Effects of certain diuretics on the electrophysiological characteristics of the nephron in the rat kidney

Electrophysiological micropuncture techniques were used to study the effect of certain diuretics on transtubular transport of electrolytes in the rat kidney. The mercurial diuretic novurite caused a reduction of active sodium transport in the proximal tubule, measured by short-circuit current and increased permeability of the tubular wall to ions which led to a considerable drop in transtubular potential and transepithelial resistance. Ethacrynic acid decreased the short-circuit current in the proximal tubule, without changing the permeability characteristics of the nephron. Xanthine diuretic euphylline did not reduce the short-circuit current in the proximal segment of the nephron; however, it increased the transepithelial potential of the renal tubule. In the distal tubule, euphylline and ethacrynic acid increased the difference in transtubular potential, whereas novurite reduced the transtubular potential. An increase in the electrical gradient of the distal tubule as a result of euphylline and ethacrynic acid action may be responsible for increasing potassium excretion. A decrease of the transtubular potential in the potassium excretion under mercurial diuretic action. The reduction of tubular reabsorption as a result of diuretic action is due to drug effect on different levels of the transtubular-ion transport system.     

Sodium transport inhibitor in proximal tubular urine during acute volume expansion

Harvested proximal tubular fluid from mannitolsaline expanded rats caused a 50% inhibition of transepithelial thelial sodium concentration difference when compared to an artifically prepared test solution used in the same and nonexpanded animals. Because of the methodology employed, none of the usual factors known to affect sodium re-absorption by the kidney could have been responsible for these changes. The factor responsible acts from the luminal side, is an inhibitor and has a short duration of action.     

Neurogenic regulation of renal tubular sodium reabsorption.

The evidence supporting a role for direct neurogenic control of renal tubular sodium reabsorption is reviewed. Electron microscopic and fluorescence histochemical studies have demonstrated adrenergic nerve terminals in direct contact with basement membranes of mammalian (rat, dog, and monkey) renal tubular epithelial cells. Low-level direct or baroreceptor reflex stimulation of renal sympathetic nerves produces an increase in renal tubular sodium reabsorption without alterations in glomerular filtration rate, renal blood flow, or intrarenal distribution of blood flow. Antinatriuresis was prevented by prior treatment of the kidney with guanethidine or phenoxybenzamine. Rat kidney micropuncture studies have localized a site of enhanced tubular sodium reabsorption to the proximal tubule. Possible indirect mediation of the antinatriuresis by other humoral agents known to be released from the kidney on renal nerve stimulation (angiotensin II, prostaglandin) was excluded by experiments with appropriate blocking agents. The possible effects of anesthesia and uncertainties about the completeness of surgical renal denervation and other tubular segmental sites of action are critically analyzed. The clinical implications of this mechanism in pathologic conditions of sodium and water retention are discussed and and a prospectus for future work is presented.     

The effect of aldosterone and the renin-angiotensin system on sodium, potassium and chloride transport by proximal and distal rat colon in vivo.

1. The roles of aldosterone and angiotensin in the direct control of epithelial sodium transport in vivo have been investigated by measurement of electrical p.d. changes and of the fluxes of sodium, potassium and chloride in rat colon, an organ actively involved in electrolyte homoeostasis. Exogenous angiotensin and aldosterone were given by both short- and long-term infusions and endogenous secretion of the hormones was varied by dietary sodium variation and by nephrectomy and/or adrenalectomy. 2. In vitro angiotensin has been shown to influence colonic salt and water absorption but in the present in vivo experiments administered angiotensin had no significant action on p.d. or on the ionic fluxes of the proximal or distal colon. The increase in p.d. produced by infusing aldosterone was unaffected by giving angiotensin concurrently. The effect of sodium depletion in stimulating sodium absorption and potassium secretion was completely abolished by adrenalectomy but was unaffected by nephrectomy. 3. During prolonged infusion of angiotensin into adrenalectomized rats, a small fall in faecal fluid and sodium content was observed, but this change would have little significance in sodium homoeostasis. 4. Aldosterone and sodium depletion stimulated sodium absorption in both proximal and distal colon but significant increase in potassium secretion was demonstrable only in the distal colon. Bicarbonate secretion (by calculation) was unaffected. In the proximal colon, the increased sodium absorption appeared to be accompanied by increased chloride absorption while in the distal colon it was principally the sodium-potassium exchange that was increased. 5. Adrenalectomy reduced potassium secretion in both proximal and distal colon but sodium absorption was only significantly reduced in the proximal colon. 6. It was concluded that there is no evidence that angiotensin in the living animal has a role as an important salt retaining hormone by direct epithelial action. Aldosterone has a considerable effect which is independent of the presence of angiotensin, and which differs in proximal and distal colon in regard to the relative effects on chloride absorption and potassium secretion.     

Effect of renal nerve activity on tubular sodium and water reabsorption in dog kidneys as determined by the lithium clearance method

The reliability of the lithium clearance method in studies of the effect of renal nerve activity upon tubular sodium and water handling in the dog kidney was investigated. Following unilateral acute surgical denervation of the kidney a significant increase in urinary flow rate (40 +/- 7%), sodium clearance (26 +/- 4%), lithium clearance (9 +/- 2%) and fractional lithium clearance (8 +/- 2%) was seen, as compared to the contralateral kidney with preserved innervation. Calculated absolute proximal reabsorption rate decreased significantly by 7 +/- 2%, while calculated absolute rates of distal reabsorption of sodium and water increased significantly by 9 +/- 2% and 8 +/- 2%. Low-frequency electrical stimulation of the distal nerve bundle of the denervated kidney caused a significant decrease in urine flow rate (37 +/- 6%), sodium clearance (31 +/- 4%), lithium clearance (17 +/- 5%) and in fractional lithium clearance (18 +/- 5%). Calculated absolute proximal reabsorption rate increased significantly by 17 +/- 3%, while calculated absolute rates of distal sodium and water reabsorption decreased significantly by 16 +/- 5% and 16 +/- 5%. These changes in tubular sodium and water reabsorption during alterations in renal nerve activity occurred without measurable changes in renal blood flow (RBF) and glomerular filtration rate (GFR). Administration of amiloride had no significant effect either on the lithium clearance, RBF or GFR, while the sodium excretion rate increased and potassium excretion rate decreased, supporting that significant distal lithium reabsorption did not occur under the present experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zur Frage einer direkten Angiotensinwirkung auf die Natriumresorption im proximalen Tubulus und in der Henleschen Schleife der Rattenniere

Summary Micropuncture experiments on the rat kidney were performed to evaluate the effect of angiotensin upon tubular sodium reabsorption.Half time reabsorption as measured by the split droplet method was constant (9.4±0.4 sec) at glomerular filtration rates ranging from 0.66–1.26 ml/g kidney/min, and remained unchanged when peritubular or tubular angiotensin concentration was elevated, indicating no direct effect of angiotensin upon transporting capacity of the proximal tubule for sodium.Sodium reabsorption in Henle's loop was studied by means of perfusing single loops with isotonic saline. From the increase in inulin concentration in the perfusat and from the sodium concentration in the early distal segment sodium reabsorption along Henle's loop was calculated. The correlation between perfusion rate and sodium reabsorption was found to be the same in the control experiments and during the perfusion with saline containing angiotensin with concentration of 0.5 and 5.0×10^–6 g/100 ml.These results together with the findings that urine volume and sodium excretion in the anaesthetized rat remain unchanged during the i.v. infusion of angiotensin indicate that angiotensin has no direct effect upon tubular transporting capacity for sodium.Changes in tubular sodium reabsorption due to angiotensin, as calculated from clearance data, are considered to be the effect of an indirect action of angiotensin upon tubular sodium reabsorption.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft und des U.S. Department of the Army, European Research Office.Auszugsweise vorgetragen auf dem IV. Symposium der Dtsch. Gesellsch. f. Nephrologie, Homburg (Saar) 1965.     

Proximal Tubular Renal Dysfunction or Damage in HIV-Infected Patients

Antiretroviral-associated toxicity, especially in the case of tenofovir plus boosted protease inhibitors, could affect different functions of the proximal renal tubule. Considering the long-term use of antiretroviral therapy and the concomitant presence of other risk factors, several degrees of proximal tubular toxicity, from chronic subclinical renal dysfunction to Fanconi syndrome, could be observed in HIV-infected patients. However, the clinical significance of isolated tubular dysfunction, in the short and long term, remains unclear. In addition, primary tubular abnormalities, even severe, may be missed until they affect the glomerular function. Therefore, there is a need for new biomarkers, not only based in serum creatinine and estimated glomerular filtration rates, that might help to identify tubular cell toxicity and predict the clinical outcome in HIV-infected patients. Increased values of urinary beta-2-microglobulin and retinol-binding protein, observed in up to 70% of patients, have been associated to tenofovir-associated mitochondrial dysfunction. Together with other tubular parameters or in isolation, both biomarkers could be useful for diagnosing proximal tubular toxicity. Other molecules, such as urinary kidney injury molecule- 1, neutrophil gelatinase associated lipocalin, or N-acetyl-b-D-glucosaminidase, could help to distinguish between tubular cell damage and dysfunction. Here, we review the current knowledge on tubular toxicity in HIV-infected patients on antiretroviral therapy.     

The effects of plasma from patients with Graves' disease on foetal mouse hearts in organ culture

Plasma, obtained during plasma exchange therapy, from 3 euthyroid patients with Graves' disease and severe progressive exophthalmos induced an increase in heart rate and then early death when applied to foetal mouse hearts maintained in isolated organ culture. All plasma samples which induced an increase in foetal heart rate had high titres of thyroid stimulating immunoglobulins. Plasma samples obtained after exchange had a much diminished effect. These studies may indicate a previously unrecognized non-thyroidal action of the abnormal immunoglobulins associated with Graves' disease and suggest that chronic thyroid heart disease may be due, at least in part, to the effect of these immunoglobulins especially when not associated with elevated thyroid hormones concentrations.     

The effects of plasma from patients with Graves' disease on foetal mouse hearts in organ culture.

Plasma, obtained during plasma exchange therapy, from 3 euthyroid patients with Graves'' disease and severe progressive exophthalmos induced an increase in heart rate and then early death when applied to foetal mouse hearts maintained in isolated organ culture. All plasma samples which induced an increase in foetal heart rate had high titres of thyroid stimulating immunoglobulins. Plasma samples obtained after exchange had a much diminished effect. These studies may indicate a previously unrecognized non-thyroidal action of the abnormal immunoglobulins associated with Graves'' disease and suggest that chronic thyroid heart disease may be due, at least in part, to the effect of these immunoglobulins especially when not associated with elevated thyroid hormones concentrations.     

Receptors and effectors in hormone action on the kidney

An analysis is presented of the similarities and differences in receptor-effector relationships in the actions of aldosterone and triiodothyronine on the kidney. Both agents augment tubular reabsorption of sodium at different sites in the nephron. Aldosterone acts primarily distally and triiodothyronine, primarily proximally. In both cases, the respective hormone-receptor complexes associate with target cell chromatin and modulate the abundance of specific coding by mRNAs for regulatory proteins. The quantitative relationships between nuclear-receptor occupancy and responses were analyzed by fractional plots in a bounded domain. When applied to the toad bladder, this analysis revealed a parabolic dependence of the increase in transepithelial Na+ transport on the abundance of nuclear of the increase in transepithelial Na+ transport on the abundance of nuclear aldosterone-receptor complexes. In contrast, four independent response parameters (QO2, QO2(t), Na-K-ATPase, alpha-glycerophosphate dehydrogenase) exhibited a hyperbolic dependence on nuclear abundance of T3 in the rat kidney. The observed cosaturation of nuclear occupancy and responses in both systems, for both hormones, implies that the respective high-affinity binding sites are authentic receptors. Further information is needed on the molecular bases of the nonlinear response-occupancy relationships in hormone action on the kidney.     

Atrial natriuretic peptide has no direct effect on proximal tubule sodium and water reabsorption

Infusion of ANP has been shown to increase the urinary excretion of sodium and water. However it is still controversial in which tubular segment sodium reabsorption is inhibited. To clarify this problem we have performed in vivo and in vitro studies to examine the direct effect of ANP on rat proximal tubules. The in vivo effect of ANP has been tested by using the micropuncture technique and in particular the shrinking droplet method that allows each investigated tubule to serve as its own control. Addition of either low (10^–9 M) or high (2×10^–6 M) concentrations of ANP to the luminal perfusate resulted in no significant change in isotonic fluid reabsorption (J _v). The same holds when the proximal tubules were perfused on both the tubular and peritubular side, with modified Ringer solution containing 10^–9 M ANP. To examine possible in vitro effects of ANP we prepared highly purified proximal tubule suspension derived from rat renal cortex and monitored oxygen consumption (QO₂) that is tightly coupled to sodium transport in this segment. Synthetic ANP, either at low (10^–9 M) or at high (10^–6 M) concentrations, did not affect basal rate of tubular respiration. Moreover the peptide hormone (10^–9 M) did not inhibit nystatin stimulated and ouabain sensitive QO₂. These results indicate that the enhancement of renal sodium excretion induced by ANP is not related to a direct inhibition of sodium transport in the proximal tubule.     

Increase of kinin in urine after partial occlusion of the renal vein and the effect of bradykinin on renal sodium excretion

A partial renal vein occlusion of one kidney, in hydropenic dogs, increases around twenty times the oxytocic activity of the urine from this kidney while in the urine from the control kidney the activity remains unchanged. This activity does not appear in urine by filtration because the glomerular filtration rate is significantly reduced by the vein occlusion. A partial occlusion of the renal artery does not increase the activity of urine. Through filtration on Sephadex LH-20 two fractions of activity in urine are obtained. The first peak appears in the same position as a standard of bradykinin appears. The second peak appears in the same position as a standard of antidiuretic hormone does. The samples of the first peak relax the rat duodenum and contract the rat uterus, the rat ileum and the guinea-pig ileum. They produce hypotension in rats and they maintain the activity after incubation with sodium thioglycolate. The activity is destroyed by chymotrypsin. As bradykinin may play a physiologic role in the regulation of renal sodium excretion, the effect of synthetic bradykinin on sodium excretions was studied in dogs using the stop-flow method (Malvin, R.L., et al. [4]). It was observed that bradykinin decrease the proximal tubular reabsorption of sodium and water but does not alter the capacity of the distal part of the nephron to lower the intratubular concentration of sodium. It was also observed that bradykinin decreases the proximal tubular secretion ofp-aminohippuric acid.Supported by Grants from CNPq and CPq. UFMG.     

Hypertrophy of basolateral Na-K pump activity in the proximal tubule of the remnant kidney

Reduction of renal mass leads to an increase in the filtration rates of the remaining glomeruli and an increased rate of sodium and water reabsorption by the proximal tubules. To define the basis for this increased tubular reabsorptive capacity, the authors studied the relationship of basolateral sodium pump activity to the process of hypertrophy in the proximal tubule. They wished to determine whether the growth of the cell is associated with an increase in the number of basolateral Na-K pumps and whether basolateral membrane hypertrophy is symmetrical with respect to overall cell growth. Normal and subtotally nephrectomized rabbits (remnant kidneys) were studied. Ouabain-sensitive potassium uptake was measured in a highly purified suspension of cortical proximal tubules using 86Rb as a tracer. In normal kidneys Km was 0.99 +/- 0.30 mM and Vmax 83.1 +/- 13.7 nmoles X mg-1 X minute-1; in remnant kidneys Km was 0.63 +/- 0.10 mM and Vmax 49.2 +/- 10.9 nmoles X mg-1 X minute-1. These values are not significantly different from each other. In a suspension of isolated cortical proximal tubular cells, protein per cell was 172 +/- 23 pg in normal kidney and 450 +/- 56 pg in remnant kidneys, representing a 2.6-fold increase. The extrapolated Vmax for K uptake per cell was thus increased approximately 2.6-fold in the remnant kidney. This was confirmed by measuring the number of specific ouabain-binding sites in proximal tubular cells. This was also found to be approximately 2.5 to 3 times greater in the remnant kidney cells, the increase being proportional to the increase in cell protein. Histomorphometric analysis of S2 proximal convoluted tubules, which comprise the bulk of the cortical tissue, revealed that basolateral membrane area per cross-sectional area of tubule was increased in the remnant kidney. The mean absolute surface area per cross-section of tubule and the surface density (surface/volume ratio) of the basolateral membrane increased by 110 and 26%, respectively, whereas these changes in the luminal membrane were only 38 and -9%, respectively. Thus, the membrane areas of the proximal tubular cell hypertrophy asymmetrically. Although mitochondrial density does not increase in remnant tubules, mitochondrial volume increases significantly, possibly providing a source for the increased ATP required by the hypertrophied basolateral Na-K pump activity. In summary, the cells of the proximal convoluted tubule of the remnant kidney undergo functional and structural hypertrophy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of the diuretic action of chloracizine

Chloracizine has a marked diuretic action and increases sodium excretion in rats although not by any direct action on the kidney. This effect is accompanied by an increase in the volume of intravascular fluid, a decrease in the total plasma protein concentration, and a decrease in the hematocrit index. A study of the endogenous creatinine clearance and transport of osmotically free water shows that the mechanism of the diuretic action of chloracizine is linked chiefly with an increase in the level of glomerular filtration and decrease in the relative proximal reabsorption.     

Effect of thyroid hormones on acetylcholinesterase mRNA levels in the slow soleus and fast extensor digitorum longus muscles of the rat

In the rat, the level of acetylcholinesterase messenger RNA in the typical slow soleus muscles is only about 20-30% of that in the fast extensor digitorum longus muscles. The expression of contractile proteins in muscles is influenced by thyroid hormones and hyperthyroidism makes the slow soleus muscle faster. The influence of thyroid hormones on the levels of acetylcholinesterase messenger RNA level in the slow soleus and fast extensor digitorum longus muscle of the rat was studied in order to examine the effect of thyroid hormones on muscle acetylcholinesterase expression. Hyperthyroidism was induced in rats by daily thyroid hormone injection or thyroid hormone releasing tablet implantation. Hind-limb suspension was applied to produce muscle unloading. Muscle denervation or reinnervation was achieved by sciatic nerve transection or crush. Acetylcholinesterase messenger RNA levels were analyzed by Northern blots and evaluated densitometrically. Hyperthyroidism increased the levels of acetylcholinesterase messenger RNA in the slow soleus muscles close to the levels in the fast extensor digitorum longus. The effect was the same in the unloaded soleus muscles. Acetylcholinesterase expression increased also in the absence of innervation (denervation), in the presence of changed nerve activation pattern (reinnervation), and under enhanced tonic neural activation of the soleus muscle (electrical stimulation). However, the changes were substantially smaller than those observed in the control soleus muscles. Enhancement of acetylcholinesterase expression in the soleus muscles by the thyroid hormones is, therefore, at last in part due to hormonal effect on the muscle itself. On the contrary, increased level of the thyroid hormones had no influence on acetylcholinesterase expression in the normal fast extensor digitorum longus muscles. However, some enhancing influence was apparent whenever the total number of nerve-induced muscle activations per day in the extensor digitorum longus muscle was increased. Thyroid hormones seem to be an independent extrinsic factor of acetylcholinesterase regulation in the slow soleus muscle.     

Effect of isotonic volume expansion on proximal tubular reabsorption of Na and fluid in the developing rat kidney1

Young rats (aged 22–24 days) and adult rats (aged 40–42 days) were studied during hydropenia (HP) and during volume expansion (VE) in order to clarify the role of the proximal tubule of the immature kidney in the blunted natriuretic response seen in young mammals during VE. The position of the last accessible site for micropuncture of the proximal tubular segment was determined. The disadvantages of using lissamine green as a marker of different tubular segments were investigated. Tubular function was ascertained by micropuncture of superficial proximal nephrons. Measurements of tubular length were made from latex casts of the proximal tubule. No side-effects of lissamine green were detected, when small quantities were used (20–30 μl) and at least 20 min elapsed between the infusions of the dye and tubular samplings. The last accessible proximal tubule available for micropunction was found to be similarly located in young and adult rats. Fractional reabsorption during HP remained constant during development. An equivalent degree of VE induced an increase in tubular load in both age groups, but it was more marked among younger rats. Absolute proximal reabsorption in both young and old rats in HP paralleled that of the tubular load. Fractional reabsorption, however, decreased slightly during VE but to the same extent in both age groups. This indicates a great flexibility in the immature proximal tubule under various tubular loads although it had been thought that this part of the nephron was in the later stages of development. The results imply that the proximal tubule does not create the blunted sodium response in the immature kidney during VE.     

Hypothyroidism and the heart

The cardiovascular system is sensitive to the action of thyroid hormones, and thyroid dysfunction causes a wide spectrum of cardiovascular changes. The effect of overt hypothyroidism on the cardiovascular system has long been recognised. Nowadays, the clinical presentation of cardiovascular symptoms related to hypothyroidism is only rarely observed due to early diagnosis of hypothyroidism by easily available thyroid-stimulating hormone assays. Overt hypothyroidism causes changes in such parameters of cardiovascular function as heart rate, left ventricular systolic and diastolic function, blood, arterial pressure and systemic vascular resistance. During the last years, there has been increasing evidence that subclinical hypothyroidism may also impair the cardiovascular system. This review discusses the effect of hypothyroidism on the cardiovascular system.     

Efffect of vasoactive intestinal polypeptide on gallbladder smooth muscle in vitro

The effect of vasoactive intestinal polypeptide (VIP) on basal and octapeptide of cholecystokinin (OP-CCK) induced tension was examined with guinea pig gallbladder smooth muscle strips in vitro. VIP alone produced dose-related decreases in resting tension and antagonized spontaneous contractile activity where present. In combination with OP-CCK, VIP decreased the expected contractile respone. The degree of antagonism depended upon the concentrations of OP-CCK and VIP. VIP had no effect on acetylcholine-induced contractions. From these observations, we propose that VIP can affect gallbladder motor activity by decreaseing smooth muscle tone and by antagonizing cholecystokinin. These findings lend further support to our proposal that gallbladder motor function may depend upon the action and interaction of the gastrointestinal hormones.     

Identification and partial characterization of a novel membrane-associated protein (MAP17) up-regulated in human carcinomas and modulating cell replication and tumor growth.

Using the differential display technique, we have recently reported the identification of a novel gene originally designated DD96. As determined by Northern blot and in situ hybridization, DD96 was expressed at significant levels only in a single epithelial cell population, the proximal tubular epithelial cells of the kidney. However, it was diffusely expressed in various carcinomas originating from kidney, colon, lung, and breast. Using a specific polyclonal antibody, we have not determined that the DD96 protein product is a 17-kd membrane-associated protein, which we have therefore redesignated MAP17. In normal tissues, MAP17 is expressed in significant amounts only in the kidney, where it was localized to the brush border of proximal tubular epithelial cells. However, MAP17 is expressed abundantly in carcinomas arising from kidney, colon, lung, and breast, in some cases with a membrane-associated apical glandular distribution. In tissue culture, MAP17 was localized to the cell membrane in areas of cell-cell contact, ie, the distribution of cell-function-associated proteins. Transfection of a full-length wild-type DD96 cDNA clone into a colon carcinoma cell line, HT-29, markedly decreased cell proliferation in vitro and tumor growth in vivo. Although the precise function of MAP17 remains to be determined, our findings suggest that this protein may play an important role in tumor biology.