Effects of lysine on bicarbonate and fluid absorption in the rat proximal tubule

The effects of lysine on bicarbonate and fluid reabsorption in the rat proximal tubule were studied by luminal and capillary perfusion in situ. The proximal tubule and peritubular capillaries were perfused with bicarbonate Ringer solution containing [14C]inulin. The rate of bicarbonate reabsorption (JHCO3) was estimated to be 124 +/- 9.5 peq.min-1.mm-1 using a pH membrane glass electrode. The rate of net fluid reabsorption (Jv) was 2.6 +/- 0.21 nl.min-1.mm-1. When 10 mM L-lysine was added to the luminal perfusate, a 35% reduction in JHCO3 and no change in Jv were observed. Increase of L-lysine concentration in the luminal perfusate to 20 mM did not reduce JHCO3 further nor did it influence Jv.l When 10 mM L-lysine was added to the capillary perfusate, a 13% reduction in JHCO3 was observed (NS). Increase of lysine concentration in the capillary perfusate to 20 mM significantly reduced JHCO3 by 26% (P less than 0.01). There was no significant change in Jv under both conditions. The effect of L-lysine in the lumen was related to its reabsorption kinetics, D-Lysine, which was not reabsorbed significantly, did not affect bicarbonate reabsorption in the proximal tubule. These results indicate that the inhibitory effect of L-lysine is related to the entry of lysine into the cell from the lumen.     

Adrenergic control of bicarbonate absorption in the proximal convoluted tubule of the rat kidney

The effects of norepinephrine and phenoxybenzamine on bicarbonate absorption in the rat proximal convoluted tubule were studied by simultaneous microperfusion of tubule and peritubular capillaries. Bicarbonate was determined by using a pH-sensitive membrane electrode system. The rates of bicarbonate absorption were examined in the same proximal tubule before and after the addition of norepinephrine or phenoxybenzamine. When the proximal tubule was perfused with Ringer solution and peritubular capillaries were perfused with albumin Ringer solution, was 145±3.3 pEq/min×mm. Addition of 2×10^–6 mol/l norepinephrine to the capillary perfusate caused a 21% increase in . Addition of 2×10^–6 mol/l phenoxybenzamine to the capillary perfusate caused a 12% decrease in . Addition of both norepinephrine and phenoxybenzamine to the capillary perfusate caused a 19% decrease in . However, there was no significant effect on observed when either norepinephrine or phenoxybenzamine was added to the luminal perfusate. These results suggest that adrenergic nerves participate in the regulation of renal tubular bicarbonate absorption through the direct action of norepinephrine on adrenergic receptors located at the basolateral side of the proximal tubule.This work was supported by American Heart and Chicago Heart AssociationPart of this work was presented in Federation Meeting of American Societies for Experimental Biology and Medicine, Anaheim, California, April 1980     

Carbonic anhydrase-dependent bicarbonate reabsorption in the rat proximal tubule.

The extent to which bicarbonate reabsorption in the rat proximal convoluted tubule depends on carbonic anhydrase has been examined by in vivo microperfusion and the measurement of total CO2 concentration by microcalorimetry. Tubules were perfused with an ultrafiltrate-like solution at 13 nl/min, and volume reabsorptive rate (JV) was measured using [14C]inulin. Addition of either 800 or 100 microM acetazolamide to the perfusion solution completely inhibited the reabsorption of total CO2. The control total CO2 reabsorptive rate (JtCO2) was 147 +/- 23 pmol/mm.min, and acetazolamide reduced JtCO2 to -3 +/- 5 pmol/mm.min. Acetazolamide reduced JV by 65% from a control of 2.3 +/- 0.4 to 0.8 +/- 0.1 nl/mm.min. The dose-response curve for acetazolamide showed that the I50 for inhibition of JtCO2 was 4 microM. The inactive congener of acetazolamide, t-butyl acetazolamide, did not reduce JV or inhibit bicarbonate reabsorption, indicating that the effect of acetazolamide on JtCO2 was specific for carbonic anhydrase inhibition. Since bicarbonate reabsorption was completely blocked by carbonic anhydrase inhibition, there is no need to postulate either carbonic acid recycling or carbonic anhydrase-independent bicarbonate reabsorption.     

Studies of the electrical potential difference in rat proximal tubule.

The electrical potential difference (PD) in the rat proximal convoluted tubule was investigated in vivo as a function of distance from the glomerulus. The PD was found to be invariably negative (up to -4.5 mV) in the earliest segments (less than 0.5 mm from the glomerulus) and rose to positive values (+2 to +4) in the later segments (1 mm beyond the glomerulus). This change in PD correlated with the bubule fluid-to-plasma (TF/P) chloride ratios, which rose from unity in the early segments to approximately 1.3 in the late. Corresponding changes in PD and chloride ratios could be elicited by single-nephron stop-flow techniques in the early segments. Luminal perfusion techniques demonstrated a direct relationship between PD and tubule fluid chloride concentration. Acetazolamide was found to significantly reduce both late proximal PD (less than +2 mV) and TF/P chloride ratios (less than 1.06). Split-drop studies demonstrated that the negative PD in the early proximal tubule was dependent on the presence of glucose and alanine and the absence of a chloride gradient, whereas in the late proximal tubule under the same conditions the PD was not significantly different from zero. In this segment of the nephron the positive PD in free flow appeared to result from the chloride diffusion potential generated by preferential HCO3 reabsorption. These results provide further demonstration of intrinsic differences in the transport properties along the length of the proximal convoluted tubule.     

Peritubular capillary control of proximal tubule reabsorption in the rat.

Changes in peritubular capillary hydrostatic and oncotic pressures, which probably affect net interstitial pressure and, thus, the force on fluid movement across the tubule basement membrane, can modulate absolute proximal reabsorption rate (APR). To examine the relationship between APR and net interstitial pressure, we measured peritubular capillary hydrostatic and oncotic pressure, single nephron filtration rate, APR, absolute distal reabsorption (ADR), and tubular hydrostatic pressure in hydropenic, saline-loaded, and plasma-loaded rats. Net interstitial pressure in saline loading was estimated from subcapsular hydrostatic pressure and lymph protein concentration measurements. The surface area-hydraulic conductivity product of the peritubular capillary network was estimated from these measurements with a model of capillary fluid exchange in which fluid uptake was defined to be APR plus ADR. The estimated value was assumed to remain constant in all three states, and was then used to estimate net interstitial pressure in hydropenic and plasma-loaded rats. APR and net interstitial pressure correlated strongly, a finding consistent with the hypothesized role for net interstitial pressure in regulating proximal reabsorption.     

Cholinergic effect on rat proximal convoluted tubule

The effect of cholinergic agents on proximal tubular absorption of bicarbonate and fluid were examined to investigate the possible role of the cholinergic receptor in the regulation of renal function. Proximal convoluted tubule (PCT) and peritubular capillaries were perfused with bicarbonate-Ringer's solution containing radioactive inulin. Bicarbonate (total CO₂) was determined by microcalorimetry. The rates of bicarbonate absorption ( ) and fluid absorption (J_v) were 143.3±7.2 pEq/min · mm and 2.52±0.23 nl/min · mm, respectively. Addition of carbachol (10⁻⁸ M) to the capillary perfusate reduced by 17% and J_v by 32%. A higher dose of carbachol (10⁻⁶ M) did not further inhibit or J_v. Simultaneous perfusion of atropine (10⁻⁵ M) together with carbachol (10⁻⁸ M) abolished the inhibitory effect of carbachol on PCT transport. Atropine itself, however, had no effect on PCT transport. The inhibitory effect of carbachol was also diminished by lanthanum chloride (10⁻⁴ M). Carbachol (10⁻⁶ M) had no effects from the luminal side. W-7, a calmodulin antagonist, inhibited carbachol-induced effects. Ionophore A-23187 also inhibited J_v and . However, there was no additive effect when A-23187 and carbachol were combined in the capillary perfusate. These results suggest that there are functional cholinergic receptors on the basolateral side of the PCT that can regulate and J_v. Calcium influx may play a role in mediating the cholinergic effects on PCT transport.     

Conductances, diffusion and streaming potentials in the rat proximal tubule.

1. Transtubular potential differences and specific resistances were measured in rat proximal tubules by means of single and double barrelled glass micro-electrodes. 2. Tip localization was made by observation of effective resistance changes measured with double barrelled micro-electrodes upon passage of oil droplets, and by perfusion with choline C1. 3. Mean early proximal p.d.s. of the order of -1 to -2 mV, and late values of +0-5 to +1mV were found. Mean specific resistances ranged from 12 to 15 omega cm2. 4. Diffusion potentials and single ion relative conductances were evaluated, perfusing the lumen with solutions differing only with respect to one salt concentration. Na and K conductances were similar and greater than those of C1. 5. Luminal and peritubular perfusions with hypotonic solutions showed the occurrence of streaming potentials in this structure suggesting the existence of pores lined with negative charges. The effective diameter of these pores appeared to be reduced by hypotonic perfusion, as evidenced by a significant increase in resistance, indicating that the main ion path across this structure is represented by intercellular spaces.     

Solvent drag of sucrose during absorption indicates paracellular water flow in the rat kidney proximal tubule

Single convoluted proximal tubules of the rat kidney were lumen perfused in situ with isosmotic solutions containing C¹⁴-sucrose and H³-inulin as tracers, to evaluate whether the extracellular marker sucrose is entrained by water during proximal tubular reabsorption. Inulin was used as volume marker. The absorptive rate was varied by using as luminal perfusion fluids either a solution made up of (in mmole/l) 120 NaCl, 5 glucose, 25 NaHCO₃ and altering the perfusion rate, or a solution containing 110 NaCl and 70 raffinose.J ^S, the net sucrose efflux is found to be a function of the net volume flow,J _V, such that atJ _V=0,J ^S is very small and at high rates ofJ _V,J ^S is over 60-fold the value observed at lowJ _V values. In addition, the transported to luminal sucrose concentrations decreased withJ _V in a hyperbolic manner.Unstirred layers affect the diffusive component ofJ ^S, but only to a small extent. Therefore, the large remaining dependency ofJ ^S withJ _V must be due to drag of sucrose by water, within the paracellular pathway. This leads to the conclusion that water flows through the paracellular pathway during absorption in the rat proximal tubule, in addition to transcellular water flow. Using equations for molecular sieving and the measured value of _s for sucrose of 0.76–0.91, it is calculated that the pathway where entrainment of solute by water occurs must be 1.0–1.1 nm wide. This calculation is only tentative since _s depends on the as yet unknown relative contribution of transcellular and paracellular pathways to transepithelial water osmotic permeability.     

Effects of mercury on lysosomal protein digestion in the kidney proximal tubule.

The effect of mercury on renal lysosomal protein digestion was studied after administration of mercury in vitro and in vivo. Mercuric chloride or methylmercury chloride was added in vitro to lysosomal enzymes isolated from normal rats, and subsequently, digestion experiments were carried out using 125I-labeled lysozyme or cytochrome c as substrate proteins. Both mercury compounds produced a concentration-dependent inhibition of the degradation of the proteins, mercuric chloride being the strongest inhibitor. Mercuric chloride was also administered to rats in vivo for 5 to 8 months. Renal lysosomal enzymes from these animals also had a decreased ability to digest for the two substrate proteins. Furthermore, the digestion of lysozyme intravenously injected into mercury-intoxicated rats was decreased in renal cortical slices incubated in vitro. Electron microscope autoradiography showed that intravenously injected labeled lysozyme was located primarily over lysosomes in proximal tubule cells 1 hour after injection in both control animals and mercury-intoxicated rats. These results suggest a decreased catabolism of low molecular weight proteins in the kidney during chronic mercury intoxication.     

Sodium, bicarbonate, and chloride absorption by the proximal tubule

Proximal tubules are lined with epithelial cells that contain Na-K-ATPase in their basolateral cell membrane. The luminal cell membrane contains transport proteins that couple movement of many solutes to the active transport of sodium. The cells are connected by low-resistance junctional complexes that permit passive movement of solutes via a paracellular shunt pathway. Acidification is mediated by a Na/H antiporter localized specifically in the luminal membrane and a chloride-independent, voltage-dependent bicarbonate exit process in the basolateral membrane. The rate of acidification is controlled by the pH of the luminal and peritubular fluids. Reabsorption of NaCl from the high-chloride, low-bicarbonate fluid in the late proximal tubule is approximately 40% passive and 60% active. In proximal straight tubules the active component is entirely by simple rheogenic sodium transport, with chloride absorption driven through the paracellular shunt pathway by the lumen-negative PD. In convoluted tubules the active component is primarily neutral, with both sodium and chloride transported in approximately equivalent amounts through the cell. The mechanisms for neutral NaCl transport across the luminal membrane and for chloride exit across the basolateral membrane are unknown. A reduction in peritubular Starling forces (hydraulic and oncotic pressures) suppresses net proximal reabsorption by two mechanisms: 1) increased paracellular permeability with modest backleak of solutes (bicarbonate, glucose, amino acids) whose luminal concentration falls below their plasma level, and 2) specific inhibition of active neutral transcellular transport of NaCl by reduced peritubular protein concentration by some mechanism other than inhibition of Na-K-ATPase.     

Heterogeneity of sodium-dependent D-glucose transport sites along the proximal tubule: evidence from vesicle studies

The glucose transport properties of brush border membrane vesicles from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) of rabbit kidney were studied. In the outer cortical preparation the behavior of the sodium-dependent component of D-glucose flux indicated the presence of a low-affinity transport system with Km congruent to 6 mM and Vmax congruent to 10 nmol.min-1.mg protein-1 as measured under zero trans conditions at 40 mM NaCl and 17 degrees C. By contrast, in the outer medullary preparation this component of flux behaved as a high-affinity system with Km congruent to 0.35 mM and Vmax congruent to 4 nmol.min-1.mg protein-1. Differences in transport specificity between the two preparations were also indicated and glucose uptake by the outer cortical vesicles was significantly more sensitive to inhibition by phlorizin. These results suggest the existence of two distinct sodium-dependent D-glucose transport systems in the renal proximal tubule brush border membrane. The kinetic studies presented here were done under zero trans sodium and glucose conditions. The rationale and methodology for carrying out these measurements reliably are discussed in detail.     

Protein kinase C activity in rat renal proximal tubule cells.

The presence of protein kinase C (PKC) in proximal tubule cells of the rat kidney is established by means of immunodetection and by the demonstration of calcium- and phospholipid-dependent, staurosporine-inhibitable histone phosphorylation. The calcium-dependence of renal PKC is described. Maximal activation of the enzyme (178.2 and 258.8 pmol P1 mg-1 min-1 for cytosol and membrane respectively) was achieved with 5 microM of Ca2+. Phorbol 12, 13 dibutyrate (PDBu) translocated PKC from cytosol to membrane in a dose- and time-dependent fashion, while 4 alpha-phorbol 12,13-didecanoate produced no significant effect on translocation. Cytosolic PKC activity was compared in immature and mature tissues (10- and 40-day-old kidneys). Basal activity was found to be significantly higher (P less than 0.05) in immature cells (272.8 vs. 157.5 pmol Pi mg-1 min-1). PDBu at 10(-6) M for 15 min reduced immunoreactivity in the soluble fraction of both groups, which was accompanied by a significant decrease in kinase activity. We speculate that the high PKC activity in the infant kidney plays a role in cell growth.     

Rat proximal tubule D-glucose transport as a function of concentration,flow, and radius

Summary From earlier microperfusion studies ofD-glucose and water reabsorption in the proximal surface nephron of the rat,D-glucose was found to be removed by a saturable carrier and by an apparent coupling with net fluid reabsorption. Equations appropriate to describe this system were developed. They incorporated carrier-mediatedD-glucose transport, net water transport, and water-coupled solute transport. Water reabsorption was assumed to be constant either per unit surface area, or per unit volume of the nephron, and the rate of carrier-transportedD-glucose was assumed constant per unit length, per unit surface area, or per unit volume of the tubule. The possibility thatD-glucose could be reabsorbed via two carrier systems was also explored analytically. It was observed from this treatment that the fraction ofD-glucose reabsorbed would change if the perfusion rate was changed. With an increase in perfusion rate, a decrease in reabsorbed fraction was seen which indicates that if net fluid reabsorption is proportional to volume, carrier-mediated sugar transport is proportional to surface area or length of the tubule. From these relationshipsJ _max, the maximal rate of carrier-transported sugar, was calculated to be 3.3×10⁻¹⁰M/cm² sec, a value comparable to that reported from other laboratories. The results of this analysis are compatible with the data obtained both by micropuncture experiments during free flow and by glucose clearance studies until theT _mG is reached. The possibility that theT _mG obtained in clearance studies is due to a decrease in the fraction of fluid reabsorbed in the proximal tubule or to a second saturable carrier is discussed. It is observed that, in either case, if load is increased by increasing the glomerular filtration rate, noT _mG would be reached, or stated another way, one would predict from the analysis thatT _mG would be proportional to glomerular filtration rate. Partially supported by NIH-AM-10779-02 and “Deutsche Forschungsgemeinschaft”. Partially presented at the XXIV International Congress of Physiological Societies, Washington, D.C., 1968. PHS post doctoral fellow 1-F2-AM-37056-01 and Dr. Henry C. and Bertha H. Buswell Fellow Dr. Henry C. and Bertha H. Buswell Fellow     

Amiloride-inhibitable Na+ conductance in rat proximal tubule

 Previous single-channel recordings from the luminal membrane of the rabbit proximal tubule have revealed amiloride-inhibitable Na⁺ channels of a characteristic conductance range. The present study aimed to pursue this issue in rat proximal tubule. Control rats were compared to those put on a low-Na⁺ diet or pretreated by triamcinolone injections (s.c.). Stimulation of Na⁺ absorption by glucocorticoids was verified by examining the transepithelial voltage in Ussing chamber studies of the distal colon. The membrane voltage (V _m) of isolated, in-vitro-perfused proximal tubule segments was measured in patch-clamp and impalement studies. It was found that amiloride hyperpolarized V _m significantly by 2.1 ± 0.9 mV (n = 26) in tubules of control rats, by 3.9 ± 0.7 mV (n = 12) in rats put on a low-Na⁺ diet and by 3.7 ± 1.0 mV (n = 17) in rats treated with glucocorticoids. The effect of amiloride was concentration dependent with a half-maximal effect at < 1 μmol/l. RT-PCR techniques were used to search for the presence of the α-, β- and γ-subunits of the epithelial Na⁺ channel in isolated oximal tubule segments. The presence of the respective mRNAs was verified. These data indicate that: (1) amiloride-inhibitable Na⁺ channels are present in rat proximal tubules; (2) the Na⁺ conductance may be upregulated by Na⁺ deprivation but is still very limited when compared to total cell conductance; (3) therefore, the contribution of Na⁺-channel-mediated absorption to total proximal Na⁺ absorption is probably small. Received: 5 August 1996 / Received after revision: 22 January 1997 / Accepted: 28 January 1997     

An analysis of glomerular-tubular balance in the rat proximal tubule

An analysis of glomerulo-tubular balance in the rat proximal tubule. Flow dependence of absolute proximal reabsorption (APR) or glomerulo-tubular balance (GTB) has been observed with spontaneous alterations in flow and attributed to both intraluminal and extraluminal factors. Flow dependent alterations in APR were demonstrated when 1. nephron filtration rate (SNGFR) was decreased by tubulo-glomerular feedback mechanisms by increasing late proximal tubular microperfusion rates, and 2. when SNGFR was increased by addition of [Sar¹, Ala⁸] angiotensin II to the adjacent peritubular capillary flow. Selective reduction in early proximal tubular flow rate by pump aspiration also resulted in flow dependent reductions in APR. However, selective additions of perfusion fluids of various native and artificial constituency to the early proximal tubule did not result in flow dependent increase in APR.Conclusions. 1. GTB with both increases and decreases in SNGFR can be demonstrated at the level of the single nephron, 2. selective reductions in luminal flow rate produces parallel reductions in APR; however, 3. increases in flow rate with either artificial or native fluids of different ionic concentrations did not result in increases in APR. This lack GTB may be due to lack of parallel changes in peritubular physical factors or that APR in the S2 segment is less sensitive to increase in flow rate.     

Contraluminal sulfate transport in the proximal tubule of the rat kidney

In order to study the specificity for the contraluminal sulfate transport system the inhibitory potency of disulfonates, di-, tricarboxylates and sulfocarboxylates on the³⁵SO ₄ ^2– influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1) Methane- and ethane-disulfonate as well as benzene-1,3-disulfonate inhibit contraluminal³⁵SO ₄ ^2– influx (with an (app.K _i of <6 mmol/l), while benzene-1,2- and 1,4-disulfonate do not. 2) The inhibitory potency of 1,3-benzene disulfonate is slightly augmented by an additional NH₂ ^– or OH-group in position 4. However, OH-groups at position 4 and 5 or 4 and 6 abolish the inhibitory potency. 3) The naphthalene disulfonates tested inhibit only if they have an OH-group in ortho-position to one SO₃H group. 4) The stilbene disulfonates H₂DIDS and DNDS inhibit the contraluminal³⁵SO ₄ ^2– influx with high (app.K _i0.8 mmol/l), DADS with lower potency (app.K _i6 mmol/l). 5) Amongst the tested aliphatic di- and tricarboxylates inhibition was exerted by oxalate (app.K _i 1.1 mmol/l) and maleate (app.K _i 3.8 mmol/l), but not by malonate, hydroxymalonate and citrate. 6) Out of the tested benzenedicarboxylates only those inhibit which have the COO^–-groups directly on the ring in 1,2 and 1,3 position (app.K _i 4.0 and 2.7 mmol/l), but not in the 1,4 position. An additional OH-group in position 4 augments the inhibitory potency of 1,3 benzene-dicarboxylates (app.K _i 0.8 mmol/l), while an OH group on position 5 abolishes it. 7) The benzene tricarboxylates (BTC) inhibit in the sequence 1,2,3-BTC>1,3,5-BTC>1,2,4-BTC (app.K _i 0.9, 1.5 and 4.2 mmol/l, respectively). 8) The carboxy-benzene-sulfonates inhibit also in the 1,2 and 1,3 position only (app.K _i 6.7 and 5 mmol/l), but not in the 1,4 position. Addition of an –OH-group to the 3-carboxy-1-benzene-sulfonate forming 4-hydroxy-3-carboxy-1-benzene-sulfate augments the inhibitory potency drastically (app.K _i 0.32 mmol/l), while a NH₂ substitution at the same position leaves it unchanged (app.K _i 4.7 mmol/l). If, however, ethylamine instead of NH₂ is used as substituent, the inhibitory potency is almost as high as of 4-hydroxy-3-carboxy-1-benzene-sulfonate (app.K _i0.6 mmol/l). Amongst the dicarboxy-benzene-sulfonates, 3,4-carboxy-benzene-1-sulfonate inhibits (app.K _i ca. 2 mmol/l), while 3,5-carboxy-benzene-1-sulfonate does not. The data indicate that a strong interaction of substrate with the sulfate transporter is given, when two charged groups (COO^– and/or SO ₃ ^– ) are present in a distance equivalent to the meta-position on the benzene ring and an additional hydrogen bond forming OH- or –NH-group. Hydrogen bond forming groups and charged groups in other positions usually abolish the inhibitory potency.