP concentration.

Mechanism of inhibition of proximal tubule fluid reabsorption after exposure of the rat kidney to the physical effects of expansion of extracellular fluid volume.

The natriuresis and concomitant decline in absolute proximal reabsorption (APR) that occur in rats in response to saline loading are blunted markedly when renal perfusion pressure is reduced immediately before, but not after, the volume load. To ascertain the mechanism responsible for these differences between early clamp (EC) vs. late clamp (LC), intracapillary and interstitial determinants of peritubular capillary uptake of APR were measured in seven LC and seven EC Munich-Wistar rats before and after isotonic saline loading (80% body wt). With volume expansion in LC animals, we observed a marked decline in APR (averaging 11+/-1 nl/min), associated with large increases in urinary sodium excretion rate, which averaged 8+/-2 mueq/min. In EC, the changes in urinary sodium excretion rate (+1+/-0 mueq/min) and APR (-3+/-1 nl/min) with volume expansion were smaller in magnitude. Since peritubular capillary reabsorption coefficient and mean peritubular transcapillary hydraulic pressure difference did not change with saline loading in LC, the marked fall in APR was attributed primarily to a measured large decline in mean peritubular transcapillary oncotic pressure difference (deltapi). Despite an equivalent mean fall in deltapi with volume expansion in EC, near-constancy of APR was found to be associated with a simultaneous and equivalent decline in mean peritubular transcapillary hydraulic pressure difference (a consequence of decreased mean peritubular capillary hydraulic pressure), which effectively offset the fall in deltapi. These results demonstrate the importance of hydraulic pressure patterns of the peritubular capillaries in modulating APR and are consistent with the view that Starling forces across the postglomerular microcirculation play a fundamental role in determining APR.

Modification of feedback influence on glomerular filtration rate by acute isotonic extracellular volume expansion.

The behavior of the feedback mechanism, that causes glomerular capillary pressure and filtration rate to decrease when tubule fluid flow rate through the loop of Henle of the same nephron is increased, was examined in rats before and during isotonic extracellular fluid volume expansion. The loop of Henle was perfused from the late proximal tubule at either 10 or 40 nl/min while proximal fluid was collected to measure single nephron filtration rate (SNGFR), while proximal stop-flow pressure (PSF) was measured, or while fluid was collected from the early distal tubule to assess reabsorption of fluid and electrolytes by the loop of Henle. During control periods increasing loop perfusion caused SNGFR to decrease 37%, PSF to decrease 19%, and absorption of fluid, sodium and chloride by the loop of Henle to increase. After 1 h of infusion of isotonic NaCl solution the same change in loop flow causes a 19% decrease in SNGFR and an 8% decrease in PSF. Fluid absorption by the loop of Henle did not increase with increased loop perfusion. Increases in Na and Cl absorption were similar to the increases in control periods. The smaller decreases in SNGFR and PSF indicate that acute volume expansion decreases the sensitivity of the feedback response. The mechanism of this decrease in gain could involve interference with local generation or action of angiotensin, or a change in the composition or pressure of interstitial fluid tending to dilate the pre-glomerular resistance vessels.

Glomerular and renal hemodynamics during converting enzyme inhibition (SQ20,881) in the dog.

It has been suggested that intrarenal levels of angiotensin II may preferentially control efferent arteriolar resistance or may influence the glomerular filtration coefficient (Kf). To examine these possibilities, micropuncture and clearance experiments were performed on nine anesthetized dogs evaluating renal and glomerular hemodynamics before and during the administration of an angiotensin converting enzyme inhibitor (SQ20,881). During the micropuncture measurements, renal arterial pressure was reduced to range of 85 to 90 mm Hg in order to maximize renin secretion and intrarenal formation of angiotensin II. Also, this procedure minimizes potential errors in the determination of single nephron glomerular filtration rate (SNGFR) and of glomerular pressure when estimated by techniques that require complete blockade of proximal tubule fluid flow. During the administration of SQ20,881, a converting enzyme inhibitor (CEI), renal blood flow increased significantly by 13%, but GFR was not altered. There were no significant alterations in SNGFR, proximal tubule pressure, peritubular capillary pressure or estimated glomerular pressure. By using the micropressure measurements in combination with the whole kidney hemodynamic data, it was estimated that afferent resistance was reduced 23%. Although significant decreases in efferent resistance could not be documented, there was a tendency for this variable to decrease also. Neither Kf nor effective filtration pressure were altered significantly by CEI. These results do not support the contention that intrarenal effects of angiotensin II are exerted predominantly on the efferent arteriolar resistance segments; rather, they suggest that angiotensin may exert a modest tonic effect on both pre- and postglomerular resistance elements in the anesthetized hydropenic dog.

Effect of parathyroid hormone on bicarbonate absorption by proximal tubules in vitro

The effect of parathyroid hormone on bicarbonate absorption was tested in rabbit proximal renal tubules perfused in vitro. In proximal straight tubules 0.05 U/ml of parathyroid hormone caused a large and reversible increase in the steady-state bicarbonate concentration in tubule fluid. Further, the rates of bicarbonate and fluid absorption (measured at faster flow rates) were inhibited approximately 50% by the hormone. We conclude that parathyroid hormone directly inhibits fluid and bicarbonate absorption by proximal straight tubules, causing an increase in the bicarbonate concentration in the tubule fluid, and we suggest that this action of the hormone contributes to the increase in renal bicarbonate excretion that is generally caused by the hormone. In proximal convoluted tubules, parathyroid hormone was previously demonstrated by other investigators to inhibit fluid and bicarbonate absorption approximately proportionally, so that there was little or no change in the bicarbonate concentration in tubule fluid. In agreement we found in the present studies that 0.05 U/ml of the hormone did not affect the steady-state bicarbonate concentration in proximal convoluted tubule fluid and that 5 U/ml caused only an equivocal increase in tubule fluid bicarbonate concentration.

Mechanism of the uricosuric activity of ticrynafen.

Studies employing pyrazinamide, which have included the measurements of clearances of pyrazinamide and pyrazinoate, as well as of ticrynafen itself and its principal metabolite, have lent support to the hypothesis that ticrynafen inhibits the tubular reabsorption of both filtered and secreted urate by nephrons of the human kidney. The inhibition of the reabsorption of secreted urate by ticrynafen appears to be quantitatively important for eliciting its uricosuric and hypouricemic responses. Because the uricosuric and natriuretic responses correlate with urinary ticrynafen concentrations, the drug appears to inhibit tubular reabsorption after gaining access to tubule fluid. As a consequence of its extensive binding to plasma proteins, clearance data suggest that tubular secretion of ticrynafen is necessary for its action.

Renal potassium transport: contributions of individual nephron segments and populations

General features of the processes that contribute to renal potassium excretion are understood from clearance, stop-flow, micropuncture, and in vitro microperfusion experiments. However, the complex architecture of the kidney has made it difficult to examine individual nephron segments in all parts of the kidney. Accordingly, the extent to which distinguishable nephron populations, such as superficial and deep, may differ in their contributions to overall potassium excretion are not known. Also, the nature of transport processes across the successive segments of the nephrons (including not only the underlying cellular mechanisms, but even the direction of transport) is not known for all segments in any one nephron population. Excreted potassium is derived both from filtered potassium that escapes reabsorption and from secreted potassium. The filtered portion is large in amphibians and may be larger than generally recognized in mammals. The remainder is secreted primarily by distal nephron segments (distal tubule and cortical collecting duct). Potassium is also secreted into descending limbs of Henle loops; apparently this fraction is recycled from collecting ducts, and so does not represent an additional quantity of potassium transferred from blood to tubule fluid. Systemic factors that affect potassium excretion (potassium intake, sodium chloride intake, mineralocorticoid hormone levels, acid-base balance, and diuretic treatments) do so by modifying the net uptake of potassium from blood to cell and by altering the rate of fluid flow through the distal nephron. Under most circumstances, the distal nephron in the cortex appears to secrete potassium and the medullary collecting duct reabsorbs potassium. Although it is clear that successive nephron segments transport potassium in different ways, evidence to date does not indicate that potassium is handled differently by superficial nephrons compared to nephrons whose glomeruli lie in the deeper levels of the cortex.

Effect of chronic potassium loading on potassium secretion by the pars recta or descending limb of the juxtamedullary nephron in the rat.

Recently we demonstrated potassium secretion by the pars recta or by the descending limb of the juxtamedullary nephron. The purpose of this present investigation is to study the effect of a chronic high-potassium intake on this phenomenon. Fractional reabsorption of water and sodium by the juxtamedullary proximal nephron was decreased when compared to that in normal hydropenic rats. There was a striking increase in the fraction of filtered potassium at the end of the juxtamedullary descending limb from 94+/11% to 180+/18%, which was principally a result of enhanced potassium secretion. When the concentration of potassium in the collecting tubule fluid of potassium-loaded rats was reduced after the administration of amiloride, a sharp fall was observed in the amount of potassium which reached the end of the descending limb (64+/8%). A direct correlation was observed between the fraction of filtered potassium at the descending limb and the potassium concentration in the final urine (P less than 0.001). The findings suggest that potassium, like urea, normally undergoes medullary recycling, which is enhanced by chronic potassium loading.