S. J. Walter

The role of vasopressin in blood pressure regulation immediately following acute haemorrhage in the rat

1. The possible pressor effect of vasopressin immediately after acute haemorrhage has been studied using anaesthetized Brattleboro rats with diabetes insipidus and rats of the Long Evans parent strain.

A comparison of micropuncture and lithium clearance methods in the assessment of renal tubular function in rats with diabetes insipidus

The lithium clearance technique has been proposed as a non-invasive method whereby fluid delivery from the pars recta and pars convoluta of proximal tubules can be measured as CLi and CIN [0.78 CLi/CIN+0.22], respectively [12], CLi being the clearance of lithium and CIN that of inulin. In the present study, fluid delivery from proximal tubules was estimated simultaneously by micropuncture and lithium clearance techniques in anaesthetized Brattleboro rats with diabetes insipidus, under control conditions and following chronic treatment with hydrochlorothiazide. Absolute deliveries from the proximal convoluted tubules as determined by the micropuncture and lithium clearance methods were 437 and 427 μl/min, respectively, in untreated animals and 348 and 355 μl/min, respectively, in thiazide-treated animals. The individual results obtained by the two methods showed a high degree of correlation (r=0.85,P<0.001).In untreated Brattleboro rats, proximal fluid delivery as estimated by both the micropuncture and lithium clearance techniques showed significant (P<0.001) correlations with urine flow rate.These results provide further evidence for the acceptance of lithium clearance as a valid estimate of proximal tubular fluid delivery.

Upregulation of H+-ATPase in the distal nephron during potassium depletion: structural and functional evidence.

In the present study, we have investigated the effects of dietary potassium depletion on the activity and distribution of the H+-ATPase in the distal nephron of the Sprague-Dawley rat. H+-ATPase activity was assessed from the change in transepithelial potential difference (Vte) in response to bafilomycin A1 during perfusion of the late distal tubule in vivo, with solutions containing inhibitors of known ion channels. Bafilomycin A1 caused a negative deflection in Vte in control animals, an effect that was significantly enhanced during potassium depletion (P < 0.01). The distribution of H+-ATPase within the population of intercalated cells was assessed using a specific monoclonal antibody (E11). Hypokalemia was associated with a highly significant redistribution of the staining pattern (P < 0. 001), with an increase in the percentage of cells displaying immunoreactivity in the apical membrane. These results indicate that dietary potassium depletion increases electrogenic H+-ATPase activity in the rat distal tubule; this may be associated with increased insertion of pumps into the apical membrane.In the present study, we have investigated the effects of dietary potassium depletion on the activity and distribution of the H+-ATPase in the distal nephron of the Sprague-Dawley rat. H+-ATPase activity was assessed from the change in transepithelial potential difference ( V te) in response to bafilomycin A1 during perfusion of the late distal tubule in vivo, with solutions containing inhibitors of known ion channels. Bafilomycin A1 caused a negative deflection in V te in control animals, an effect that was significantly enhanced during potassium depletion ( P < 0.01). The distribution of H+-ATPase within the population of intercalated cells was assessed using a specific monoclonal antibody (E11). Hypokalemia was associated with a highly significant redistribution of the staining pattern ( P < 0.001), with an increase in the percentage of cells displaying immunoreactivity in the apical membrane. These results indicate that dietary potassium depletion increases electrogenic H+-ATPase activity in the rat distal tubule; this may be associated with increased insertion of pumps into the apical membrane.

Effects of the potassium channel blocker barium on sodium and potassium transport in the rat loop of Henle in vivo.

In vitro evidence suggests that the ‘recycling’ of K+ ions through luminal K+ channels in the thick ascending limb of the loop of Henle (TALH) is essential for the normal operation of the luminal Na+‐K+‐2Cl− co‐transporter. In the present study these channels were investigated in vivo by perfusing superficial loops of Henle in anaesthetised rats with and without the K+ channel blocker barium. Using a standard perfusate, intraluminal barium (5 mmol l−1) reduced sodium reabsorption (JNa) from 1887 ± 50 to 1319 ± 53 pmol min−1 (P < 0.001). When the experiment was repeated using a low‐Na+ perfusate, designed to inhibit reabsorption in the pars recta (the initial segment of the loop of Henle), a similar reduction in JNa was observed (from 698 ± 47 to 149 ± 23 pmol min−1, P < 0.001), strongly suggesting that the effect of barium is localised to the TALH. The magnitude of the reduction in JNa during blockade of K+ channels confirms the importance of K+ recycling in facilitating Na+ reabsorption in the TALH in vivo. However, the reduction in JNa was not associated with a fall in the K+ concentration of the fluid collected at the early distal tubule. When bumetanide, an inhibitor of the Na+‐K+‐2Cl− co‐transporter, was included in the low‐Na+ perfusate, net K+ secretion was observed. Addition of barium to this perfusate reduced, but did not abolish, the secretion, suggesting that bumetanide‐induced K+ secretion results partly from paracellular transport.

TRANSEPITHELIAL ELECTROCHEMICAL GRADIENTS IN THE PROXIMAL CONVOLUTED TUBULE DURING POTASSIUM DEPLETION IN THE RAT

1 In order to examine the electrochemical gradient for potassium reabsorption across the S2 segment of the proximal convoluted tubule, transepithelial potential differences and transepithelial potassium concentrations were measured in anaesthetized potassium‐replete and potassium‐depleted rats. 2 Potassium‐depleted rats were markedly hypokalaemic (plasma potassium, 1.4 ± 0.1 vs. 4.1 ± 0.1 mmol l−1 in potassium‐replete rats) and had a significantly reduced muscle potassium content. In confirmation of previous reports, glomerular filtration rate was slightly reduced, while fractional reabsorption in the proximal convoluted tubule was enhanced. 3 In potassium‐replete animals, the transepithelial potential difference (PD) at the late proximal convoluted tubule was +2.1 ± 0.3 mV (lumen positive) and the tubular fluid to plasma ultrafiltrate concentration ratio for potassium (TFK/UFK) at the same site was 1.03 ± 0.01. In potassium‐depleted rats, there was a striking reversal of the transepithelial PD (to −4.0 ± 0.4 mV), while the TFK/UFK was increased to 1.19 ± 0.03. 4 The data from both potassium‐replete and potassium‐depleted animals are consistent with accumulating evidence that potassium reabsorption in the proximal convoluted tubule is passive in nature and depends partly on diffusion down an electrochemical gradient.

Renal aluminium handling in the rat: a micropuncture assessment

Uncertainties exist over the glomerular filtration of aluminium and virtually nothing is known about its segmental handling along the nephron. The present study has used micropuncture, combined with electrothermal atomic absorption spectroscopy, to determine directly the aluminium content of glomerular filtrate and of late PCTs (proximal convoluted tubules) and early distal tubules in anaesthetized Munich-Wistar rats infused with three different doses of aluminium citrate (plasma aluminium concentrations, 2.9+/-0.1, 5.2+/-0.4 and 10.0+/-0.9 microg.ml(-1) respectively). Aluminium filtration into Bowmans space was found to be considerably greater than that predicted by an in vitro filtration system: in all three groups it was essentially filtered freely. No significant aluminium reabsorption took place along the PCT, but with every dose the FD(Al) (fractional delivery of aluminium; tubular fluid:plasma aluminium/inulin concentration ratio) was lower at the early distal site than at the late PCT (P<0.001 in each case), indicating net aluminium reabsorption in the loop of Henle. This reabsorption amounted to 19-26% of the filtered aluminium load. In the low- and medium-dose groups, there was no significant difference between FD(Al) at the early distal site and that in the final urine; however, in the high-dose group, FD(Al) in the urine (1.02+/-0.06) exceeded that at the early distal tubule (0.75+/-0.04; P<0.001), suggesting aluminium secretion in the distal nephron. The results indicate that aluminium loads, when complexed with citrate, are excreted efficiently owing to a combination of glomerular filtration and minimal reabsorption.

Contribution of Na+‐H+ exchange to sodium reabsorption in the loop of Henle: a microperfusion study in rats

1 The contribution of apical Na+‐H+ exchange to sodium reabsorption in the thick ascending limb of the loop of Henle (TALH) in vivo was examined in anaesthetized rats by perfusing loops of Henle of superficial nephrons with solutions containing the Na+‐H+ exchange inhibitor, ethyl isopropyl amiloride (EIPA). 2 Using a standard perfusate, no statistically significant effect of EIPA on net sodium reabsorption (JNa) was detected. However, when sodium reabsorption in the pars recta of the proximal tubule was minimized by using a low‐sodium perfusate, EIPA reduced JNa from 828 ± 41 to 726 ± 37 pmol min−1 (P < 0.05), indicating that apical Na+‐H+ exchange can make a small contribution to net sodium reabsorption in the TALH in vivo. This contribution appears to be dependent on the bicarbonate load, since an increase in the latter led to an enhancement of EIPA‐sensitive sodium transport. 3 Addition of the Na+‐K+‐2Cl− cotransport inhibitor, bumetanide, to the low‐sodium perfusate reduced baseline JNa to 86 ± 27 pmol min−1. In this setting, EIPA reduced JNa further, to −24 ± 18 pmol min−1 (P < 0.05), an effect similar to that seen in the absence of bumetanide. This finding argues against previous suggestions (based on in vitro evidence) that inhibition of the Na+‐K+‐2Cl− cotransporter leads to an increase in apical Na+‐H+ exchange in the TALH.

The effect of vasopressin on extracellular cation concentrations and muscle resting potentials in the rat.

1. The Na+ and K+ concentrations in plasma and cerebrospinal (c.s.f.), resting potentials in skeletal muscle fibres, cardiac beat to beat intervals and 90% repolarization times were measured in Long Evans rats (parent strain controls) and in Brattleboro rats with hypothalamic diabetes insipidus (DI). 2. Cation concentration measurements confirmed previous observations that Brattleboro DI rats are mildly hypokalaemic compared with rats of the parent Long Evans strain, and indicated that the c.s.f. [Na+] is significantly raised in the former group of animals while the [K+] in the c.s.f. samples is similar in the two groups. 3. The mean resting potential of deep skeletal muscle fibres in Brattleboro DI rats was significantly more negative than the corresponding value in the Long Evans rats, and this finding was in close agreement with the difference observed for the calculated K+ equilibrium potentials in the two groups of animals. 4. The beat to beat intervals and the 90% repolarization times of cardiac action potentials were also determined in Brattleboro DI and Long Evans rats, and the mean values for both variables were significantly shorter in the former group of animals. 5. The administration of Pitressin by subcutaneous injection (500‐100 mu./24 hr) to Brattleboro rats abolished the hypokalaemia and the hyperpolarization of skeletal muscle fibre membranes but had no significant effect on c.s.f. cation concentrations. 6. The present findings suggest that the absence of vasopressin in the Brattleboro DI rats results in a hyperpolarization of muscle cell membranes, and in changes in the cardiac action potential. These effects may be partly related to the mild hypokalaemia present in these animals.

Selective blockade of oxytocin and vasopressin V(1a) receptors in anaesthetised rats: evidence that activation of oxytocin receptors rather than V(1a) receptors increases sodium excretion.

Background: Although it is known that moderate-to-high doses of the neurohypophysial hormones oxytocin and vasopressin are natriuretic, doubts remain over the identity of the receptors responsible. To address this issue, we have used highly selective antagonists of oxytocin and vasopressin receptors in animals with elevated endogenous circulating levels of the 2 hormones. Methods: Rats were anaesthetised and prepared surgically for clearance studies, thereby raising plasma oxytocin and vasopressin concentrations. Sodium excretion, glomerular filtration rate and lithium clearance (an index of end-proximal fluid delivery) were measured: first during a control period, then after administration of the selective oxytocin receptor antagonist desGly-NH2,d(CH2)5[D-Trp2,Thr4,Dap5]OVT, the selective vasopressin V1a receptor antagonist d(CH2)5[Tyr(Me)2,Dab5]AVP, or vehicle alone. Results: Absolute and fractional sodium excretion fell in rats given the oxytocin antagonist (by 32 and 27%, respectively, compared with corresponding values in vehicle-infused rats), but not in those given the V1a antagonist or vehicle. Antinatriuresis was associated with a small reduction in the ratio of sodium clearance to lithium clearance (an index of the fraction of distally delivered sodium that escapes reabsorption in the distal nephron). Conclusions: These results corroborate previous studies showing that activation of oxytocin receptors increases sodium excretion and imply that the natriuretic effect of elevated plasma vasopressin concentrations results from stimulation of oxytocin receptors.

Sodium balance and antidiuresis in thiazide-treated rats with diabetes insipidus.

A sodium deficit was induced in Brattleboro rats by lowering the dietary sodium content. Urine volume was only slightly reduced. Addition of hydrochlorothiazide to the food caused a small, transient sodium deficit but led to a marked, sustained antidiuresis. The acute administration of a large dose of hydrochlorothiazide produced a larger, sustained sodium deficit but no lasting antidiuresis. These results indicate that sodium depletion cannot account for the antidiuresis of hydrochlorothiazide treatment in diabetes insipidus.