Nl Min Research Articles

Effects of indomethacin on glomerular hemodynamics in experimental diabetes

The glomerular hemodynamics were studied in streptozotocin diabetic rats 3 months after the induction and in age-matched normal animals. Indomethacin infusion failed to cause changes in glomerular hemodynamics in normal rats but produced striking effects in the diabetic rats: The afferent arteriolar hydraulic resistance increased substantially while the efferent resistance rose moderately causing large reductions in single nephron blood flow and in the hydraulic pressure in the glomerular capillaries. Consequently, single nephron glomerular filtration rate (SNGFR) was reduced significantly below normal. The ultrafiltration coefficient of the glomerular membrane was significantly lower in the diabetic than in the normal animals (2.8 versus 5.0 nl min-1 mm Hg-1) and remained unchanged during indomethacin infusion. Our results suggest that the prostaglandin system compensates for the changes in the glomerular hemodynamics induced by diabetes by reducing the arteriolar resistances to increase the glomerular capillary pressure making it possible to maintain normal values of SNGFR and nephron blood flow.

Effects of renal arterial angiotensin I infusion on glomerular dynamics in sodium replete dogs

During intrarenal infusion of angiotensin I (AI), conversion to angiotensin II (AII) within the kidney has been shown to occur early enough to decrease glomerular filtration rate (GFR). To evaluate further the mechanism by which AI decreases GFR, micropuncture studies were conducted in sodium replete dogs. Feedback-mediated alterations in glomerular function were minimized by reducing renal arterial pressure to 90 mm Hg. During infusion of AI (0.82 +/- 0.01 micrograms min-1), renal blood flow (3.91 +/- 0.25 ml min-1 g-1) and GFR (0.63 +/- 0.04 ml min-1 g-1) decreased by 36.7 +/- 6.1% and 18.9 +/- 6.1%, respectively. Similarly, single nephron GFR decreased from 66.4 +/- 3.8 to 40.0 +/- 3.2 nl min-1 and estimated glomerular plasma flow (280 +/- 49 nl min-1) decreased by 55 +/- 6%. Stop-flow pressure (40.5 +/- 3.6 mm Hg) did not change significantly, while proximal tubular (21.8 +/- 1.4 mm Hg) and peritubular capillary pressures (13.2 +/- 1.8 mm Hg) decreased by 25.5 +/- 2.8% and 49.4% +/- 5.1%, respectively. Glomerular capillary and effective filtration pressures were not altered significantly. There were increases in both preglomerular (168%) and efferent (203%) arteriolar resistances, along with a decrease in the glomerular filtration coefficient (Kf) from 4.6 +/- 0.6 to 2.5 +/- 0.5 nl mm Hg-1 min-1. These data indicate that augmented intrarenal conversion of circulating AI reduces GFR as a consequence of decreases in Kf as well as in glomerular plasma flow, the latter being due to concomitant increases in preglomerular and efferent arteriolar resistances.

Effect of cAMP and calmodulin inhibitors on water absorption in rat proximal tubule.

The possible role of calmodulin in solute transport was examined in the kidney of the rat. Utilizing a radioimmunoassay, calmodulin was identified and quantitated in homogenates of the cortex of the kidney. The physiologic significance of these findings was examined utilizing in vivo microperfusion techniques applied to the proximal convoluted tubule of the thyroparathyroidectomized rat. The addition of dibutyryl cyclic adenosine monophosphate (cAMP) to the luminal perfusion solution resulted in a lower rate of water absorption of 1.67 +/- 0.09 nl min-1 mm-1 as compared to 2.46 +/- 0.11 in controls. The addition of either of two compounds with affinity for calmodulin, trifluoperazine (TFP) or W-13, reversed the cAMP-induced inhibition of water absorption. In the absence of cAMP, neither agent affected water absorption. Analogs of TFP and W-13 with lower binding affinities for calmodulin had no effect on water absorption and did not reverse the cAMP effect. None of the above experimental maneuvers affected the absorption of phosphate. These results demonstrate the presence of calmodulin in the kidney of the rat and suggest that calmodulin may be involved in cAMP-associated inhibition of water and electrolyte transport in the proximal tubule of the rat.

Effect of loop of Henle flow rate on glomerular capillary pressure.

Studies were performed in male Munich-Wistar rats weighing a mean of 100 g to evaluate further the hemodynamic effects of changing loop of Henle flow rate. When late proximal flow rate was varied by microperfusion from the late proximal tubule, single nephron glomerular filtration rate (SNGFR) measured in the early proximal tubule fell from 17.2 +/- 0.86 nl min-1 at a perfusion rate of zero to 11.8 +/- 0.88 nl min-1 at a perfusion rate of 14 nl min-1. Further increases in loop flow did not further depress SNGFR. Glomerular capillary pressure (Pgc) was measured directly in superficial glomeruli while loop of Henle flow was varied. Elevating loop flow was followed by a rapid decrease in Pgc, which was reversible when flow was reduced. The mean response to maximum stimulation was a decrease in Pgc of 6.6 +/- 0.7 mm Hg, and the response was close to maximum at a perfusion rate of 14 nl min-1. Parallel changes in stop-flow pressure were observed. These findings, together with previous observations that increasing loop of Henle flow results in decreased single nephron blood flow, permit the conclusion that the dominant vascular response to elevated loop of Henle flow is constriction of the afferent arteriole.

Diuresis in newly emerged, unfed mosquitoes. I. Fluid loss in normal females and males during the first 20 hours of adult life.

Unfed mosquitoes (Aedes aegypti) decapitated soon after emergence from the pupa survive better than those decapitated later. In a test involving more than 700 females and more than 600 males, 80% of the mosquitoes decapitated during the first hour of adult life survived a period of 45 h post emergence (p.e.), whereas about 80% of those decapitated at 12 h or later failed to survive the same period. There was a linear fall in the rate of survival between 1 and 10 h p.e., after which the rate fluctuated between 10 and 30%. The mechanism accounting for this phenomenon seemed most likely to be loss of fluid under the control of a diuretic hormone released from the head, early decapitation protecting the insects from excess loss of fluid. To test this explantation the rate of excretion was measured in normal unfed mosquitoes at regular intervals during the first 20 h of adult life. In females the rate of excretion was shown to rise from 10 nl min-1 during the first hour of adult life to each 27 nl min-1 12 h later. In males the rate of excretion fell from 7 to 5 nl min-1 during the first 3 h before rising to 12 nl min-1 by 9 h p.e. The rate of excretion decreased in both sexes from 15-16 h onwards. Newly emerged mosquitoes thus increase their preparedness for flight by loss of water through enhanced excretion during the first day.

Further evidence for an inverse relationship between macula densa NaCl concentration and filtration rate

It has been concluded that tubulo-glomerular feedback mechanism is triggered by changes in NaCl concentration ([NaCl]) at the macula densa. This conclusion is based on the demonstration that changes in filtration rate produced during retrograde perfusion of the loop of Henle depend upon the perfusate [NaCl]. Experiments were performed to evaluate whether the effect on glomerular function of orthograde perfusion of the loop of Henle is consistent with this conclusion. Early proximal flow rate (VEP), stop-flow pressure (PSF), early distal chloride concentration ([C]), and flow rate were measured during perfusion of the loop of Henle with mannitol solution (300 mosm kg-1), 30mM NaCl + mannitol (300 mosm kg-1), 140 mM Na isethionate and artificial tubular fluid. When distal flow exceeded 10 nl min-1, the magnitude of the glomerular response was predictable from the [Cl]. The linear regression line, delta VEP = -0.27 [Cl] + 4.3, did not differ from that obtained previously with the retrograde technique. Retrograde perfusion with 140 mM Na isethionate was without effect on VEP. We conclude that the effect on glomerular function of perfusion of the loop of Henle in either an orthograde or a retrograde direction with these solutions depends upon the chloride concentration at the macula densa.

Skin water uptake and renal function in the toad

1. 1. Water balance in the toad Bufo marinus ictericus was studied by evaluating cutaneous water uptake and renal excretion. 2. 2. The permeability of the skin to water was 78 ± 6 nl min −1 cm −2 atm −1 in 60 toads anaesthetized by chloralose and preincubated in water for 3 hr. Injection of Ringer's solution did not reduce the water uptake by the skin, while hemorrhage, or injection of vasopressin, or oxytocin approximately doubled the rates of water transport. In vivo values of skin water permeability were similar to those observed in vitro. 3. 3. The renal parameters of the water balance were significantly reduced by hemorrhage and by injection of vasopressin. Injection of Ringer's solution promoted a significant increase of the urinary flow and osmolar clearance. 4. 4. It is suggested that the fight against water deprivation could proceed initially by the triggering of the urinary mechanisms of water retention, while the increase of the skin water permeability would occur in a later stage.

Volume absorption in the pars recta. II. Hydraulic conductivity coefficient.

We evaluated the hydraulic conductivity (Pf, micron s-1) of superficial proximal straight tubules isolated from rabbit kidney. Tubules were perfused with hypotonic (270 mosmol/kg H2O) and bathed with isotonic (290 mosmol/kg H2O) NaCl buffers at 25 degrees C. Due to the tendency of transepithelial osmosis plus solute entry to produce osmotic equilibrium along the perfused length, we observed that the total net volume absorption ('JV, nl min-1) increased from 0.64 to 2.21 when the perfusion rate (VO, nl min-1) was increased from 11 to 45 in a group of tubules with an average length of 0.86 mm. From a 'JV of 2.21 nl min-1 at VO = 45 nl min-1 we computed a minimum Pf of 2,200 micron s-1. And extrapolation of the data to VO leads to infinity gave a Pf value of 5,200-7,600 micron s-1. The same perfusion rate dependence of 'JV in a group of tubules with an average length of 3.29 mm gave quantitatively similar results. A theoretical analysis of radial osmosis occurring simultaneously with axial osmotic equilibration showed that Pf values in the range of 3,000-4,000 micron s-1 accurately predicted the observed relations between VO, 'JV, and tubule length.

Volume absorption in the pars recta. III. Luminal hypotonicity as a driving force for isotonic volume absorption.

This paper examines the possibility that osmotic disequilibrium between luminal and bathing solutions may account for isotonic fluid absorption coupled to active Na+ absorption observed when superficial proximal straight tubules isolated from rabbit kidney are perfused and bathed with NaCl solutions in the absence of CO2, HCO3-, and luminal organic solutes. If luminal hypotonicity provides a driving force for isotonic fluid absorption under these conditions, the luminal fluid must be nearly isotonic; and steady-state luminal hypotonicity should develop sufficiently rapidly that the absolute rate of volume absorption ('JV, nl min-1) coupled to active Na+ transport is relatively independent of perfusion rate, so that the normalized rate of fluid absorption (JV, nl min-1 mm-1) is approximately constant. Our theoretical calculations indicate that these expectations are fulfilled. A 0.42-0.56 mM reduction in luminal NaCl concentration adequately accounts for the JV observed under such conditions, because of the high hydraulic conductivity of these tubules; and within the range of tubule lengths normally employed with isolated proximal straight tubules, JV is relatively indepedent of perfusion rate within the generally observed range of experimental error.

Lack of Effect of Potassium on Fluid Absorption in Isolated, Perfused Snake Proximal Renal Tubules

Effects of alterations in potassium concentrations on net fluid absorption (J<sub>v</sub>) by isolated, perfused snake <i>(Thamnophis </i>spp.) proximal renal tubules were studied. With standard (3 mmol/l potassium, 150 mmol/l sodium) Ringer’s solution in perfusate and bath, J<sub>v</sub> was about 1.3 nl min<sup>-1</sup> mm<sup>-1</sup>. Replacing potassium with sodium in perfusate or in both perfusate and bath had no significant effect on J<sub>v</sub>. Increasing potassium concentration to 100 mmol/l in place of sodium in perfusate or in both perfusate and bath also had no effect on J<sub>v</sub>. Findings that potassium is not critical for maintaining J<sub>v</sub> are also consistent with previous findings that sodium is not necessary for maintaining J<sub>v</sub>.

Fluid absorption with and without sodium in isolated perfused snake proximal tubules

Net fluid absorption (JV) was studied in isolated, perfused snake (Thamnophis spp.) proximal renal tubules. With standard (150 mmol/liter Na+) bicarbonate-buffered Ringer in perfusate and standard Ringer plus dextran (4 g/100 ml) in bath, JV was about 0.9 nl min-1 mm-1. Removing dextran from bath reduced JV by about 20 percent. When sodium in perfusate was replaced with choline JV approached zero. However, when sodium in bath as well as perfusate was replaced with choline, JV returned to control level. Results were the same when sodium was replaced with tetramethyl-ammonium, sodium chloride was replaced with sucrose or lactose, or chloride was replaced with methyl sulfate. In contrast, replacing sodium in perfusate or in both perfusate and bath with lithium did not reduce JV. Fluid absorption was always isosmotic. Replacing bicarbonate with phosphate or Tris in sodium-containing media had no effect on JV, but the presence of buffer in sodium-free or low-chloride media may have been important for JV. Reducing temperature 10 degrees C reduced JV by about 35 percent with either sodium chloride or sucrose in both perfusate and bath. The results indicate that isosmotic fluid absorption can occur when lithium is substituted for sodium or when some other substitution is made for sodium, chloride, or both in perfusate and bath simultaneously.

Calcium and phosphate transport in isolated segments of rabbit Henle's loop.

Calcium and phosphate transport was examined in rabbit thin descending, thin ascending, and thick ascending limbs of Henle by in vitro perfusion of isolated tubular segments. Permeability coefficients for these segments with 45Ca and 32PO4 were determined for both lumen-to-bath and bath-to-lumen directions. Both the thin descending and thin ascending limbs were found to be relatively impermeable to both 45Ca and 32PO4. In neither segment were we able to show evidence for net transport of calcium or phosphate. In contrast, the thick ascending limb of Henle showed a decrease in calcium lumen-to-bath concentration from 0.97 +/- 0.02 to 0.88 +/- 0.02 when perfused at 4.8 nl min-1. 45Ca lumen-to-bath and bath-to-lumen fluxes were 19.96 +/- 1.05 and 9.89 +/- 0.02 peq-min-1-cm-1, respectively, and the potential difference was +3.8 +/- 0.3 mV (lumen positive). The observed calcium flux ratio was significantly higher than that predicted by Ussing's equation. When ouabain was added to the bath the potential difference fell to +1.1 +/- 0.3 mV, whereas the calcium efflux was only slightly diminished (29.5 +/- 5.3-23.7 +/- 5.1 peq-cm-1-min-1). Ouabain had no effect on the influx of Ca across the thick ascending limb of Henle. There was no net transport of phosphate across the thick ascending limb. Phosphate permeability was exceedingly low bidirectionally across the thick ascending limb. Our findings indicate: (a) all segments of Henle's loop are relatively impermeable to calcium and phosphate; (b) net transport of phosphate seems to be absent in Henle's loop; (c) net calcium reabsorption, which cannot be explained by passive mechanisms, occurs in the thick ascending limb.

PAH transport and fluid absorption by isolated perfused frog proximal renal tubules

Para-aminohippurate (PAH) transport and fluid absorption were studied in isolated perfused frog (Rana catesbeiana) proximal renal tubules. With 2 X 10(-5) M PAH in the bath, tubule fluid-to-bath (TF/B) concentration ratios averaged 3.0 and net secretion averaged 746 X 10(-15) mol min(-1) mm(-1) in the proximal tubule. Net PAH secretion did not vary with perfusion rate. During PAH secretion, cell water PAH concentration exceeded that in the tubular fluid or bath, suggesting active transport into cells and subsequent diffusion into lumen. In accordance with this concept, luminal membrane permeability (3.8 X 10(-5) cm s(-1) calculated from perfusion studies was about 6 times greater than peritubular membrane permeability (0.66 X 10(-5) cm s(-1)) determined from studies of PAH efflux from tubules with oil-filled lumens. Net transepithelial PAH transport saturated at bath concentration of about 6 X 10(-5) M. Addition of 20 mM urea to PAH bath concentration of 2 X 10(-5) M reduced net PAH secretion by 32%. Fluid absorption in proximal tubules averaged 0.34 nl min(-1) mm(-1). Ouabain (10(-4), 10(-5), or 10(-6) M) added to bath blocked fluid absorption. Fluid absorption was partially restored following removal of ouabain.

A component of fluid absorption linked to passive ion flows in the superficial pars recta.

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.

Volume reabsorption, transepithelial potential differences, and ionic permeability properties in mammalian superficial proximal straight tubules.

This paper describes experiments designed to evaluate Na(+) and Cl(-) transport in isolated proximal straight tubules from rabbit kidneys. When the perfusing solution was Krebs-Ringer buffer with 25 mM HCO(3) (-) (KRB) and the bath contained KRB plus 6% albumin, net volume reabsorption (J(v), nl min(-1) mm(-1) was -0.46 +/- 0.03 (SEM); V(e), the spontaneous transepithelial potential difference, was -1.13 +/- 0.05 mV, lumen negative. Both J(v), and V(e), were reduced to zero at 21 degrees C or with 10(-4) M ouabain, but J(v), was not HCO(3) (-) dependent. Net Na(+) reabsorption, measured as the difference between (22)Na(+) fluxes, lumen to bath and bath to lumen, accounted quantitatively for volume reabsorption, assuming the latter to be an isotonic process, and was in agreement with the difference between lumen to bath (22)Na(+) fluxes during volume reabsorption and at zero volume flow. The observed flux ratio for Na(+) was 1.46, and that predicted for a passive process was 0.99; thus, Na(+) reabsorption was rationalized in terms of an active transport process. The Cl(-) concentration of tubular fluid rose from 113.6 to 132.3 mM during volume reabsorption. Since V(e), rose to +0.82 mV when tubules were perfused with 138.6 mM Cl(-) solutions, V(e) may become positive when tubular fluid Cl(-) concentrations rise during volume reabsorption. The permeability coefficients P(Na) and P(Cl) computed from tracer fluxes were, respectively, 0.23 x 10(-4) and 0.73 x 10(-4) cm s(-1). A P(Na)/P(Cl) ratio of 0.3 described NaCl dilution potentials at zero volume flow. The magnitudes of the potentials were the same for a given NaCl gradient in either direction and P(Na)/P(Cl) was constant in the range 32-139 mM NaCl. We infer that the route of passive ion permeation was through symmetrical extracellular interfaces, presumably tight junctions, characterized by neutral polar sites in which electroneutrality is maintained by mobile counterions.

Fluid secretion by in vitro preparations of the Malpighian tubules of the desert locust Schistocerca gregaria

In vitro preparations of locust Malpighian tubules can conveniently be made by a new technique in which the alimentary canal to which the tubules attach is removed from the insect and set up in Ringer's solution under liquid paraffin. Such Malpighian tubules will secrete a fluid iso-osmotic to the bathing fluid at a steady rate of about 1 to 2 nl min −1 for some hours. The secreted fluid is rich in potassium ions, the lumen is at a potential positive to that of the bathing solution, and the rate of secretion can be controlled by changing the potassium concentration of the bathing fluid. It seems likely, therefore, that an active transport of potassium drives secretion ny locust Malpighian tubules. The secreted fluid contains an elevated concentration of phosphate ions. The Malpighian tubules will secrete at a high rate in a chloride-free phosphate-based solution. The rate of fluid secretion can be increased by treatment with cyclic AMP but 5-hydroxytryptamine has no such effect.

The nature of transtubular Na and K transport in isolated rabbit renal collecting tubules.

In order to investigate the mechanism of Na and K transport, rabbit cortical collecting tubules were perfused in vitro and the concentrations of Na and K in lumen and bathing fluid and the transtubular electrical potential difference (PD) were measured. When the perfusate and external bath contained 150 Na-5 K (mEq liter(-1)), the sodium concentration decreased and the potassium concentration increased by an approximately equal amount in collected tubular fluid. The transtubular electrical potential was equal at both ends of the tubule in the steady state and ranged between 21 and 67 mv, lumen negative. In all tubules perfused at rates less than 0.5 nl min(-1), the K concentration of the collected fluid was higher and the Na concentration lower than that predicted for electrochemical equilibrium between lumen fluid and external bath, evidence for active transtubular transport of both cations. These results differ from those observed in rat distal tubule in which potassium secretion is passive. Active Na and K transport and the transtubular PD were decreased by (a) ouabain, (b) removal of sodium from the perfusate, or (c) removal of potassium from the external bath, evidence of interdependence of Na and K transport. The dependence of active K secretion on intraluminal Na concentration accounts for the phenomenon of "distal" Na-K exchange noted previously in clearance and stop-flow studies. The mechanism of Na transport may in part be electrogenic since the rate of decline of the transtubular PD in low K media was faster than could be accounted for on the basis of a reduction in cell potassium concentration.