Production and functional roles of nitric oxide in the proximal tubule

A significant role for nitric oxide (NO) in proximal tubule physiology and pathophysiology has been revealed by a series of in vivo and in vitro studies. Whether the proximal tubule produces NO under basal conditions is still controversial; however, evidence suggests that the proximal tubule is constantly exposed to NO that might include NO from nonproximal tubule sources. When challenged with a variety of stimuli, including hypoxia, the proximal tubule is able to produce large quantities of NO. In vivo studies generally indicate that NO inhibits fluid and sodium reabsorption by the proximal tubule. However, the final effect of NO on proximal tubular reabsorption appears to depend on the concentration of NO and involve interaction with other regulatory mechanisms. NO regulates Na(+)-K(+)-ATPase, Na(+)/H(+) exchangers, and paracellular permeability of proximal tubular cells, which may contribute to its effect on proximal tubular transport. Enhanced production of NO, perhaps depending on macrophage type inducible NO synthase, participates in hypoxic/ischemic proximal tubular injury. In conclusion, NO plays a fundamental role in both physiology and pathophysiology of the proximal tubule.     

Regulation of renal proximal and distal tubule transport: sodium, chloride and organic anions

Renal tubular transport and its regulation are reviewed for Na(+) (and Cl(-)), and for fluid and organic anions (including urate). Filtered Na(+) (and Cl(-)) is reabsorbed along the tubules but only in mammals and birds does most reabsorption occur in the proximal tubules. Reabsorption involves active transport of Na(+) and passive reabsorption of Cl(-). The active Na(+) step always involves Na-K-ATPase at the basolateral membrane, but the entry step at luminal membrane varies among tubule segments and among vertebrate classes (except for Na(+)-2Cl(-)-K(+) cotransporter in diluting segment). Regulation can involve intrinsic, neural and endocrine factors. Proximal tubule fluid reabsorption is dependent on Na(+) reabsorption in all vertebrates studied, except ophidian reptiles. Fluid secretion occurs in glomerular and aglomerular fishes, reptiles and even mammals, but its significance is not always clear. A non-specific transport system for net secretion of organic anions (OAs) exists in the proximal renal tubules of almost all vertebrates. Net transepithelial secretion involves: (1) transport into the cells at the basolateral side against an electrochemical gradient by a tertiary active transport process, in which the final step involves OA/alpha-ketoglutarate exchange and (2) movement out of the cells across the luminal membrane down an electrochemical gradient by unknown carrier-mediated process(es). Regulation may involve protein kinase C and mitogen-activated protein kinase. Urate is net secreted in the proximal tubules of birds and reptiles. This process is urate-specific in reptiles but in birds, it may involve both a urate-specific system and the general OA system.     

Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR

The effect of acute arterial hypertension on proximal tubular fluid reabsorption was investigated in Sprague-Dawley rats and spontaneously hypertensive rats (SHR) by measuring proximal tubular flow with a nonobstructive optical method. Under control conditions, spontaneous tubular flow was oscillating at 0.02-0.03 Hz in Sprague-Dawley rats. Acute hypertension induced an immediate increase of mean tubular flow (50% increase after 20 min of hypertension) and augmentation of oscillatory amplitude. Acute hypertension did not alter single-nephron blood flow as measured by laser-Doppler velocimetry (n = 12), suggesting that the increase of tubular flow was due to inhibition of reabsorption but not increase of filtration. By contrast, spontaneous tubular flow was fluctuating aperiodically in SHR. Acute hype tension did not induce a continuous increase of tubular flow or an increase in amplitude of fluctuations (n = 15). When apical Na(+)/H(+) exchange activity of proximal tubule was monitored, acute hypertension did not alter the activity in SHR (n = 8), while similar procedures had been shown to inhibit apical Na(+)/H(+) exchange activity of proximal tubules by more than 40% in Sprague-Dawley rats. These observations suggest that acute hypertension inhibits proximal tubular fluid reabsorption by inhibiting apical Na(+)/H(+) exchange activity in Sprague-Dawley rats and that this mechanism is impaired in SHR.     

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.     

Quantitative intravital microscopy using a Generalized Polarity concept for kidney studies

In this article, we describe a ratiometric intravital two-photon microscopy technique for studying glomerular permeability and differences in proximal tubule cell reabsorption. This quantitative approach is based on the Generalized Polarity (GP) concept, in which the intensity difference between two fluorescent molecules is normalized to the total intensity produced by the two dyes. After an initial intravenous injection of a mixture of 3-, 40-, and 70-kDa fluorescently labeled dextrans into live Munich-Wistar-Frömter (MWF) rats, we were able to monitor changes in the GP values between any two dyes within local regions of the kidney, including the glomerulus, Bowman's capsule, proximal tubule lumens and proximal tubule cells, and individual capillary vessels. We were able to quantify accumulations of different dextrans in the Bowman's space and in tubular lumens as well as reabsorption by proximal tubular cells at different time points in the same rat. We found that for 6- to 8-wk-old MWF rats that developed spontaneous albuminuria, the 40- and 70-kDa dextrans, with hydrodynamic radii larger than albumin, were differentially filtered, but both were able to pass the glomerular filtration barrier and enter into the urinary space of the Bowman's capsule within a few seconds after intravenous infusion. Using GP image analysis, we found that negatively charged dextrans of both 40 and 70 kDa were better reabsorbed by the proximal tubule cells than the neutrally charged 40-kDa dextran. These results demonstrate the potential power of the GP imaging technique for quantitative studies of glomerular filtration and tubular reabsorption. glomerular permeability; tubular reabsorption; charge selectivity; two-photon excitation; multiphoton     

Effect of ACE inhibition on glomerular permselectivity and tubular albumin concentration in the renal ablation model

Despite the central role of tubular plasma proteins that characterize progressive kidney diseases, protein concentrations along the nephron in pathological conditions have not been quantified so far. We combined experimental techniques and theoretical analysis to estimate glomerular and tubular levels of albumin in the experimental model of 5/6 nephrectomy (Nx) in the rat, with or without angiotensin-converting enzyme (ACE) inhibition. We measured glomerular permselectivity by clearance of fluorescent Ficoll and albumin and used theoretical analysis to estimate tubular albumin. As expected, 5/6 Nx induced an elevation of the fractional clearance of the largest Ficoll molecules (radii >56 ?, P < 0.05), increasing the importance of the shunt pathway of the glomerular membrane and the albumin excretion rate (119 ± 41 vs. 0.6 ± 0.2 mg/24 h, P < 0.01). ACE inhibition normalized glomerular permselectivity and urinary albumin (0.5 ± 0.3 mg/24 h). Theoretical analysis indicates that with 5/6 Nx, an increased albumin filtration overcomes proximal tubule reabsorption, with a massive increase in average albumin concentration along the tubule, reaching the highest value of >2,500 μg/ml at the end of the collecting duct. ACE inhibition improved glomerular permselectivity, limiting albumin filtration under proximal tubule reabsorption capacity, with low albumin concentration along the entire nephron, averaging <13 μg/ml at the end of the collecting duct. These results reinforce our understanding of the mechanisms of renal disease progression and the effects of angiotensin II antagonism. They also suggest that evaluation of tubular protein concentration levels could help to identify patients at risk of kidney disease progression and to improve clinical management.     

Regulation of glomerulotubular balance. I. Impact of dopamine on flow-dependent transport

In response to volume expansion, locally generated dopamine decreases proximal tubule reabsorption by reducing both Na/H-exchanger 3 (NHE3) and Na-K-ATPase activity. We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na(+) and HCO(3)(-) reabsorption and have suggested that this observation underlies glomerulotubular balance. In the present work, we investigate the impact of dopamine on the sensitivity of reabsorptive fluxes to changes in luminal flow. Mouse proximal tubules were microperfused in vitro at low and high flow rates, and volume and HCO(3)(-) reabsorption (J(v) and J(HCO3)) were measured, while Na(+) and Cl(-) reabsorption (J(Na) and J(Cl)) were estimated. Raising luminal flow increased J(v), J(Na), and J(HCO3) but did not change J(Cl). Luminal dopamine did not change J(v), J(Na), and J(HCO3) at low flow rates but completely abolished the increments of Na(+) absorption by flow and partially inhibited the flow-stimulated HCO(3)(-) absorption. The remaining flow-stimulated HCO(3)(-) absorption was completely abolished by bafilomycin. The DA1 receptor blocker SCH23390 and the PKA inhibitor H89 blocked the effect of exogenous dopamine and produced a two to threefold increase in the sensitivity of proximal Na(+) reabsorption to luminal flow rate. Under the variety of perfusion conditions, changes in cell volume were small and did not always parallel changes in Na(+) transport. We conclude that 1) dopamine inhibits flow-stimulated NHE3 activity by activation of the DA1 receptor via a PKA-mediated mechanism; 2) dopamine has no effect on flow-stimulated H-ATPase activity; 3) there is no evidence of flow stimulation of Cl(-) reabsorption; and 4) the impact of dopamine is a coordinated modulation of both luminal and peritubular Na(+) transporters.     

Detection of apical Na(+)/H(+) exchanger activity inhibition in proximal tubules induced by acute hypertension

We previously showed that acute arterial hypertension induces an inhibition of fluid and NaCl reabsorption in proximal tubules of Sprague-Dawley rats, which is associated with a rapid reversible internalization of apical Na(+)/H(+) exchanger in brush border. To determine whether there is a corresponding inhibition of apical Na(+)/H(+) exchanger activity in proximal tubules to account for the reduced tubular reabsorption, an instrument capable of measuring intracellular pH (pH(i)) ratiometrically and repeatedly on the surface of kidney with high temporal resolution is required. We report the design and validation of such a fluorimetric system based on two ultraviolet nitrogen-pulsed lasers and a photomultiplier. pH(i) of proximal tubules in situ was measured with pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein at 5 Hz. Using the initial rate of change of pH(i) (dpH(i)/dt) during luminal Na(+) removal as an index of apical Na(+)/H(+) exchanger activity, the exchanger activity was found to be reduced by 52 +/- 11% (n = 14, P < 0.05) compared with the baseline after 20 min of induced acute hypertension. The inhibition of Na(+)/H(+) exchange activity was alleviated when the blood pressure was returned to prehypertensive level. These observations indicate that acute changes in arterial pressure can reversibly inhibit apical Na(+)/H(+) exchanger activity, which might contribute to pressure natriuresis in proximal tubule.     

Water-soluble o-hydroxycinnamate as an efficient photoremovable protecting group of alcohols with fluorescence reporting

Two new o-hydroxycinnamates have been prepared for photoremovable protecting groups, and their photochemistry has been investigated. The photolysis of two caged compounds can efficiently release the corresponding alcohol in aqueous solutions, and the uncaging reaction proceeds with large one-photon excitation cross sections (1919 and 1535 M(-1) cm(-1)). The uncaging process has been observed by NMR spectroscopy. The caged compounds exhibit good aqueous solubility and excellent resistance to hydrolysis in a buffer solution.     

Glomerulotubular balance, dietary protein, and the renal response to glycine in diabetic rats

The glomerular filtration rate (GFR) normally increases during glycine infusion, which is a test of "renal reserve." Renal reserve is absent in diabetes mellitus. GFR increases after protein feeding because of increased tubular reabsorption, which reduces the signal for tubuloglomerular feedback (TGF). Dietary protein restriction normalizes some aspects of glomerular function in diabetes. Renal micropuncture was performed in rats 4-5 wk after diabetes was induced by streptozotocin to determine whether renal reserve is lost as a result of altered tubular function and activation of TGF, whether 10 days of dietary protein restriction could restore renal reserve, and whether this results from effects of glycine on the tubule. TGF activation was determined by locating single-nephron GFR (SNGFR) in the early distal tubule along the TGF curve. The TGF signal was determined from the ionic content of the early distal tubule. In nondiabetic rats, SNGFR in the early distal tubule increased during glycine infusion because of primary vasodilation augmented by increased tubular reabsorption, which stabilized the TGF signal. In diabetic rats, glycine reduced reabsorption, thereby activating TGF, which was largely responsible for the lack of renal reserve. In protein-restricted diabetic rats, the tubular response to glycine remained abnormal, but renal reserve was restored by a vascular mechanism. Glycine affects GFR directly and via the tubule. In diabetes, reduced tubular reabsorption dominates. In low-protein diabetes, the vascular effect is enhanced and overrides the effect of reduced tubular reabsorption.     

Proximal and distal tubular activity in chronically catheterized fetal sheep compared with the adult

Renal function was studied in unanaesthetized fetal sheep aged 112-120 and 126-132 days and in adult nonpregnant ewes. The clearance of lithium was used to measure proximal and distal fractional sodium reabsorption. In five nonpregnant adult sheep, 80.6 +/- 1.7% (SE) of the filtered sodium load was reabsorbed proximally and 18.2 +/- 1.53% distally. This was different from all groups of fetal sheep (p less than 0.001). In younger fetuses, proximal fractional sodium reabsorption was less (51.3 +/- 2.3% (SE), p less than 0.05) and distal fractional sodium reabsorption greater (42.4 +/- 2.3% (SE), p less than 0.05) than older fetuses (126-132 days old) in which 61.4 +/- 2.4% (SE) was reabsorbed proximally and 33.6 +/- 2.5% (SE) distally. In another group of fetuses aged 125-137 days, in which proximal tubular sodium reabsorption was measured after distal tubular blockade, proximal fractional sodium reabsorption was 57.8 +/- 2.95% (SE) and distal fractional sodium reabsorption, 38.7 +/- 2.64% (SE). In adult sheep there was no relationship between distal tubular sodium reabsorption and glomerular filtration rate, i.e., proximal tubular function was responsible for glomerulotubular balance. However, in the fetuses, both proximal and distal tubular sodium reabsorption contributed to glomerulotubular balance. Thus in fetal life, the proximal tubule participates to a lesser extent in reabsorbing the filtered sodium load possibly because its function is suppressed by its relatively "volume-expanded" state or because it is functionally immature. Therefore, a greater proportion is reabsorbed distally and the distal nephron participates under physiological conditions in glomerulotubular balance.     

Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone

Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7-8 wk of age compared with vehicle-treated controls: 131 +/- 3 vs. 115 +/- 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl.mm(-1).min(-1) in control rats and 0.93+ 0.07 nl.mm(-1).min(-1) in rats that received prenatal dexamethasone (P < 0.05). Na(+)/H(+) exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na(+)/H(+) exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7-8 wk old, in part by stimulating Na(+)/H(+) exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.     

Luminal flow regulates NO and O2(-) along the nephron

Urinary flow is not constant but in fact highly variable, altering the mechanical forces (shear stress, stretch, and pressure) exerted on the epithelial cells of the nephron as well as solute delivery. Nitric oxide (NO) and superoxide (O(2)(-)) play important roles in various processes within the kidney. Reductions in NO and increases in O(2)(-) lead to abnormal NaCl and water absorption and hypertension. In the last few years, luminal flow has been shown to be a regulator of NO and O(2)(-) production along the nephron. Increases in luminal flow enhance fluid, Na, and bicarbonate transport in the proximal tubule. However, we know of no reports directly addressing flow regulation of NO and O(2)(-) in this segment. In the thick ascending limb, flow-stimulated NO and O(2)(-) formation has been extensively studied. Luminal flow stimulates NO production by nitric oxide synthase type 3 and its translocation to the apical membrane in medullary thick ascending limbs. These effects are mediated by flow-induced shear stress. In contrast, flow-induced stretch and NaCl delivery stimulate O(2)(-) production by NADPH oxidase in this segment. The interaction between flow-induced NO and O(2)(-) is complex and involves more than one simply scavenging the other. Flow-induced NO prevents flow from increasing O(2)(-) production via cGMP-dependent protein kinase in thick ascending limbs. In macula densa cells, shear stress increases NO production and this requires that the primary cilia be intact. The role of luminal flow in NO and O(2)(-) production in the distal tubule is not known. In cultured inner medullary collecting duct cells, shear stress enhances nitrite accumulation, a measure of NO production. Although much progress has been made on this subject in the last few years, there are still many unanswered questions.     

Isosmotic volume reabsorption in rat proximal tubule

A theoretical model incorporation both active and passive forces has been developed for fluid reabsorption from split oil droplets in rat intermediate and late proximal tubule. Of necessity, simplifying assumptions have been introduced; we have assumed that the epithelium can be treated as a single membrane and that the membrane "effective" HCO3 permeability is near zero. Based on this model with its underlying assumptions, the following conclusions are drawn. Regardless of the presence or absence of active NaCl transport, fluid reabsorption from the split oil droplet is isosmotic. The reabsorbate osmolarity can be affected by changes in tubular permeability parameters and applied forces but is not readily altered from an osmolarity essentially equal to that of plasma. In a split droplet, isosmotic flow need not be a special consequence of active Na transport, is not the result of a particular set of permeability properties, and is not merely a trivial consequence of a very high hydraulic conductivity; isosmotic flow can be obtained with hydraulic conductivity nearly an order of magnitude lower than that previously measured in the rat proximal convoluted tubule. Isosmotic reabsorption is, in part, the result of the interdependence of salt and water flows, their changing in parallel, and thus their ratio, the reabsorbate concentration being relatively invariant. Active NaCl transport can cause osmotic water flow by reducing the luminal fluid osmolarity. In the presence of passive forces the luminal fluid can be hypertonic to plasma, and active NaCl transport can still exert its osmotic effect on volume flow. There are two passive forces for volume flow: the Cl gradient and the difference in effective osmotic pressure; they have an approximately equivalent effect on volume flow. Experimentally, we have measured volume changes in a droplet made hyperosmotic by the addition of 50 mM NaCl; the experimental results are predicted reasonably well by our theoretical model.     

培养的大鼠海马神经元胞内Ca2＋和 NO 双标记方法研究

以培养 8 ～ 10 天的大鼠海马神经元为对象,选择 Calcium Orange AM 和 DAF-FM diacetate 为 Ca2＋和一氧化氮 (NO) 的荧光指示剂,建立了基于激光扫描共聚焦显微技术的细胞内 Ca2＋和 NO 双标记检测方法 . 此方法对 Ca2＋和 NO 进行分步染色,然后应用激光扫描共聚焦显微镜 (LSCM) 的双轨迹 (Two Track) 模式,通过快速切换激光实现对细胞内 Ca2＋和 NO 的同时检测 . 实验结果显示,两种染料之间无串扰现象;在 N- 甲基 -D- 天冬氨酸 (NMDA) 刺激下,海马神经元胞内 Ca2＋快速升高,随后达到平台期并有波动, NO 则稳定持续升高,这些变化过程与单标记的结果一致;双标记层切序列图像显示细胞内 Ca2＋和 NO 都较集中分布于细胞中部,但在细节上两者的分布存在差异 . 此双标记方法能同时检测培养的海马神经元胞内 Ca2＋和 NO ,为研究神经元胞内 Ca2＋和 NO 的相互调控作用提供了一种新的手段 .     

Contraluminal uptake of serine in the proximal nephron

Rabbit proximal nephron segments were microperfused in vitro to determine whether active contraluminal uptake of serine occurs in the renal proximal tubule during bath-to-lumen transport (influx) of the L- and D-isomers in the convoluted (pars convoluta) and straight (pars recta) segments. It is known that several amino acids are actively reabsorbed in the proximal nephron by a mechanism involving co-transport with sodium at the luminal membrane. There is some evidence that certain amino acids may also be accumulated across the contraluminal membrane by an energy-dependent mechanism, indicating that net reabsorption is the result of two oppositely directed active transport processes. During in vitro microperfusion of rabbit proximal nephron segments in this study, inward movement of L- and D-serine occurred in a bath-to-cell direction against a concentration gradient in the range 305-2735:1, indicating active uptake at the contraluminal membrane. The concentration gradients were maintained during influx of both isomers of serine in the proximal tubule. L-Serine accumulation by tubular cells was similar in the pars convoluta and recta, and significantly greater than that of D-serine, which was the same in both regions of the proximal tubule. The data support the conclusion that renal handling of serine involves active contraluminal uptake of the L- and D-isomers in both regions of the proximal tubule, and suggest that contraluminal events play an important role in renal handling of amino acids.     

Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat

To determine the tubular sites and mechanisms involved in enhanced renal phosphate (P(i)) reabsorption seen in the juvenile animal, renal micropuncture experiments were performed in acutely thyroparathyroidectomized adult (>14 wk old) and juvenile (4 wk old) male Wistar rats fed either a normal P(i) diet (NPD, 0.6% P(i)) or low P(i) diet (0.07% P(i)) for 2 days, in the presence and absence of parathyroid hormone (PTH). P(i) reabsorption was greater in proximal convoluted (PCT) and straight tubules (PST) of the juvenile compared with adult rats fed NPD, whether or not PTH was present. These findings were consistent with a greater P(i) uptake in brush-border membrane (BBM) vesicles from both superficial (SC) and outer juxtamedullary (JMC) cortices of juvenile animals. Western blot analysis revealed a 2- and 1.8-fold higher amount of NaPi-2 protein in the SC and JMC, respectively, in juvenile rats. Immunofluorescence microscopy also indicated that NaPi-2 protein expression was present in the proximal tubule (PT) BBM to a greater extent in juvenile rats. Dietary P(i) restriction in juvenile rats resulted in a significant increase in P(i) reabsorption in the PCT and PST segments. NaPi-2 expression in the PT BBM was also increased, as was the expression of intracellular NaPi-2 protein. These studies indicate that P(i) reabsorption in both the PCT and PST segments of the renal tubule contributes to the attenuated response to PTH in the normal juvenile animal. In addition, dietary P(i) restriction in the juvenile rat upregulates BBM NaPi-2 expression, which is associated with a further increase in proximal tubular P(i) reabsorption.     

Acute Functional Adaptation to Nephron Loss: Micropuncture Studies

The renal and proximal tubule response to contralateral kidney exclusion was studied in a variety of circumstances. Recollection micropuncture studies were performed to assess the response to contralateral kidney clamping in the normal or a remnant kidney of the dog. Acute clamping of the contralateral kidney for a normal and unilateral remnant kidney resulted in marked reduction in proximal TF/P inulin ratios in the experimental kidney reflecting a 15 percent reduction in fluid reabsorption. Mean fractional excretion of sodium, potassium and water increased significantly in remnant kidney dogs but no significant change was observed in normal dogs except for potassium excretion. The marked reduction in proximal reabsorption occurred as soon as 5-15 minutes after contralateral kidney clamping and was compensated by distal reabsorption. Acute obstruction of the contralateral ureter results in a similar markedly reduced proximal tubular reabsorption. The reduction in proximal reabsorption induced by contralateral clamping occurred in the presence of reduced perfusion pressure and volume expansion and to some extent with renal denervation. When prostaglandin E₂ or acetycholine were infused prior to contralateral kidney clamping, proximal reabsorption remained at control levels and the contralateral clamping response was blocked. Similar blockade occurred after treatment with indomethacin. Acute reduction in nephron mass causes a marked depression of proximal tubular sodium and fluid absorption not obviously accounted for by hemodynamicphysical factors and humoral factors may be involved. The level of distal reabsorption to increased proximal delivery following contralateral clamping, determines the net urinary excretion.     

Kinetics of Na(+) transport in necturus proximal tubule

The dependence of proximal tubular sodium and fluid readsorption on the Na(+) concentration of the luminal and peritubular fluid was studied in the perfused necturus kidney. Fluid droplets, separated by oil from the tubular contents and identical in composition to the vascular perfusate, were introduced into proximal tubules, reaspirated, and analyzed for Na(+) and [(14)C]mannitol. In addition, fluid transport was measured in short-circuited fluid samples by observing the rate of change in length of the split droplets in the tubular lumen. Both reabsorptive fluid and calculated Na fluxes were simple, storable functions of the perfusate Na(+) concentration (K(m) = 35-39 mM/liter, V(max) = 1.37 control value). Intracellular Na(+), determined by tissue analysis, and open-circuit transepithelial electrical potential differences were also saturable functions of extracellular Na(+). In contrast, net reabsorptive fluid and Na(+) fluxes were linearly dependent on intracellular Na(+) and showed no saturation, even at sharply elevated cellular sodium concentrations. These concentrations were achieved by addition of amphotericin B to the luminal perfusate, a maneuver which increased the rate of Na(+) entry into the tubule cells and caused a proportionate rise in net Na(+) flux. It is concluded that active peritubular sodium transport in proximal tubule cells of necturus is normally unsaturated and remains so even after amphotericin-induced enhancement of luminal Na(+) entry. Transepithelial movement of NaCl may be described by a model with a saturable luminal entry step of Na(+) or NaCl into the cell and a second, unsaturated active transport step of Na(+) across the peritubular cell boundary.     

Solvent drag measurement of transcellular and basolateral membrane NaCl reflection coefficient in kidney proximal tubule

The NaCl reflection coefficient in proximal tubule has important implications for the mechanisms of near isosmotic volume reabsorption. A new fluorescence method was developed and applied to measure the transepithelial (sigma NaClTE) and basolateral membrane (sigma NaClcl) NaCl reflection coefficients in the isolated proximal straight tubule from rabbit kidney. For sigma NaClTE measurement, tubules were perfused with buffers containing 0 Cl, the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl] quinolinium and a Cl-insensitive indicator fluorescein sulfonate, and bathed in buffers of differing cryoscopic osmolalities containing NaCl. The transepithelial Cl gradient along the length of the tubule was measured in the steady state by a quantitative ratio imaging technique. A mathematical model based on the Kedem-Katchalsky equations was developed to calculate the axial profile of [Cl] from tubule geometry, lumen flow, water (Pf) and NaCl (PNaCl) permeabilities, and sigma NaClTE. A fit of experimental results to the model gave PNaCl = (2.25 +/- 0.2) x 10(-5) cm/s and sigma NaClTE = 0.98 +/- 0.03 at 23 degrees C. For measurement of sigma NaClbl, tubule cells were loaded with SPQ in the absence of Cl. NaCl solvent drag was measured from the time course of NaCl influx in response to rapid (less than 1 s) Cl addition to the bath solution. With bath-to-cell cryoscopic osmotic gradients of 0, -60, and +30 mosmol, initial Cl influx was 1.23, 1.10, and 1.25 mM/s; a fit to a mathematical model gave sigma NaClbl = 0.97 +/- 0.04. These results indicate absence of NaCl solvent drag in rabbit proximal tubule. The implications of these findings for water and NaCl movement in proximal tubule are evaluated.