Tubular Volume Research Articles

Possible role of basolateral cell membrane in proximal renal tubule osmoregulation

The cellular response to hypotonic stimulation was studied with videometric methods in 266 proximal renal tubules dissected from Carassius auratus (goldfish). In hypotonic solutions (low NaCl), cells underwent rapid swelling followed by gradual shrinking toward isotonic volume (volume-regulatory decrease phase, VRD). Hypothermia (8 degrees C), increased extracellular potassium (15, 25, and 40 mM), quinine (0.1 mM), barium (0.5 mM), 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS; 0.02 mM), acetazolamide (0.1 mM), decrements in extracellular bicarbonate, and increases in extracellular chloride impaired VRD. Ouabain (1.0 mM), furosemide (0.1 mM), and the chloride channel blocker 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB; 0.001 mM) had no effect. While VRD occurred in the absence of extracellular calcium influx, addition of the calcium ionophore A23187 (0.01 mM) in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 2.0 mM) impaired this process both in acidic and alkaline media. Trifluoroperazine (0.01 mM) reversibly inhibited VRD. The effect of this calmodulin inhibitor could not be overridden with the cationic ionophore gramicidin (0.5 microM). The data suggest that Carassius proximal renal tubular cells volume regulate in hypotonic solutions by the loss of KCl and osmotically obligated water. We postulate that the main efflux of potassium is through a calcium-gated potassium channel with its counter ion extruded through a calmodulin-regulated Cl(-)-HCO3- exchanger.

Neonatal rabbit juxtamedullary proximal convoluted tubule acidification.

The present in vitro microperfusion study examined apical membrane Na+/H+ antiporter and basolateral membrane Na(HCO3)3 symporter activity in newborn and adult juxtamedullary proximal convoluted tubules. Proton fluxes were determined from the initial rate of change of intracellular pH after a change in the luminal or bathing solution, buffer capacity, and tubular volume of newborn and adult tubules. Intracellular pH (pHi) was measured fluorometrically using the pH-sensitive dye (2',7')-bis (carboxyethyl)-(5,6)-carboxyfluorescein (BCECF). Apical Na+/H+ antiporter proton flux, assayed by the effect of sodium removal (147----0 meq/liter) on pHi, was one-third the adult level for the first 2 wk and doubled in the 3rd wk of life. Adult levels were achieved by 6 wk of age. Na+/H+ antiporter activity was not detected on the basolateral membrane of 1-wk-old newborns, indicating that polarity of this transporter was already present. Basolateral membrane Na(HCO3)3 proton flux, assayed by the effect of a bath bicarbonate change (25----5 meq/liter) and by a bath sodium change (147----0 meq/liter) on pHi, was 50-60% of adult values in 1-wk-old newborns. Basolateral membrane Na(HCO3)3 proton flux assayed by a bath bicarbonate change (25----5 meq/liter) remained at 50-60% of adult values for the 1st mo of life and increased to adult levels by 6 wk of age. This transporter not only plays a role in net acidification, but is an important determinant of cell pH in newborn juxtamedullary proximal convoluted tubules.

Effect of cell sodium on [formula omitted] sodium efflux in cortical collecting tubule of rabbits under different aldosterone status

Aldosterone increased the tubular volume in cortical collecting tubules (CCD) of rabbit kidney. It modulated the rate of cell sodium accumulation, under condition of ATPase inhibition ( 4° C, in the absence of K + ). In contrast, the relationship between Na +/K +-ATPase-dependent Na + extrusion rate and intracellular Na + concentration ( Na + i ) was similar in control, adrenalectomized, and aldosterone-treated adrenalectomized animals; Na + extrusion rate increased with Na + i , up to 70 mM Na + i, and then plateaued. This indicates that aldosterone does not modify the characteristics of Na + i-dependent Na + extrusion rate by the Na +/ K +- ATPase pump in CCD.

Effect of thyroid hormone on gentamicin accumulation in rat proximal tubule lysosomes

T4 decreases gentamicin nephrotoxicity, but the mechanism is unknown. This study investigated whether T4 affects renal processing of gentamicin by examining renal cortical gentamicin accumulation and proximal tubular lysosomal volume after 48 h of gentamicin (30 mg/kg twice daily) in rats (GT4) that had previously received 10 days of T4 (10 micrograms/100 g body wt). The pair-fed control group received only gentamicin. A T4-treated control group and an untreated control group were also pair fed and studied. At the end of the 12-day study the four groups did not differ in food intake, body weight, urine volume, urinary Na+ excretion, or urinary K+ excretion. Plasma creatinine, inulin clearance, and serum clearance of gentamicin were not different among the groups. Gentamicin accumulation was higher in G vs. GT4 (2.1 +/- 0.1 vs. 1.7 +/- 0.1 micrograms/mg protein, P less than 0.01). Blinded, computer-assisted electron microscopic analysis of outer cortical proximal tubules showed that lysosomes occupied a significantly smaller fraction of the cell volume in GT4 (8.1 +/- 0.5 vs. 11.6 +/- 1.1%, P less than 0.02). Thyroxine had no effect on lysosomal volume in non-gentamicin-treated rats. Except for scatter foci of apical vacuolization, proximal tubular cells of both groups were otherwise normal. The results show that chronic T4 administration decreases renal tubular accumulation of gentamicin, and this effect may in part explain its protective effect against gentamicin nephrotoxicity.

Relationship between cell volume and cation content in thick ascending limb of rat kidney.

We examined the relationship between the cell volume and cation concentration ([Nai] and [Ki]) of isolated segments of rat medullary thick ascending limb (MAL) after incubation at 30 degrees C in various isotonic solutions. When the tubules were incubated in a normal NaCl solution containing 5 mmol/l K+, addition of 1 mmol/l of ouabain increased [Nai] and decreased [Ki] but did not change the total ([Nai] + [Ki]) concentration (about 90 mEq/l) or tubular volume. After incubation in various K+-free solutions, the tubules were almost fully K+-depleted; their volume per unit of length was similar in the three solutions, although the choline Cl-treated tubules had a very low sodium content compared to the NaCl- and Na2SO4-treated tubules (8 vs. 97 and 95 mEq/l respectively). Ouabain altered neither volume nor [Nai] of tubules incubated in choline Cl or Na2SO4 solution. Transfer of tubules from K+-free Na2SO4 or K+-free choline Cl solution into K+-free NaCl solution resulted in an increase in [Nai] (by 29 and 97 mEq/l respectively) without much increase in tubular volume. A marked swelling of the tubules was only observed when the K+-free NaCl solution contained also ouabain. Under this condition, [Nai] was comparable to the Na+ concentration of the incubation medium. After washing and incubation in a normal NaCl solution containing K+, the swollen tubules recovered their initial volume and restored Na+ and K+ concentration gradients across the cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of Na and Cl in ouabain-induced cell swelling in thick ascending limb of rat kidney.

Changes in the volume of isolated segments of rat medullary thick ascending limb (MAL) were studied by a photographic technique, after tubule incubation in isotonic solutions in the absence or presence of ouabain and/or K. When segments were incubated at 30 degrees C in NaCl solution, their volume increased by 75% after removal of external K, and by 170% after removal of external K plus addition of 1 mmol/l ouabain. At steady state, tubular volume was a function of the external K concentration. Resting volume was obtained with external K concentrations higher than 0.1 and 1.0 mmol/l in the absence and presence of ouabain respectively. When MAL samples were incubated in isotonic K-free Na2SO4 or K-free choline Cl solution, their volume per unit of length was similar to that determined in NaCl medium, but there was no swelling after the addition of ouabain. The ouabain-induced swelling was shown to depend on both the Na and Cl concentrations in the incubate (apparent Km of 87 and 80 mmol/l for Na and Cl respectively). Swollen tubules recovered their resting volume when ouabain, Na or Cl was removed from the incubation medium. Recovery of resting volume was also observed after addition of K into the incubation medium. These observations indicate that rat MAL cell volume is the result of coupled passive net fluxes of Na and Cl, which depend on the respective electrochemical gradients for Na or Cl across the cell membranes and the Na-pump activity which continuously extrudes Na.

Effect of high protein intake on sodium, potassium-dependent adenosine triphosphatase activity in the thick ascending limb of Henle's loop in the rat.

1. We have previously shown that the hypertrophy of the kidney induced by a high protein diet consists of a preferential hypertrophy of the thick ascending limb (TAL) of Henle's loop. This might be related to an increase in the active salt transport by this segment. Sodium, potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) activity was measured in TAL from kidneys of rats fed either a low (LP) or a high (HP) protein diet for several weeks. 2. Enzymatic activity was measured by microdensitometry, after appropriate cytochemical reaction, for an adenosine 5'-triphosphate (ATP) concentration of 0-66 mmol/l. Both activity per unit tubular length and mean activity per unit tissue volume were recorded. A calibration was designed to convert usual microdensitometry units (extinction) into conventional biochemical units (mol of product formed). 3. For non-limiting substrate concentrations, the Na+,K+-ATPase activity, expressed per unit length of tubule on the sections, was 50% higher in HP than in LP rats, an increase proportional to that of the simultaneously measured tubule diameter. When expressed per unit tubular volume, Na+,K+-ATPase activity was similar in both groups of rats. The dissociation constant for ATP was also similar in both groups. 4. Results show that a high protein diet induces an increase in Na+,K+-ATPase activity in TAL, thus enabling an enhanced NaCl transport in this segment. This increase in transport capacity is not due to an increase in the density of enzymatic units but to an increase in their number, in relation to the hypertrophy of the TAL.

Alterations of enzymatic activities along rat collecting tubule in potassium depletion

This study was designed to correlate morphological alterations induced in rat collecting tubule by potassium depletion with changes in the activity of enzymatic markers of the cell basolateral membrane. Results show the following responses. 1) Potassium depletion induced a huge and progressive hypertrophy of the outer medullary collecting tubule (MCT). Hypertrophy was paralleled by enhancements of vasopressin- and forskolin-dependent adenylate cyclase (AC) activities. Glucagon-sensitive AC was also increased, but with a different kinetics, whereas isoproterenol-dependent AC was only modestly stimulated. 2) In cortical (CCT) and papillary collecting tubules, AC response to hormones did not change. The concentrating defect of K-deprived rats, therefore, does not appear to result from an intrinsically defective adenylate cyclase system in any portion of the collecting tubule. Decreased AC response of the medullary thick ascending limb to vasopressin and glucagon, observed after 3-5 wk of K depletion, might account, at least in part, for reduced hypertonicity of medullary tissue. 3) Na+-K+-ATPase activity fell in CCT, probably in relation to decreased K secretion. Conversely, in MCT, Na+-K+-ATPase rose much more than tubular volume. The physiological significance of this latter observation remains to be established.

Measurement of flow rate in rat proximal tubules with a nonobstructing optical method.

A method was developed for measuring volume flow rate in free-flowing proximal tubules. Multiple injections of a solution containing fluorescein isothiocyanate-dextran (mol wt 20,000) were made into the tubular fluid stream while fluorescence was detected by videomicroscopy. Progression of the dye bolus between two points along the tubule was measured to determine fluid velocity. Tubule diameter was measured by video image shearing and flow rate was calculated from the product of velocity and cross-sectional area. The technique was validated by simultaneous collections in the same tubule just downstream. There was good correlation between the results obtained with the two methods (r = 0.89). The estimated average injection volume was 0.0165 +/- 0.00016 nl. An increase in tubular volume flow caused by repetitive injections during micropuncture collections was too small to be detected. This new method should be useful in experimental situations that require uninterrupted delivery of fluid to the macula densa, or when better temporal resolution is required than can be obtained with the conventional method.

Testicular involution in elderly men: comparison of histologic quantitative studies with hormone patterns

An endocrinologic and quantitative histologic study was carried out in 64 elderly men who underwent orchidectomy owing to prostatic carcinoma. The men were classified into age groups (decade of life), and each group was subdivided into group A (testes with complete spermatogenesis in most tubules) and group B (testes showing maturation arrest of spermatogenesis in most tubules). Up to 80 years of age, men of group A showed hormone levels and testicular parameters similar to those of young control men. From 50 to 60 years of age, men of group B showed a significant decrease in testicular volume, tubular volume, tubular length, number of germ cells, Sertoli cells and Leydig cells per testis, and plasma testosterone levels, whereas the tunica propria thickness and plasma levels of both follicle-stimulating hormone and luteinizing hormone were increased.

Seminiferous tubules and daily sperm production in older adult men with varied numbers of Leydig cells.

Previous studies of adult men have failed to reveal a relationship between numbers of Leydig cells in the testes and rates of sperm production, perhaps because of a functional excess of these cells in younger men. Hence, a possible relationship between Leydig cell numbers and sperm production was sought in 50 older men, aged 50-90 years, in whom the Leydig cell population had been depleted by age-related attrition. When these men were sorted by increasing numbers of Leydig cells per man into two, three, or five groups, no difference could be found between or within these groups when daily sperm production per man (DSP); seminiferous tubular volume, diameter, or length; or seminiferous epithelial volume was examined. Furthermore, no significant correlation could be detected between Leydig cell numbers and DSP in these 50 men. The only relationship between numbers of Leydig cells and spermatogenesis appeared to be a threshold effect, in that men with fewer than 60 million Leydig cells (4 in this study) had drastically reduced DSP. Men with few Leydig cells tended to have larger Leydig cells, and the increased size was due to more cytoplasm instead of nucleoplasm. There were weak but significant positive correlations between total Leydig cell cytoplasm per man and DSP and between average size of a Leydig cell and DSP. These findings suggest that a relationship may exist between sperm production and the amount of cytoplasm containing testosterone-producing organelles in surviving Leydig cells of older men.

Selective luminal absorption of L-carnitine from the proximal regions of the rat epididymis. Possible relationships to development of sperm motility.

The absorption of L-carnitine from the duct of the proximal regions of the rat epididymis was investigated using a stopped-flow, split-droplet microperfusion technique. L-carnitine was absorbed from the duct of the proximal caput epididymidis by a time-dependent and saturable transport system (Km = 25 micron; Vmax = 0.65 pmoles absorbed/min/mm3 tubular volume). Furthermore, absorption appeared to be primarily sodium-independent, although the existence of a minor sodium-dependent pathway cannot be ruled out. A similar transport system was not evident along the distal caput epididymidis, where absorption of L-carnitine was attributable to passive diffusion only. The inward and outward movement of L-carnitine across the epithelium of the proximal and distal caput epididymidis appears to be regulated so that the spermatozoa come into contact with high levels of L-carnitine in the distal caput region.

Calcium channel inactivation in frog (Rana pipiens and Rana moctezuma) skeletal muscle fibres.

The decay of the Ca2+ current (ICa) during a maintained depolarization was studied in intact twitch skeletal muscle fibres of Rana pipiens and Rana moctezuma with the three-micro-electrode voltage-clamp technique. ICa was recorded at 23 degrees C, after blocking K+ currents, in TEA methanesulphonate saline with 10 mM-Ca2+ made hypertonic by adding 350 mM-sucrose. In two-pulse experiments, ICa during the test pulse was reduced to about 80% (R. pipiens) or 50% (R. moctezuma) of the control value, without any detectable inward ICa during 7 s conditioning pre-pulses. The experimental points of the steady-state inactivation curve (h infinity) were fitted to h infinity = (1 + exp [Em - Vh)/kh]-1, where Em is the membrane potential and with Vh = -33 +/- 3 mV and kh = 6 +/- 1 mV for R. pipiens, and Vh = -44 +/- 3 mV and kh = 9.5 +/- 1.0 mV for R. moctezuma. The rate constant of decay for inactivated currents (range -8 to -47 mA cm-3) and for control currents (range -23 to -62 mA cm-3), was independent of ICa amplitude. The average rate constant of decay at 0 mV was 1.18 +/- 0.02 s-1 (66). These results indicate that in intact fibres under hypertonic solution ICa decay can be explained by a voltage-dependent inactivation process and not by depletion of tubular Ca2+. The absence of depletion could be due to a large fractional tubular volume or to the presence of a Ca2+ pump in the tubular system.

The continuous measurement of tubular volume changes in response to step changes in contraluminal osmolality.

A new method to measure time dependent (t) volume (V) changes in proximal straight tubules (PST) is described. V is calculated from diameter (d) measurements for which a video camera and an integrating circuit are used. A tubular image of high optical contrast is recorded with the TV camera such that the scan lines run crosswise to the tubule. The video signal is analyzed by a special processor which adds 225 tubular diameters of each TV frame and feeds this analog signal to a pen recorder. The fractional error in d measurements is 10(-3). Diameter changes of less than 0.05 micron can be detected, as compared to the usual error of a single measurement of about 0.4 micron. Pcbos, the osmotic water permeability of the contraluminal cell membrane was measured by setting up osmotic steps across it in less than 0.1 s and following the ensuing delta d/delta t. The time delay between solution change and the linear part of the osmotic response was 0.51 +/- 0.05 s. Pcbos was found to be 50.4 (+/- 8.7) X 10(-4) cm3.cm-2 of basement membrane area .s-1.osmolar-1.

MATURATION OF THE CHEMICAL FORCE DRIVING K SECRETION IN THE CORTICAL COLLECTING TUBULE (CCT)

The serum K+ concentration in the newborn is higher than in the adult, possibly resulting from a diminished K+ secretory rate in the immature nephron. Since K+ secretion is driven, in part, by the cell-to-lumen concentration gradient, a diminished intracellular K+ concentration (Ki) may limit K+ secretion in the newborn. We therefore measured Ki in rabbit CCTs at three developmental stages. We dissected CCTs in an isotonic choline chloride solution, measured length and outer diameter, and placed them in 0.1 M trichloroacetic acid for 24 hours. K+ and Na+ concentrations of the tubular extract were measured with a helium glow photometer. In order to calculate Ki, the volume of water in 1 mm of CCT was assumed to be 80% of tubular volume. Ki in the CCT is, if anything, higher in the newborn than in the adult. In addition, the cellular Na+/K+ ratio does not change during development (newborn=0.11 ± .04 vs. adult=0.16 ± .05, p=0.3). Assuming that K+ activity bears a constant relationship to Ki during development, we suggest that changes in the tubular flow rate, transepithelial voltage, and/or membrane K+ permeability, rather than in Ki, mediate the maturation of K+ secretion by the CCT.

Intracellular pH in intact rat renal cortex estimated with [14C]-5,5-dimethyl-2,4-oxazolidinedione.

The distribution of [14C]-5,5-dimethyl-2,4-oxazolidinedione ([14C]-DMO) was measured in intact rat renal cortex using 22Na+ to estimate extracellular fluid volume. Results of two different methods to obtain tissue were compared, a cortical slice technique and a whole kidney frozen technique. In normal animals with a mean blood pH of 7.41 +/- 0.006 (SEM) the cortical slice method measured an intracellular pH of 6.97 +/- 0.03 (SEM). Corrections are described which can be used to compensate for varying volumes of tubular fluid in the slice and for differing concentrations of intracellular sodium. Using reasonable estimates for tubular fluid volume and intracellular sodium these corrections increase the value of intracellular pH to about 7.20. The results of the study indicate that the cortical slice [14C]-DMO method provides a satisfactory technique to obtain baseline values for intracellular pH in intact renal cortex, which can be used to detect changes in intracellular pH produced by experimental manipulations.

Studies of the human testis. XVII. Gonadotropin regulation of intratesticular testosterone and estradiol in infertile men.

Serum levels of LH and FSH and the intratesticular concentrations of testosterone (T) and estradiol (E) were measured in biopsy tissue from 40 infertile men, aged 21-36 yr, of whom 21 were oligospermic men with varicocele and 19 were men with idiopathic oligospermia. Intratesticular T and E concentrations were negatively correlated (P less than 0.001) with testicular volumes, as measured with a calibrated orchidometer, suggesting that differences in measured intratesticular steroid levels in part reflect altered relative Leydig cell density as seminiferous tubular volume changes. To gather information about the regulation of intratesticular T and E by gonadotropins, we calculated an index of intratesticular steroid content by multiplying steroid concentration by testicular volume and compared these values with circulating LH and FSH levels. Highly significant positive correlations were found between both serum LH and FSH and intratesticular E content and between LH and FSH and intratesticular T content. Multivariant stepwise regression analysis revealed that while serum FSH is a strong predictor of intratesticular E (r = 0.72; P less than 0.001), serum LH is not (partial r = 0.00 when controlling for the influence of FSH). Instead, the apparent relationship between Serum LH and intratesticular E results from the highly positive correlation between serum LH and FSH in the patients studied (r = 0.71; P less than 0.001). Similarly, circulating LH levels are independently related to intratesticular T content (r = 0.67; P less than 0.001), whereas the relationship between FSH and T is indirect (partial r = 0.06 when controlling for the influence of LH). We believe that these associations suggest that the major regulator of intratesticular T content is LH and that FSH may be the important gonadotropin regulating intratesticular E.

Functional and morphologic patterns of renal maturation in the developing guinea pig.

Clearance experiments were carried out in fetal, young, and adult pigs undergoing salt loading. Kidney sections were histologically examined and the proximal tubular length, glomerular volume, and number of glomeruli were estimated. Between days 48 and 55 of gestation, the significant increase in glomerular filtration rate was proportionately higher than the concentration increase in water, total solutes, and sodium reabsorption. During this period, neither the proximal tubular length nor the glomerular volume of the juxtamedullary nephrons changed significantly. The main morphologic change took place in the superficial cortex, where new glomeruli attached to very short tubules differentiated. From day 55 to birth, the increase in water, total solutes, and sodium filtered loads was proportionately smaller than the concomitant increase in tubular reabsorption. During this phase, the major morphologic change was the significant lengthening of the proximal tubules of the juxtamedullary and superficial nephrons. These findings suggest that for water, sodium, and total solutes the maturation of superficial nephrons is accompanied by a phase of functional glomerular preponderance. Despite the differentiation of the superficial nephrons, renal handling of normally filtered glucose did not change significantly. Functional glomerulotubular coupling for water, sodium, and total solutes took place soon after birth.

A Quantitative Structural Model of the Testis of Fertile Males with Normal Sperm Counts

The morphology of testicular biopsies of 8 healthy fertile men with proven normal sperm counts was studied using stereological techniques. Quantitative estimations were made of different cellular compartments in the testicular biopsies. The experimental errors of observation, homogeneity of the section, accuracy of the measurements and errors caused during the preparation of the sections were studied. A stereological model of the human testis comprising all compartments was designed. The present observations on the stereological parameters were compared with the results in literature; generally a good agreement was found. This stereological model enabled the authors to establish the quantitative correlations that exist between the separate compartments of the human testis. The tubular length density was positively correlated with the tubular surface density. The tubular surface density showed a negative correlation with the volume density of the germinal cell nuclei. The tubular volume density showed a negative correlation with the volume density of the Leydig cells. The intra-tubular volume density correlated negatively with the volume density of the Leydig cells and with the volume density of the remaining extra-tubular tissue. The intra-tubular tissue density correlated negatively with the volume density of the intra-tubular space.

Postnatal development of the canine kidney: quantitative and qualitative morphology.

The normal postnatal development of the canine kidney was investigated utilizing qualitative and quantitative histologic methods. Kidneys were examined at 2, 4, 8, 14, 22, 70, and 200 days of age. A subcapsular nephrogenic zone was present in the kidneys until approximately eight days of age. This zone contained tissues which interacted to produce new nephrons and interstitial tissues. Several developmental stages of forming nephrons were identified in this zone. Beneath the nephrogenic zone, renal corpuscles of increasing maturity were located at successively deeper cortical levels. The total number of nephrons was estimated to be 445,000 per kidney. This number did not vary significantly during growth. The corpuscular volume per nephron increased 249% from 14 to 200 days of age. During the same period there was a 303% increase in the tubular volume per nephron. Although the developing kidney differed anatomically from the adult kidney, the individual nephrons maintained volumetric corpuscular-tubular balance during growth.