Secretion of prostaglandin E2 by rabbit proximal tubules.

This study was designed to evaluate prostaglandin secretion from bath to urine in isolated perfused rabbit proximal tubules. Active prostaglandin E2 (PGE2) secretion occurred along the entire length of the proximal tubule, but the rate of net secretion was highest in the S2 segment of the proximal straight tubule. Sixteen percent of the PGE2 secreted in the proximal straight tubule was metabolized to other products. The PGE2 cell-to-bath ratio averaged 40 and the tubule fluid-to-bath ratio averaged 3.4. These findings suggest active transport of PGE2 across the peritubular membrane and passive movement across the luminal membrane. Indomethacin, probenecid, para-aminohippurate, and ouabain partially inhibited PGE2 cell accumulation and net secretion. PGE2 entered the urine of the perfused descending limb of Henle (DLH), but at a rate two orders of magnitude below that observed in the S2 segment of the proximal tubule. No evidence of active PGE2 secretion was observed in the DLH. These results suggest that PGE2 is secreted into the urine at substantial rates by the organic anion transport system of renal proximal tubules.     

Carbon dioxide permeability of rabbit proximal convoluted tubules

CO2 kinetics were studied under conditions in which CO2 partial pressure and either the bicarbonate or hydrogen ion concentrations were unequal in perfusate and bath. A glass pH microelectrode and microcalorimeter were used to measure pH and total CO2 in perfused and collected fluids. Luminal appearance or disappearance of CO2 was determined from the change in concentrations of CO2 and acidic moieties of the buffers between perfused and collected fluids. The pH was not measurably affected by the tubule processes of metabolism, hydrogen ion secretion, and buffer transport because of relatively high flow rates. Since CO2 appearance and disappearance were directly related to CO2 partial pressure differences across the tubule (r = 0.94), this technique provides a valid estimate of transepithelial CO2 flux in response to the driving force of a CO2 partial pressure difference. From flux per unit of driving force and from estimates of resistances to CO2 diffusion in both internal and external unstirred layers, we obtained a transepithelial CO2 permeability of approximately 10(-4) cm3 . s-1 . cm tubule length-1. This corresponds to a diffusion coefficient through the tubule epithelium about half that through an equivalent thickness of water. We conclude that rabbit proximal convoluted tubules are so highly permeable to CO2 that even if all the filtered bicarbonate were reabsorbed by the generation of CO2 in the lumen, the tubular fluid CO2 partial pressure would exceed that of the peritubular blood by less than 4 mmHg.     

Ultrastructure of isolated perfused proximal tubules from rabbit kidney. A comparison with proximal tubules fixed by perfusion in vivo

This study was carried out to determine whether the ultrastructure of proximal tubules isolated and perfused in vitro was comparable to the ultrastructure of tubules fixed by perfusion in vivo. The kidneys of female white rabbits were either fixed by perfusion in vivo with glutaraldehyde (controls) or removed for dissection (experimentals) of proximal convoluted tubules and late proximal straight tubules. The isolated tubules were perfused in vitro with Krebs-Ringers bicarbonate solution for 1 hour after which the tubules were fixed with glutaraldehyde. The experimental tubules and the control tubules were processed for electron microscopy, compared qualitatively, and analyzed morphometrically to evaluate the volume densities and surface densities of different cell organelles and the cell membrane. Qualitatively, there were no differences in the appearance of cell organelles in experimental tubules and control tubules, except that cells in some experimental tubules accumulated small lipid droplets which were located close to mitochondria. Only a few quantitative changes were found, the most noteworthy being a 40% decreased surface density of the brush border in experimental proximal convoluted tubules and late proximal straight tubules. Damaged experimental tubules showed an increased number of vacuoles in the inverted microscope. In the electron microscope the vacuoles corresponded to swollen and disintegrated mitochondria and enlarged endocytic vacuoles. However, small degrees of tubular damage were not observable in the inverted microscope, as very small vacuoles could only be seen in the electron microscope. The results show that proximal tubules can be dissected and perfused in vitro for 1 hour without major ultrastructural changes. It should be emphasized that tubules showing an increased vacuolization in the inverted microscope may be damaged and, thus, not function optimally, and even if the tubules appear undamaged in the light microscope they may show signs of cellular degeneration in the electron microscope.     

Basolateral choline transport in isolated rabbit renal proximal tubules

 Choline can undergo both net secretion and net reabsorption by renal proximal tubules, but at physiological plasma levels net reabsorption occurs. During this process, choline enters the cells at the luminal side down an electrochemical gradient via a specific transporter with a high affinity for choline. It appeared likely that choline was then transported out of the cells against an electrochemical gradient at the basolateral membrane by countertransport for another organic cation. This possibility was examined by studying net transepithelial reabsorption and basolateral uptake and efflux of [¹⁴C]choline in isolated S2 segments of rabbit renal proximal tubules. Basolateral uptake, which was inhibited by other organic cations such as tetraethylammonium (TEA), appeared to occur by the standard organic cation transport pathway. However, the addition of TEA to the bathing medium not only failed to trans-stimulate net transepithelial reabsorption and basolateral efflux of [¹⁴C]choline but it actually inhibited transepithelial reabsorption by @60%. The results do not support the presence of a countertransport step for choline against an electrochemical gradient at the basolateral membrane. Instead, they suggest that choline crosses this membrane by some form of carrier-mediated diffusion even during the reabsorptive process. Received: 24 March 1998 / Received after Revision: 15 June 1998 / Accepted: 2 July 1998     

Secretion of proteins and glycoproteins by perifused rabbit corpus epididymal tubules: Effect of castration

Protein synthesis in epididymal tissue of intact and castrated rabbits was studied after incubation of epididymal minces with [³⁵S]-cysteine or [³⁵]-methionine and protein separation by two-dimensional gel electrophoresis. Regional differences in the pattern of protein synthesized were observed. Castration did not change overall protein synthesis, but it reduced these regional differences. The presence of 5α-DHT in the culture medium of the proximal corpus epididymidis perfused for 24 hr did not increase overall protein synthesis in tubules from intact or castrated rabbits and did not reinitiate synthesis of the proteins that had disappeared after castration. The kinetics of glycoprotein synthesis and secretion were studied by light and electron microscopy autoradiography at 0.5, 2, 6, and 24 hr after exposure to [³H]-mannose, [³H]-fucose, and [³H]-glucosamine. Changes in the distribution of msinnose- and glucosamine-labeled material indicated that the decline in grain density over the epithelium from 30 min to 24 hr coincided with an increasing reaction over the stereocilia border from 30 min to 2 hr and in thejumen from 2 to 24 hr. The distribution of fucose-labeled material indicated that the grain reaction over the epithelium declined more rapidly than with the mannose label. When the glucosamine-labeled sperm mass was released from the tubules, the labeled material was lost after the first washing, indicating that the glucosamine-labeled glycoproteins did not bind firmly to corpus spermatozoa within 24 hr. After castration, both mannose- and fucose-labeled materials migrated to the cell apex more rapidly than in the intact animal, but they were not released as readily into the lumen. The culture of epididymal tubules from castrated males with 5a-DHT for 24 hr did not promote the release of either mannose- or fucose-labeled material into the lumen. However, testosterone given in vivo for 2 weeks restored secretion of mannose-labeled material into the lumen.     

Basolateral membrane properties in proximal convoluted tubules of the newborn rabbit

We examined the properties of basolateral cell membranes in "immature" and "mature" proximal tubules and evaluated the possibility that changes in those membranes might contribute to the reported maturation of tubule function. Proximal convoluted tubules were dissected in connection with superficial glomeruli, crimped at both ends, and induced to swell by use of ouabain, low protein bathing medium, and/or collagenase. Tubules from adult animals swelled faster than tubules from 2- to 5- or 14- to 17-day-old animals. However, the basolateral cell membranes of tubules from adult animals were three- to fourfold larger in surface area than those from 2- to 17-day animals, so that the ratio of the swelling rates to the measured basolateral membrane areas remained constant during the period from newborn to adult. We conclude that the basolateral membranes "mature" only in their surface areas and that little change occurs in their intrinsic properties such as hydraulic conductivity or ion permeability.     

Mechanisms of transjunctional transport of NaCl and water in proximal tubules of mammalian kidneys

Tight junctions and the intercellular space of proximal tubules are not accessible to direct measurements of fluid composition and transport rates, but morphological and functional data permit analysis of diffusion and osmosis causing transjunctional NaCl and water transport. In the S2 segment NaCl diffuses through tight junctions along a chloride gradient, but against a sodium gradient. Calculation in terms of modified Nernst-Fick diffusion equation after eliminating electrical terms shows that transport rates (300-500 pmol min-1 mm-1 tubule length) and transepithelial voltage of +2 mV are in agreement with observations. Diffusion coefficients are Dtj=1500 microm2 s-1 in the S1 segment, and Dtj=90-100 microm2 s-1 in the S2 segment where apical intercellular NaCl concentration is 132 mM, 1 mM below complete stop (Dtj=0 and Donnan equilibrium). Tight junctions with gap distance 6 A are impermeable to mannitol (effective molecular radius 4 A); reflection coefficients are sigma=0.92 for NaHCO3 and sigma=0.28 for NaCl, because of difference in anion size. The osmotic force is provided by a difference in effective transjunctional osmolality of 10 mOsm kg-1 in the S1 segment and 30 mOsm kg-1 in the S2 segment, where differences in transjunctional concentration contribute with 21 mOsm kg-1 for NaHCO3 and -4 mOsm kg-1 for NaCl. Transjunctional difference of 30 mOsm kg-1 causes a volume flow of 2 nL min-1 mm-1 tubule length. Luminal mannitol concentration of 30 mM stops all volume flow and diffusive and convective transport of NaCl. In conclusion, transjunctional diffusion and osmosis along gradients generated by transcellular transport of other solutes account for all NaCl transport in proximal tubules.     

Analysis of reabsorbate concentrations in the proximal tubules of dog kidneys during osmotic diuresis

To examine to what extent the reabsorbate concentrations, calculated as the flux ratios between solutes and water, represent the fluid composition in the lateral intercellular space (LIS) in the proximal tubules, reabsorption was stimulated by elevating PCO2 from 5 to 13 kPa before and during infusion of mannitol to a plasma concentration of 70 mM in volume-expanded dogs receiving ethacrynic acid. The reabsorbate concentration of NaHCO3 increased by 50 mM during mannitol infusion. The real concentration of NaHCO3 in LIS could not, however, be elevated by this amount, since the driving forces for fluid reabsorption then would have increased during osmotic diuresis due to diffusion of mannitol into LIS from plasma. A model analysis of diffusion in LIS showed that transcellular transport can only lead to trivial increases of LIS concentrations compared to plasma, whereas diffusion across tight junctions can increase LIS concentrations by several mM. NaCl diffusion and coupled transcellular water transport may therefore represent a significant contribution to total bicarbonate-dependent NaCl and water reabsorption in the proximal tubules.     

Mechanisms of intercellular hypertonicity and isotonic fluid absorption in proximal tubules of mammalian kidneys

The main purpose of this theoretical analysis (second of two articles) is to examine whether transjunctional diffusion of NaCl causes intercellular hypertonicity, which permits transcellular water transport across solute-impermeable lateral cell membranes until osmotic equilibration. In the S2 segment with tubular NaCl concentration 140 mM, the calculated apical intercellular NaCl concentration is c0 approximately 132 mM, which exceeds peritubular NaCl concentration by 12 mM or 22 mOsm kg-1. Variations in volume flow, junctional reflection coefficient (sigmaNaCl = 0.25-0.50), gap distance (g = 6-8 A), junctional depth (d = 18-100 A), intercellular diffusion coefficient (DLIS=500-1500 microm2 s-1) and hypothetical active NaCl transport alter c0 only by a fraction of 1 mM. However, dilution and back-leakage of NaHCO3 lower apical intercellular hyperosmolality to approximately 18 mOsm kg-1. Water transport through solute-impermeable lateral cell membranes continues until intercellular and cellular osmolalities are equal. Transcellular and transjunctional volume flow are of similar magnitude (2 nL min-1 mm-1 tubule length) in the S2 segment. Thus, diffusion ensures isotonic absorption of NaCl. Two-thirds of NaHCO3 and other actively transported sodium salts are extruded into the last third of the exponentially widening intercellular space where the exposure time is only 0.9 s. Osmotic equilibration is dependent on aquaporins in the cell membranes. If permeability to water is low, transcellular water transport stops; tubular fluid becomes hypotonic; NaCl diffusion diminishes, but transjunctional water transport remains unaltered as long as transcellular transport of NaHCO3 and other solutes provides the osmotic force.     

Effect of gentamicin on isolated glomeruli and proximal tubules of the rabbit

Alterations in both glomerular filtration rate and tubular transport occur in clinical gentamicin nephrotoxicity. We have studied the function of isolated tubules and glomeruli from rabbits treated with gentamicin. Gentamicin was administered subcutaneously to sexually immature (1400 to 1800 gm) or sexually mature (3800 to 4600 gm) New Zealand White rabbits in a dose of 15 mg/kg twice a day. Immature rabbits were treated for 28 to 31 days and developed only minimal renal insufficiency. About one-half of the mature rabbits developed azotemia. The mature rabbits that did not become azotemic were sacrificed after 28 to 30 days, and those that became azotemic were killed when their serum creatinine reached 2.5 mg/dl or higher (10 to 24 days). Animals were anesthetized and kidneys were removed for histologic examination and isolation of tubules and glomeruli. The ratio of p-aminohippuric acid (PAH) concentration in isolated tubule cells to that in medium after incubation in 3H-PAH (1 microM) at 37 degrees C for 30 minutes (T/M PAH) was used as an indicator of transport capacity of tubules. T/M PAH ratios averaged 196 +/- 18 and 111 +/- 21 for control immature and mature rabbits, respectively, and 135 +/- 22, 80 +/- 16, and 9 +/- 2 for gentamicin-treated immature and mature nonazotemic and mature azotemic rabbits, respectively. Glomeruli were isolated and filtration induced in vitro by a transcapillary oncotic gradient. Ultrafiltration coefficient, Kf, of glomeruli of immature and mature control rabbits averaged 3.78 +/- 0.29 and 5.84 +/- 0.51 nl/minute X mm Hg. Kf from gentamicin-treated immature rabbits averaged 2.82 +/- 0.20 and from mature azotemic rabbits 3.14 +/- 0.44 nl/minute X mm Hg. Kf of both mature and immature rabbits were decreased compared with controls (p less than 0.01). When all animals were considered, relative glomerular filtration rate, estimated from 1/serum creatinine, was positively correlated with the T/M PAH and Kf. When only experimental animals were studied, 1/serum creatinine and T/M PAH were also correlated. Decreased glomerular filtration rate and dysfunction of proximal tubules were also correlated with abnormal tubule histology. We suggest that injury to glomeruli and tubules may represent independent manifestations of gentamicin toxicity. Dysfunction may be present even when there are only mild histologic changes and glomerular filtration rate is near normal. Kf does not appear to limit glomerular filtration rate after treatment with gentamicin; rather, some direct or indirect effect of tubular injury may determine the decrement in glomerular filtration rate.     

Temperature dependence of transepithelial potential in isolated perfused rabbit proximal tubules.

The response of the transepithelial potential to rapid cooling in isolated perfused proximal convoluted (PCT) and proximal straight (PST) tubules has been studied. Tubules were perfused with solutions which simulated glomerular filtrate (A), filtrate minus glucose and alanine (B), and late proximal tubular fluid (C). The values of the potentials at 37 and 10 degrees C as well as the temperature-sensitive component were found to vary with perfusate composition. Temperature sensifound to vary with perfusate composition. Temperature sensitivity in PCT was seen only when glucose and alanine were present in the perfusate. In contrast, a portion of the potential difference in PST was temperature dependent under each of the perfusion conditions. Temperature sensitivity in PST was inhibited by 10(-5) M ouabain in the bath. It is concluded that the lumen-negative, temperature-sensitive component of PCT potential may reflect the coupled luminal entry of Na+ along with glucose and alanine. In PST, the temperature-sensitive component can be associated with Na+ transport but is not dependent on luminal concentrations of glucose, alanine, HCO3- or Cl- over the ranges examined. Potentials in both segments at 10 degrees C are interpreted as resulting from processes that are passive in nature.     

Ca2+ and Ba2+ effects on basolateral tetraethylammonium transport in isolated snake renal proximal tubules

 Previous work on snake renal tubules suggested that basolateral transport of tetraethylammonium (TEA) is symmetrical. To examine regulation of this transport step more closely, we determined the effects of (1) reductions in the extracellular Ca²⁺ concentration and basolateral Ca²⁺ entry, and (2) the presence of extracellular Ba²⁺ on TEA uptake and efflux across the basolateral membrane of isolated snake renal proximal tubules. Removal of extracellular Ca²⁺ reduced initial TEA uptake and enhanced TEA efflux. Blocking Ca²⁺ entry also reduced initial TEA uptake. Extracellular Ba²⁺ depolarized the basolateral membrane and reduced both TEA uptake and efflux. Inhibition of basolateral TEA uptake with a reduced membrane potential supports previous data indicating that uptake involves potential-driven facilitated diffusion. Inhibition of basolateral TEA efflux by Ba²⁺ even with a reduced membrane potential not only supports previously obtained data indicating that efflux is not influenced by the potential difference and that basolateral TEA transport is asymmetrical, but also suggests that TEA uptake and efflux may occur by separate pathways. Received: 22 May 1997 / Received after revision: 24 July 1997 / Accepted: 28 July 1997     

Lack of solvent drag of NaCl and NaHCO3 in rabbit proximal tubules

Using in vitro microperfusion of rabbit nephron segments we measured the effects of osmotically induced water flow on net transport of HCO3 and Cl. Measurements were made in superficial and juxtamedullary proximal convolutions and in superficial pars recta. In addition, measurements were taken in the presence and absence (hypothermia) of active transport. Using osmotic gradients of 25 mM raffinose in superficial and 50 mM in juxtamedullary segments, we observed increases in water flow equal to or greater than the normal rates of volume reabsorption observed in these tubule segments. However, there were no significant changes in HCO3 and Cl flux. This lack of significant solvent drag was seen both when osmotic water flow was in the lumen-to-bath direction and when osmotic flow was in the bath-to-lumen direction. The results of these studies suggest that solvent drag does not contribute significantly to NaCl and NaHCO3 reabsorption in proximal tubules. The lack of significant solvent drag of these salts can be interpreted as indicating either that osmotically induced transepithelial water flow in proximal tubules almost exclusively traverses transcellular pathways or that proximal tubule tight junction reflection coefficients for these salts are close to unity.     

Giant mitochondria in the epithelial cells of the proximal convoluted tubules of diseased human kidneys.

Electron microscopy of 100 renal biopsies obtained at random from patients with various renal disorders revealed gigantic mitochondria in the cytoplasm of the epithelial cells of the proximal tubules in 36 cases (36 per cent). These giant mitochondria are usually round, ovoid, spindle-shaped or rod-shaped often reaching the size of the cell nucleus, and appear singularly and sporadically in the epithelial cell lining of the tubules. Once recognized by electron microscopy they are easily identified in routine histologic preparations and show the same staining characteristics as other mitochondria. In electron microscopy, giant mitochondria have a double limiting membrane, abundant matrical substance, and few remnants or cristae. In addition, they are characterized by (1) intramatrical irregularly branching fibrils approximately 35 A thick, (2) intramatrical bundles of straight filaments approximately 60 to 70 A thick, (3) intramatrical electron-opaque globules up to 0.6 mum., and (4) helical filaments in focally dilated spaces of the outer compartment. The significance of such a remarkable morphologic change of tubular mitochondria and the nature of the unusual components are at present unknown, but an abnormal metabolic condition is considered in its pathogenesis. Nothing specific has yet been found in the relationship between the positive appearance of giant mitochondria and types of renal disorders or clinical and laboratory data. It may be postulated from the present study that "giant mitochondrial tubulopathy" is a rather common, characteristic, and noteworthy change in the histopathology of renal tubule.     

Active amino acid absorption by proximal convoluted and proximal straight tubules.

Transport of glycine and alpha-aminoisobutyric acid (AIB) was studied in proximal convoluted (PCT) and proximal straight (PST) tubules isolated from rabbit kidney. In both segments, unidirectional lumen-to-bath fluxes (J1 leads to b) (pmol min-1mm-1) of glycine and AIB exceeded corresponding bath-to-lumen fluxes (Jb leads to 1), which demonstrated that both were actively absorbed. During J1 leads to b measurements, intracellular concentrations of both amino acids were greater than the luminal concentration, indicating that the site of active transport was the luminal membrane. Replacement of Na+ by choline in both perfusate and bath (PCT) or perfusate alone (PST) reduced J1 leads to b for glycine to equal Jb leads to 1. Nonlinear fitting of the relationship between J1 leads to b and the mean luminal glycine concentration according to Michaelis-Menten kinetics gave Jmax values of 28.5 (PCT) and 2.5 (PST) pmol min-1 mm-1, and Km values of 11.8 (PCT) and 0.7 (PST) mM. There was a parallel, Na+-independent, nonsaturable component of J1 leads to b characterized by an apparent permeability coefficient of 0.19 micron/s in the PCT and 0.04 micron/s in the PST.