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M A Knepper

Publications and source records attributed to M A Knepper.

At least 199 records · Page 11Linked to original sources
PubMed1986

Spontaneous luminal disequilibrium pH in S3 proximal tubules. Role in ammonia and bicarbonate transport.

We determined whether a spontaneous luminal disequilibrium pH, pHdq (pH measured - pH equilibrium), was present in isolated perfused rabbit S2 and S3 proximal tubules. Luminal pH was measured by perfusing with the fluorescent pH probe 1,4-DHPN, and the equilibrium pH was calculated from the measured collected total CO2 and dissolved CO2 concentrations. S2 tubules failed to generate a spontaneous pHdq. S3 tubules generated a spontaneous acidic pHdq of -0.46 +/- 0.15 (P less than 0.05), which was obliterated following the addition of carbonic anhydrase (0.1 mg/ml) to the perfusate. In S3 tubules perfused and bathed in 4 mM total ammonia, luminal total ammonia rose from 4.08 +/- 0.05 mM (perfusate) to 4.95 +/- 0.20 mM (collected fluid) (P less than 0.02). Carbonic anhydrase added to the perfusate prevented the rise in the collected total ammonia concentration. We conclude that the rabbit S3 proximal tubule lacks functional luminal carbonic anhydrase. The acidic pHdq in the S3 segment enhances the diffusion of NH3 into the lumen. In contrast, the S2 segment has functional luminal carbonic anhydrase.

Ammonia↗
PubMed1985

Ammonia transport in the mammalian kidney.

Ammonia, an important urinary buffer in mammals, is synthesized primarily in the proximal tubules and is transferred to the final urine by a sequence of specialized transport processes. The pathway of ammonia transfer to the urine involves secretion into the proximal tubules, absorption from the loops of Henle, accumulation in the renal medullary interstitium, and secretion into the collecting ducts. Ammonia is transported as NH3 at some nephron sites and as NH+4 at others. In this paper, we discuss the physical basis of NH3 and NH+4 transport in epithelia and then describe ammonia transport mechanisms in individual nephron segments. Information about ammonia transport in individual nephron segments from isolated perfused tubule studies is integrated with data from in vivo studies to obtain an expanded overall model of renal ammonia handling.

Acids↗
PubMed1985

Deoxycorticosterone-stimulated bicarbonate secretion in rabbit cortical collecting ducts: effects of luminal chloride removal and in vivo acid loading.

To assess the role of cortical collecting duct bicarbonate secretion in the regulation of net acid excretion, we have sought to identify what factors influence the secretion rate. Net and unidirectional bicarbonate fluxes were measured in isolated perfused cortical collecting ducts from deoxycorticosterone-treated rabbits. The collecting ducts secreted bicarbonate at 11-24 pmol X mm-1 X min-1, confirming the high rate seen in earlier studies. Oral acid loading (50 mM NH4Cl drinking water) completely inhibited the net bicarbonate secretion. The bath-to-lumen flux was markedly reduced with acid loading, but the lumen-to-bath flux changed very little. In tubules from rabbits treated with deoxycorticosterone (but not NH4Cl), luminal chloride replacement with either sulfate or gluconate completely and reversibly inhibited the net bicarbonate secretion. The bath-to-lumen flux was greatly inhibited, but there was little change in the lumen-to-bath flux. We conclude: 1) High rates of bicarbonate secretion can be induced in rabbit cortical collecting ducts by chronic treatment of the animals with deoxycorticosterone. 2) When deoxycorticosterone-treated rabbits were made acidotic by oral administration of NH4Cl, the bicarbonate secretion was prevented, indicating that the systemic acid-base state of the animal may be an important factor regulating bicarbonate secretion. 3) Replacement of chloride in the lumen with sulfate inhibits bicarbonate secretion in the cortical collecting duct, an effect which may explain in part the decrease in urinary pH in response to sulfate infusions in mineralocorticoid-stimulated animals.

Acid-Base Equilibrium↗
PubMed1985

Role of inner medullary collecting duct NaCl transport in urinary concentration.

Mathematical modeling and simulation techniques were used to analyze the role of medullary collecting duct NaCl transport in the urinary concentrating process. The mathematical model incorporated experimentally determined epithelial transport parameters and anatomical parameters obtained chiefly from experiments in rabbit kidneys. The simulations predict that solute concentration profiles along the medullary collecting ducts are highly sensitive to the rate and pattern of active NaCl absorption along the length of the collecting duct system. When active NaCl absorption was assumed to be zero in the outer medullary collecting duct and to increase along the inner medulla to a very high value at the papillary tip, the simulated solute concentration profiles in the medullary collecting ducts as well as relative concentrations between different inner medullary structures agreed well with experimental data. However, despite optimal choice of collecting duct transport parameters and the use of experimentally determined permeability coefficients, only modest total solute gradients could be generated axially in the inner medullary interstitium, and passive luminal dilution did not occur in the thin ascending limb. We conclude: 1) Axial heterogeneity of transport properties along the inner medullary collecting duct must be assumed to explain in vivo findings from micropuncture and microcatheterization studies. 2) Active NaCl transport from the inner medullary collecting ducts is important chiefly for efficient conservation of NaCl rather than for concentration of solutes in the renal inner medulla. 3) Important inconsistencies exist between several previously reported experimental observations and the theoretical requirements for passive luminal dilution in the thin ascending limb of Henle's loop.

Animals↗
PubMed1985

Ammonium replaces potassium in supporting sodium transport by the Na-K-ATPase of renal proximal straight tubules.

Ammonium is capable of replacing potassium to support the hydrolysis of ATP by the Na-K-ATPase in many tissues. Whether ammonium supports the transport function of the Na-K-ATPase in the kidney (where such a substitution could be physiologically important) has not been studied, however. To address this issue, we determined the rates of fluid absorption and bicarbonate absorption (measured as total carbon dioxide) in the proximal straight tubule of the rabbit where both processes are dependent on sodium transport by the Na-K-ATPase. Both fluid absorption and total carbon dioxide absorption were significantly inhibited by potassium removal from the bath and perfusate. When ammonium was included in the perfusate and bath (replacing potassium completely), both fluid absorption and bicarbonate absorption occurred at rates indistinguishable from the rates observed with potassium present (and ammonium absent). We conclude that ammonium can replace potassium in supporting sodium transport by the Na-K-ATPase of the proximal straight tubule. Interaction between ammonium and potassium on the Na-K-ATPase could be important in this and other nephron segments.

Absorption↗
PubMed1985

Ammonia and bicarbonate transport by rat cortical collecting ducts perfused in vitro.

We measured bicarbonate and ammonia transport by isolated perfused cortical collecting ducts from deoxycorticosterone-treated rats. With no ammonia in the perfusate and bath solutions, the collecting ducts secreted bicarbonate. The bicarbonate secretion was prevented when the rats were given 40 mM NH4Cl to drink. When 4 mM total ammonia was added to the perfusate and bath, the collecting ducts secreted ammonia and the direction of bicarbonate transport reversed toward absorption. Under those conditions the collected total ammonia concentration exceeded the value predicted by the diffusion-trapping model, assuming pH equilibrium. However, when carbonic anhydrase was added to the perfusate (to assure pH equilibrium), the collected total ammonia concentration decreased to the level predicted by the diffusion-trapping model. We conclude that rat cortical collecting ducts can secrete bicarbonate at substantial rates; the rate of bicarbonate secretion is modified by changes in the acid-base intake of the rats; ammonia secretion occurs by simple nonionic diffusion in this segment; the ammonia secretion is enhanced by the presence of acidic pH disequilibrium in the lumen; and ammonia in the perfusion and bath solutions inhibits bicarbonate secretion by rat cortical collecting ducts, a response that may be important for the regulation of renal bicarbonate excretion.

Ammonia↗
PubMed1985

Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, vasopressin, and deoxycorticosterone.

Several factors interact to maintain precise control of electrolyte transport in the mammalian cortical collecting duct. We have studied the effects of deoxycorticosterone, arginine vasopressin, and bradykinin on net transepithelial sodium and potassium transport in isolated, perfused rat cortical collecting ducts. Chronic administration of deoxycorticosterone to rats increased both sodium absorption and potassium secretion above very low basal levels. Consequently, deoxycorticosterone-treated rats were used for all remaining studies. Arginine vasopressin (10(-10) M in the bath) caused a sustained fourfold increase in net sodium absorption and a sustained threefold increase in net potassium secretion. Bradykinin (10(-9) M in the bath) caused a reversible 40-50% inhibition of net sodium absorption without affecting net potassium transport or the transepithelial potential difference. In the perfusate, up to 10(-6) M bradykinin had no effect. We conclude: As in rabbits, chronic deoxycorticosterone administration to rats increases sodium absorption and potassium secretion in cortical collecting ducts perfused in vitro. Arginine vasopressin causes a reversible increase in net potassium secretion and net sodium absorption. Bradykinin in the peritubular bathing solution reversibly inhibits net sodium absorption, possibly by affecting an electroneutral sodium transport pathway.

Animals↗
PubMed1985

Bicarbonate secretion and chloride absorption by rabbit cortical collecting ducts. Role of chloride/bicarbonate exchange.

Cortical collecting ducts (CCD) from rabbits treated with deoxycorticosterone (DOC) actively secrete bicarbonate at high rates. To investigate the mechanism of bicarbonate secretion, we measured bicarbonate and chloride transport in CCD from rabbits treated with DOC for 9-24 d. Removal of chloride (replaced with gluconate) from both perfusate and bath inhibited bicarbonate secretion without changing transepithelial voltage. Removal of chloride only from the bath increased bicarbonate secretion, while removal of chloride only from the perfusate inhibited secretion. In contrast to the effect of removing chloride, removal of sodium from both the perfusate and bath (replacement with N-methyl-D-glucamine) did not change the rate of bicarbonate secretion. The rate of bicarbonate secretion equaled the rate of chloride absorption in tubules bathed with 0.1 mM ouabain to inhibit any cation-dependent chloride transport. Under these conditions, chloride absorption occurred against an electrochemical gradient. Removal of bicarbonate from both the perfusate and bath inhibited chloride absorption. Removal of bicarbonate only from the bath inhibited chloride absorption, while removal of bicarbonate from the lumen stimulated chloride absorption. We conclude that CCD from DOC-treated rabbits actively secrete bicarbonate and actively absorb chloride by an electroneutral mechanism involving 1:1 chloride/bicarbonate exchange. The process is independent of sodium.

Absorption↗
PubMed1984

Ammonia and bicarbonate transport by thick ascending limb of rat kidney.

Ammonia and bicarbonate transport by the thick ascending limb of rat kidney was studied to determine whether this segment contributes to the regulation of renal ammonia and net acid excretion. Cortical and medullary thick ascending limbs were perfused in vitro at 1.0-1.5 nl X min-1 X mm-1 with HCO3-buffered solutions. There was no significant net fluid transport. With 4 mM ammonia in bath and perfusate, transepithelial voltage averaged 6-9 mV, lumen positive, and did not differ between the two segments. The mean ammonia concentration in collected tubule fluid was 2.8 mM with cortical segments and 2.3 mM with medullary segments, indicating net absorption of ammonia. Furosemide (10(-4) M) in the perfusate eliminated ammonia absorption in medullary thick ascending limbs and converted net absorption to net secretion in cortical thick ascending limbs. Furosemide reduced transepithelial voltage to near zero in every tubule. Cortical and medullary thick ascending limbs also absorbed bicarbonate, indicating that their tubule fluid was acidified relative to the bath. Therefore, absorption of ammonia could not have occurred by nonionic diffusion. The absorption most likely was due to direct transport of NH4+. The possible mechanisms involved are discussed, and it is proposed that absorption of ammonia by thick ascending limbs provides a source for its accumulation in the renal medulla and secretion into the collecting ducts.

Acid-Base Equilibrium↗
PubMed1984

Mechanism of ammonia secretion by cortical collecting ducts of rabbits.

The collecting duct system is a major site of ammonia addition to the tubule fluid. To study the mechanisms involved, we measured total ammonia and total CO2 transport in isolated, perfused cortical collecting ducts (CCD) from deoxycorticosterone-(DOC) treated rabbits. Perfusate and bath solutions contained 25 meq/liter HCO3 and 4 mM total ammonia. Net fluid transport was not significantly different from zero. Net secretion of total CO2 occurred in all tubules (mean collected concentration, 44.2 mM). Despite bicarbonate secretion, there was net secretion of total ammonia (mean collected concentration, 6.4 mM). There was no detectable ammonia addition to the collected fluid when ammonia was excluded from the perfusate and bath, ruling out a major contribution from synthesis. Ouabain did not significantly affect net transport of total ammonia or total CO2. To test the hypothesis that an acid pH disequilibrium may lower the luminal pH enough to drive ammonia secretion by nonionic diffusion, we perfused CCD from DOC-treated rabbits with carbonic anhydrase (CA) (0.1 mg/ml). Without CA, there was net total ammonia secretion (-2.2 pmol X min-1 X mm-1) and net total CO2 secretion (-16.6 pmol X min-1 X mm-1). Luminal CA converted the net total ammonia secretion to net absorption (1.0 pmol X min-1 X mm-1) while the bicarbonate secretion persisted (-11.2 pmol X min X mm-1). We conclude that total ammonia secretion in these tubules occurs primarily by diffusion of NH3 and is dependent on a luminal acid pH disequilibrium.

Ammonia↗
PubMed1983

Nitrogen-14 nuclear magnetic resonance spectroscopy of mammalian tissues.

The use of 14N nuclear magnetic resonance (NMR) to monitor the concentrations of nitrogenous compounds in biological tissues was investigated. 14N-NMR spectra were collected in vivo and in vitro from several tissues of the rat and rabbit. Many nitrogen-containing compounds were detected, including urea, NH+4, trimethylamines, and several amino acids. In general, the compounds detected had approximately tetrahedral symmetry about the 14N nucleus and concentrations in the millimolar range. A problem that may limit the usefulness of 14N-NMR in intact tissue is the rapid exchange of nitrogen compounds in solution with those bound to sites on macromolecules. Such interactions tend to broaden the 14N resonance signal considerably. The binding of urea to intra- and extracellular proteins is presented as a specific example of this phenomenon. A particularly interesting finding was the high concentration (approximately 90 mM) of trimethylamine compounds in the renal inner medulla. We propose that 14N-NMR is a potentially useful technique for noninvasive detection of specific nitrogen-containing compounds in intact biological tissues.

Amino Acids↗
PubMed1983

Distribution of immunoreactive kallikrein along the rat nephron.

We have used a direct radioimmunoassay to localize kallikrein along the corticomedullary axis of the rat kidney and in microdissected rat cortical nephron segments. As previously seen in rat and dog, kallikrein content was highest in the outer cortex and decreased progressively toward the papillary tip. In microdissected cortical nephron segments, the highest kallikrein content was found in the connecting tubule [176 +/- 16 (SE) pg/mm tubule length (n = 12)]. In addition, a significant amount of kallikrein was found in distal convoluted tubules [53 +/- 4 pg/mm (n = 14)], initial collecting tubules [75 +/- 10 pg/mm (n = 14)], and cortical collecting ducts [37 +/- 4 pg/mm (n = 13)]. Little or no kallikrein was found in glomeruli, proximal convoluted tubules, proximal straight tubules, and cortical thick ascending limbs. These results are compatible with a site of action of the kallikrein-kinin system in the cortical segments of the rat distal nephron.

Animals↗
PubMed1983

Urea transport in nephron segments from medullary rays of rabbits.

To evaluate possible routes of urea delivery to the renal medulla, urea transport was studied in cortical thick ascending limbs and proximal straight tubules dissected from inner cortical medullary rays of rabbit kidneys. Urea was measured colorimetrically in the perfused, collected, and bath fluids. No evidence for active transport of urea was found in either segment. With imposed urea concentration differences between perfusion and bath fluids, there were significant passive fluxes of urea in both segments. The magnitude of the flux was independent of the direction of the concentration difference. Apparent permeability coefficients (X10(-5) cm/s) for urea were 2.0 for the cortical thick ascending limbs and 1.5 for the proximal straight tubules. Based on the measured permeability in the cortical thick ascending limb, substantial passive absorption of urea is predicted in vivo. This will contribute to the dilution of tubular fluid in this segment. The results in proximal straight tubules are compatible with passive urea secretion but not with a significant degree of active secretion.

Animals↗
PubMed1983

Urea transport in isolated thick ascending limbs and collecting ducts from rats.

The use of pathogen-free rats allows the dissection and in vitro perfusion of several rat nephron segments not previously studied. In the present experiments, net urea fluxes were measured in isolated perfused cortical and medullary thick ascending limbs and cortical collecting ducts from pathogen-free Sprague-Dawley rats. No evidence for active transport of urea was found in either cortical or medullary thick ascending limbs. Permeabilities were calculated from urea fluxes measured with 5 mM urea either in the bath or perfusate and with no urea on the opposite side of the epithelium. Permeability coefficients (cm/s X 10(-5) +/- SE) in different portions of the thick ascending limb were: inner stripe, short-looped nephrons, 0.9 +/- 0.2; inner stripe, long-looped nephrons, 0.6 +/- 0.2 (not significantly different vs. short loops); outer stripe, 1.4 +/- 0.3 (P less than 0.05 vs. inner stripe); and cortical, 1.5 +/- 0.3 (P less than 0.05 vs. inner stripe). The relatively high urea permeability of thick ascending limbs in the outer stripe of the outer medulla and medullary rays is likely to permit substantial passive absorption of urea from these segments in vivo. This will contribute to dilution of the tubule fluid in thick ascending limbs and thus indirectly enhance urinary concentrating ability. In cortical collecting ducts, the urea permeability was relatively low both in the presence of 100 microU/ml arginine vasopressin in the bath (0.5 +/- 0.1 X 10(-5) cm/s) and in its absence (0.4 +/- 0.1). These permeability values are similar to values previously measured in rabbit cortical collecting ducts.

Animals↗
PubMed1982

Measurement of osmolality in kidney slices using vapor pressure osmometry.

A method is described for the measurement of tissue fluid osmolality in intact kidney slices using a vapor pressure osmometer (Wescor). The intact tissue slices were placed directly into the equilibration chamber of the instrument. A high degree of accuracy and reproducibility were obtained for both renal cortical and medullary tissue when care was taken to adhere to specific requirements for tissue slice geometry and size. The measurements proved to be technically easier than using the freezing point depression method, and better spatial resolution was obtained.

Animals↗
PubMed1982

A colorimeter for measurement of picomole quantities of urea.

We described a new colorimeter for the measurement of picomole quantities of urea in nanoliter volume fluid samples. The diacetyl monoxime reaction was used to produce a colored product from urea. The method is capable of resolving differences of 10 pmoles between samples containing 0 to 225 pmoles.

Colorimetry↗