Innervation of the renal proximal convoluted tubule of the rat

Experimental data suggest the proximal tubule as a major site of neurogenic influence on tubular function. The functional and anatomical axial heterogeneity of the proximal tubule prompted this study of the distribution of innervation sites along the early, mid, and late proximal convoluted tubule (PCT) of the rat. Serial section autoradiograms, with tritiated norepinephrine serving as a marker for monoaminergic nerves, were used in this study. Freehand clay models and graphic reconstructions of proximal tubules permitted a rough estimation of the location of the innervation sites along the PCT. In the subcapsular nephrons, the early PCT (first third) was devoid of innervation sites with most of the innervation occurring in the mid (middle third) and in the late (last third) PCT. Innervation sites were found in the early PCT in nephrons located deeper in the cortex. In juxtamedullary nephrons, innervation sites could be observed on the PCT as it left the glomerulus. This gradient of PCT innervation can be explained by the different tubulovascular relationships of nephrons at different levels of the cortex. The absence of innervation sites in the early PCT of subcapsular nephrons suggests that any influence of the renal nerves on the early PCT might be due to an effect of neurotransmitter released from renal nerves reaching the early PCT via the interstitium and/or capillaries.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V _BL) and Cl-selective electrode PD (V _BL ^Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate _i ^Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl^– transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^Cl ; both results are consistent with basolateral membrane uphill Cl^– transport from interstitium to the cell. TMA⁺ for Na⁺ substitutions in either luminal or peritubular perfusate had no effect on _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^Cl , presumably related to enhanced Cl^– cell influx: we infer that Cl^– is exchanged against HCO ₃ ^– at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl^– concentration above equilibrium values: intracellular CO₂ hydration gives rise to cell HCO ₃ ^– concentrations above equilibrium. The passive exit of HCO ₃ ^– at the basolateral membrane energizes an uphill entry of Cl^– into the cell. The resulting increase of _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Ultrastructural localization of glucose-6-phosphatase activity in proximal convoluted tubule cells of rat kidney

Summary The ultrastructural localization of glucose 6-phosphatase activity was investigated in the proximal convoluted tubule cells of the rat kidney. The reaction product for the enzyme activity was present in the endoplasmic reticulum and nuclear envelope, as reported for the hepatic enzyme and others, but was absent from the brush border, plasma membrane and other organelles. The metabolic significance of the association of this enzyme with the endoplasmic reticulum and the role of the enzyme in the active reabsorption and transport of glucose in the renal tubules are discussed.     

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture

The mechanisms and control of transepithelial inorganic sulfate (Si) transport by primary cultures of chick renal proximal tubule monolayers in Ussing chambers were determined. The competitive anion, S2 O 3 2- (5 mM), reduced both unidirectional reabsorptive and secretory fluxes and net Si reabsorption with no effect on electrophysiological properties. The carbonic anhydrase (CA) inhibitor ethoxzolamide decreased net Si reabsorption approximately 45%. CAII protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Cortisol reduced net Si reabsorption up to approximately 50% in a concentration-dependent manner. Thyroid hormone increased net Si reabsorption threefold in 24 h, and parathyroid hormone (PTH) acutely stimulated net Si reabsorption approximately 45%. These data indicate that CA participates in avian proximal tubule active transepithelial Si reabsorption, which cortisol directly inhibits and T3 and PTH directly stimulate.     

Osmotic gradient dependence of osmotic water permeability in rabbit proximal convoluted tubule

Summary To assess steady-state transepithelial osmotic water permeability (P _f ), rabbit proximal convoluted tubules were perfused in vitro with the impermeant salt, sodium isethionate at 26°C. Osmotic gradients () were established by varying the bath concentration of the impermeant solute, raffinose. When lumen osmolality was 300 mOsm and bath osmolality was 320, 360 and 400 mOsm, apparentP _f decreased from 0.5 to 0.10 to 0.08 cm/sec, respectively. Similar data were obtained when lumen osmolality was 400 mOsm. Five possible causes of the dependence of apparentP _f were considered experimentally and/or theoretically: (1) external unstirred layer (USL); (2) cytoplasmic USL; (3) change in surface area; (4) saturation of water transport; (5) down-regulation ofP _f . ApparentP _f was inhibited 83% byp-chloromercuribenzene sulfonate (pCMBS) at 20 mOsm, but not at 60 mOsm , suggesting presence of a serial barrier resistance to water transport. Increases in perfusate or bath solution flow rate and viscosity did not alter apparentP _f , ruling out an external USL. A simple cytoplasmic USL, described by a constant USL thickness and solute diffusion coefficient, could not account for the dependence of apparentP _f according to a mathematical model. The activation energy (E _a ) for apparentP _f increased from 7.0 to 12.5 kcal/mol when was increased from 20 to 60 mOsm, not consistent with a simple USL or a change in membrane surface area with transepithelial water flow. These findings are most consistent with a complex cytoplasmic USL, where the average solute diffusion coefficient and/or the area available for osmosis decrease with increasing . These results (1) indicate that trueP _f (at physiologically low ) is very high (>0.5 cm/sec) in the rabbit proximal tubule; (2) provide an explanation for the wide variation inP _f values reported in the literature using different , and (3) suggest the presence of a flow-dependent cytoplasmic barrier to water flow.     

The electron density of light and dark lysosomes in the proximal convoluted tubule of the rat kidney

The present paper deals with the electron density of the lysosomal matrices in the renal proximal-convoluted-tubule cells of Wistar rats (20-60 days of age). Its purpose was to determine which physicochemical factors influence the electron density of lysosomes, and how their electron density is affected by various methods of fixation.--3 types of lysosomes can be distinguished in the proximal-convoluted-tubule cell; namely light, intermediate and dark lysosomes, which have an electron density lower, equal to or higher than the surrounding cytoplasm. All 3 types of lysosomes were invariably present after all methods of fixation tested. Light, intermediate and dark lysosomes differ in several respects. Staining intensity with uranyl acetate, lead citrate and potassium permanganate, osmiophilia, basophilia (in semithin sections) and --probably most importantly--the physical mass density of the lysosomal matrix are all low in light lysosomes, higher in intermediate lysosomes and highest in dark lysosomes. Light, intermediate and dark lysosomes of the proximal convoluted tubule do not form discrete classes, but one continuous spectrum of lysosomes of increasing electron density.     

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.     

Bicarbonate-water interactions in the rat proximal convoluted tubule. An effect of volume flux on active proton secretion

The effect of volume absorption on bicarbonate absorption was examined in the in vivo perfused rat proximal convoluted tubule. Volume absorption was inhibited by isosmotic replacement of luminal NaCl with raffinose. In tubules perfused with 25 mM bicarbonate, as raffinose was increased from 0 to 55 to 63 mM, volume absorption decreased from 2.18 +/- 0.10 to 0.30 +/- 0.18 to -0.66 +/- 0.30 nl/mm X min, respectively, and bicarbonate absorption decreased from 131 +/- 5 to 106 +/- 8 to 91 +/- 13 pmol/mm X min, respectively. This bicarbonate-water interaction could not be attributed to dilutional changes in luminal or peritubular bulk phase bicarbonate concentrations. Inhibition of active proton secretion by acetazolamide abolished the effect of volume flow on bicarbonate absorption, which implies that the bicarbonate reflection coefficient is close to 1 and eliminates the possibility of solvent drag across the tight junction. When the luminal bicarbonate concentration was varied, the magnitude of the bicarbonate-water interaction increased with increasing luminal bicarbonate concentration. The largest interaction occurred at high luminal bicarbonate concentrations, where the rate of proton secretion has been previously shown to be independent of luminal bicarbonate concentration and pH. The results thus suggest that a peritubular and/or cellular compartment exists that limits bicarbonate diffusion, and where pH changes secondary to bicarbonate-water interactions (solute polarization) alter the rate of active proton secretion.     

Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')- bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.     

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.     

Lipids in the proximal convoluted tubule of the cat kidney and the reabsorption of cholesterol

Summary Lipid deposits in the cat kidney are mainly located in the epithelium of the proximal tubuli contorti, particularly in the pars contorta. As the amount of fatty acids in the blood of renal arteries is higher than in renal veins, the lipid inclusions are likely to be formed in the proximal convoluted tubule. Whether fat occurring in the urine has been released from the nephron epithelium and the mode of this release remains obscure. The structural equivalent of lipid extrusion into the tubules has not been observed.Components of the tubular lipids include triglycerides, phosphoglycerides and cholesterol. The results of the digitonin-cholesterol reaction favour the assumption that cholesterol is eliminated in the glomeruli and pinocytotically reabsorbed by the brush border cells, this process possibly serving recycling of this compound. The dilated basal labyrinth and intercellular space contain perpendicularly oriented lipid accumulations that reach the basal lamina. The ultrastructure of the lipid storing cells of pars contorta reacting positively for phosphoglyceride and cholesterol is characterised mainly by bodies with marginal plates. As far as can be judged from their morphology, these bodies are interpreted as large peroxisomes. A special feature of the pars recta are dumbbell shaped bodies and elongated or cup-like mitochondria concentrically surrounding cytoplasmic areas, as well as a well-developed smooth ER. In what way the organelles of the brush border cells are involved in catabolic and anabolic processes as far as renal lipid metabolism is concerned remains to be answered.This investigation was supported by a grant from the Deutsche ForschungsgemeinschaftThis paper is dedicated in friendship to Professor Berta Scharrer (New York) on the occasion of her 70th birthday