Pfl~gers Arch. 360, 81 --89 (1975) 9 by Springer-Verlag 1975
Effects of Certain Diuretics on the Electrophysiological Characteristics of the Nephron in the Rat Kidney V. A. K a n t a r i y a a n d A. A. L e b e d e v The Ulyanov Memorial Kuibyshev Medical Inst.itu~, Chair of Pharmacology, Kuibyshev, USSR Received April 12, 1975
Summary. 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 potentiM and transepithelial resistance. Ethacrynic acid decreased the shortcircuit cttrrent in the proximal tubule, without changing the permeability characteristies 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 distal tubule under the action of novurite may serve to explain a lack of significant 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. Key words: Micropuncture -- Active Transport -- Sodium Transport -- Potassium Transport -- Diuretics. A l t h o u g h a g r e a t n u m b e r of e x p e r i m e n t s to d e t e r m i n e the a c t i o n of m o d e r n diuretics have b e e n carried out, the cellular m e c h a n i s m of m o s t of said drugs is n o t clear. The m e t h o d of the eleetrophysiological s t u d y of tile n e p h r o n plays a definite role i n the u n d e r s t a n d i n g of the cellular a n d m e m b r a n e aspects of the action of diuretics. The p r e s e n t paper deals with the cellular m e c h a n i s m of the action of diuretics d e t e r m i n e d b y m e a n s of the electrophysiological s t u d y of the n e p h r o n i n the r a t kidney.
Methods Experiments were performed on both male and female albino rats weighing 180 to 250 g, anesthetized by intraperitoneal injection of Thiopentalum (50 mg/kg body weight). The left kidney was placed into a plastic holder-cup. The Kidney surface was con~stantly washed by warm physiological solution. A Traeheotomy 6 PfltigersArch., Vol. 360
82
V. A. K a n t a r i y a and A. A. Lebedev
was performed and catheters were inserted into the external jugular veins for infusion of saline and diuretics solution. Polyethilene catheter was inserted into the left kidney ureter for free urine flow and sampling. Urine samples were collected in glass mieroeylinders and analyzed for electrolyte concentration b y flame photometer FPL-1. The eleetrophysiological characteristics of nephron were studied in eontrols and at peak diuresis during diuretic action. Diuretics were injected i.v. a t the rate of 12.5 mg/kg of euphylline, 25 mg/kg of novurite and 50 mg/kg of ethaerynic acid. A solution of sodium chloride was injected i.v. during the experiment at the rate of 0.05 ml/min. Mieropuneture of superfieial cortical tubules was performed under the stereoscopie microscope MBS-2 (90--120 magnification). Microeleetrodes 2 o. d. filled with 3 M KCL according to the method of Tasaki et al. [15] with the use of fiberoptics were used in our experiment. The use of such microeleetrodes facilitates visual control of microcleetrode position in the tubule lumen and eliminates recording of the electro-physiological cell activities, which lead to errors in the study of t r a n s t u b u l a r electrical properties. The diffusion potential of such microelectrodes is less t h a n 5 mv, the resistance--no more t h a n 500 K ohms. A V 2--11 high-resistance D.C. amplifier was used to measure t r a n s t u b u l a r potential. Stable potential in proximal tubule for at least 1 rain, on condition t h a t change in the diffusion potential of the microelectrodes did not exceed 1 m y was considered favorable for adequate reeording of the process under study. Several supeifieial tubules were punctured during the experiment. I n the distal tubule it is possible to take dynamic recordings of the stable potential for at least 20 to 30 rain, whereas the same t r a n s t u b u l a r potential in the proximal tubule tends to decrease spontaneously with time. EPP-09 D.C. electronic potentiometer with pen recorder was used to take dynamic recordings of the t r a n s t u b u l a r potential in the distal nephron. The early parts of the distal tubules were punetured, evidenced b y the lissamine green t h a t appeared after passing the loop of I-Ienle [14]. The use of low-resistance microeleetrodes 2 ~zo.d. helps to measure the effective electrical resistance of the nephron wall aeeording to the method of Karger et al. [7] and carry out a study of the short-circuit e u r r e n t - - a criterion of the active sodium transport. This method was ruled out in the distal tubule because of the high concentration gradients across the tubule wall which prevented the use of this method in the distal p a r t of the nephron. The experimental segment of the nephron was localized visually by a distinct bright b a n d inside the proximal tubule which is absent in the distal segment [3]; i.v. injection of lissamine green or indigocarmine helps to identify the tubule. Because of the inhibitory effect of lissamine green on the proximal tubule, reabsorption dye was injected either at the very end of the experiment or a t half-hour intervals [6]. I n a n u m b e r of experiments we used microelectrodes filled with 3 ~ solution of E v a n s blue, which was injeeted into the lumen b y iontophoresis, identified the segment of the nephron, and localized the tip of the mieroeleetrode in the tubule lumen (Fig. 1). Itistologieal slices were prepared to check localization of the dye. I t is very convenient to use microeleetrodes filled with Evans blue to record tubule potential. However, they are not of much use when the study of the effective electrieal resistance of the nephron wall and the short-eireuit current is carried out because they are. high-resistant and their eleetrieal characteristics are less stable over a period of time. We usually used mieroelectrodes filled with 3 M KCL solution. A very careful selection of microeleetrodes is imperative. They should be low-resistant, their diffusion potential at the tip should be small, their electrical characteristics should be maintained stable t h r o u g h o u t micropuneture and when current is passed.
Diuretics and Electrophysiology of the Nephron
83
Fig. 1. E v a n s blue into the tubule lumen identify microelectrode position inside the nephron. Photomicrograph of a histological section of superficial tubules of the r a t kidney. Sections were fixed in Bowin fixation and stained with hematoxylin a n d eosin. Magnification 400
Results
In the present study, ethacrynic acid, euphylline and novurite produced a marked diuretic and saluretic reaction (Table l). When ethacrynic acid and euphylline were administrated they produced a Table 1. Effect of i.v. injected diuretics on the urine flow and electrolyte excretion in the r a t kidney. Means • S. E. Group
l~enal excretion V ~zliter/min
Control Ethaerynic acid Control Novurite Control Euphylline
6*
U~AV [zEq/min
UKV ~zEq/min
2.7 • 0.9
0.83 ~ 0.23
0.16 ~ 0.05
65.5 i 16.0 P < 0.001
11.52 • 2.90 P < 0.001
1.08 • 0.25 P < 0.001
4.5 :j: 0.8 13.5 :[: 1.9 P < 0.01
0.50 i
0.10
0.49 • 0.10
2.81 ~ 0.70 P < 0.001
0.70 ~ 0.13 P > 0.05
3.6 • 0.3
0.74 • 0.15
0.28 • 0.01
19.0 :t: 2.8 P < 0.001
8.00 ~_ 1.70 P < 0.01
0.85 ~ 0.18 P < 0.01
84
V.A. Kantariy~ and A. A. Lebedev
pharmacological effect which was evidenced directly after the infusion of diuretics. Xanthine diuretic euphylline and ethacrynic acid significantly increased potassium exretion; mercurial diuretic novurite did not produce the same effect in our studies. The diuretic effect of euphylline as a rule persisted only 30 to 40 rain, then diminished considerably, and finally dropped to its original level. Conversely, the diuretic and saluretie effect of novurite developed only by the end of the 30 to 40-rain test period after i.v. inihsion. The study of etectrophysiologieM characteristics of the nephron at peak diuresis revealed discrepancies in the changes produced by different drugs. Novnrite decreased the transtubular potential and transepithelial resistance of the proximal tubule and reduced the short-circuit current (Table 2). Ethacrynic acid, on the other hand, caused a sharp drop in the short-circuit current without affecting the transepithelial resistance or transtubular potential of the proximal tubule. Euphylline caused the tubule wall to become hyperpolarized and a definite rise in the transepithelial resistance was recorded. The difference in the effect of diuretics on electrophysi01ogicaI characteristics of the nephron has been revealed in the distal tubules as well. Dynamic recording has shown that novurite decreased transtubular potential from 19.8 m y to 12.0 mv (A = 6.6 :~ 1.6 my, P < 0.05) and reduced the effective transepithelial resistance from 0.84 d: 0.07 M ohms to 0.60 ~ 0.05 M ohms (P < 0.01) ~. Ethacrynie acid has increased transtubutar potential from 17.6 my to 26.3 mv (A == 8.6 ~ 1.1 my, P < 0.02); resistance of the tubule wall in presence of diuretic --0.74 ~ 0.07 M ohms (N.S.). Euphylline increased tubular potential from 22.4 my to 30.0 my (A -- 7.6 ~= 1.1 my, P < 0.01), transepithelial resistance -- 0.90 =j= 0.09M ohms (N. S.) and under the action of diuretics has been done to study transepithelial resistance in the distal tubule. 0.84 =j= 0.07 M ohms-common controls.
Discussion We may assume from the data obtained that different diuretics tend to block the tubular reabsorption of electrolytes affecting the work of certain stages of transtubular ion transport. Mercurial diuretic novurite, on the one hand, inhibits the active sodium transport,, reducing the short-circuit current in the proximal tubule, on the other hand, it changes permeability characteristics of the nephron wall. I t is well known that transtubular potential and transepithelial resistance are values that in general reflect the degree of renal epithelium permeability to ions [1,5, t8]. Transepithelial resistance in the proximal segment of the nephron is due to extracelln!ar shunts for fluid and electrolyte transDiscrete mierol0uneture of several tubules in controls.
Diuretics and Electrophysiology of the Nephron
85
Table 2. Effect of i.v. injected diuretics on the eIectrophysiological characteristics of the proximal tubule in rat kidney Group
Transtubular potential (my)
Transepithelial resistance (~ ohms)
Short-circuit current (10 --9 A)
Control
--9.4 4- 1.0 (27) 0.24 4- 0.02 (25)
8.0 4- 0.7 (t4)
Novurite
-- 6.9 ~: 0.5 (32) 0.12 4- 0.02 (24) P < 0.05 P < 0.001
4.4 ~ 0.5 (22) P < 0.001
Control
--8.5 ~ 0.7 (37) 0.24 • 0.04 (36)
6.8 ~ 0.9 (15)
Ethaerynic acid
--9.3 ~_ 0.8 (31) 0.22 4- 0.03 (23) P > 0.05 P > 0.05
2.7 ~ 0.2 (t9) P < 0.001
Control
--5.7 ~ 0.4 (54) 0.31 4- 0.03 (25)
4.0 • 0.3 (13)
Euphylline
-- 8.7 4- 0.4 (52) 0.40 • 0.04 (29) P < 0.05 P > 0.05
4.0 ~ 0.2 (15) N.S.
Figures in brackets denote number of punctured tubules. port (I). In our studies a decrease of transepithelial resistance and transtubular potential under the action of novurite is a result of high-tubulewall ion permeability to electrolyte diffusion. Increase of the passive ion permeability of the skin, bladders, cellular membranes of the mammalian kidney preparation under lbhe action of mercurial substances is noted in a nmnber of studies [8, i0,13]. White et al. [17] have revealed an increase of radioactive sodium diffusion into the proximal tubule lumen of kidney in the presence of mercurhydrine. We m a y assume that sodium diffusion into the nephron lumen along with the high permeability of paraeellular pathway to ions is responsible to a certain extent for an increase in sodium excretion caused by mercurial diuretics. We cannot rule out the inhibitory effect of mercurial drugs on the functioning of the "sodium pump". The electrophysiological study of Necturus kidney has revealed a drop in the transtubular potential under the action of 5Iereurhydrine and a marked decrease of peritubular-membrane potential due to the action on sodium pump "built in" the basal membrane of the renal epithelium [5]. Thus, we m a y assume that sodium-pump blockade and an increase of ion diffusion into the nephron lumen play a certain role in effecting sodium excretion by mercurial drugs. Ethaerynie acid in our studies did not affect permeability characteristics of the renal tubules, nor did it change the transtubular potential and transepitheliM resistance of the nephron wall. The pharmacological effect of ethacrynic acid was manifest in a definite increase of the width of tubule lumen as a result of high intratubular pressure due to an
86
V. A. Kantariy~ and A. A. Lebedev
increased amount of nonreabsorbed fluid which lead to the danger of a "leak" around the mieroelectrode tip and a drop in transtubular potential and transepithclial resistance. Nontheless, we did not find any changes in permeability characteristics of the nephron under the action of ethacrynic acid. An increase in the width of the tubule lumen, on the one hand, hampered localization of the microelectrode position in the nephron lumen; on the other hand, however, it helped perform micropuncture of dilatated renal tubules and reduce possible recordings of eleetrophysiologieal cell activities. Blockade of active sodium transport is imperative for the pharmacological effect of ethacrynie acid. Ethaerynie acid was mainly responsible for a sharp drop in short-circuit current of the proximal tubule. Macknight [11] revealed a depression of cell metabolism under the action of ethaerynic acid. Landon and Forte [9] emphasize the blocking action of diuretic on the energy supply system of the active sodium transport in renal cells. Thus, the inhibition of active ion transport and energy supply is mainly responsible for the development of the ethaerynic acid effect. Contrary to cell phenomena of novurite and ethacrynie acid, xanthine diuretic euphylline did not inhibit active sodium transport in the proximal tubule nor did it increase ion "leakage" via the extraeel]ular pathway. Diuretic hyperpolarized the tubule wall and caused a slight increase in the renal epithelial resistance. The hyperpolarization of the renal tubule may be due to an increased permeability of the luminal membrane to sodium. When sodium enters the cells, it depolarizes the luminal membrane and increases transtubular potential. This is evidenced by an increased radioactive sodium uptake across the mucosal membrane of the amphibian bladder under the action of theophylline [8], and also by the action of amiloride which blocks this membrane effect of theophylline [12]. Amiloride is known to reduce the permeability of the luminal membrane to sodium. Xanthinc does not seem in any way to block biochemical processes of sodium transport. There is an evident increase of the activities of the certain encymes: suceinate dehydrogenase, Na-K stimulated ATPase in the renal epithelium, a high rate of oxidative phosphorylation, and 02 consumption of the kidney in the presence of xanthine derivates [16]. The changes of electrophysiological characteristics of the nephron wall under the euphylline action evidenced in the present study are in no way proof of the inhibitory effect of diuretic on the functioning of the active ion transport system. We understand that main points of the pharmacological effect of euphyllinc include the action of this drug on intrarenal hemodynamies, which is evidenced on the level of renal microcirculation and hydrodynamics of the nephron and may not necessarily be connected with an increase of the total glomerular filtration and general renal blood flow.
Diuretics and Eleet,rophysiology of the Nephron
87
Euphylline and ethacrynie acid were remarkable for producing an increase of potassium excretion by the kidney. Novurite, however, did not increase potassium excretion. It is well known, that potassium excretion depends on the level of ion nltrafiltration, reabsorption along the nephron length, and secretion in the distal tubule. The electrical gradient across the nephron wall is the main characteristics of the distal potassium secretion ae~ing as an electromotive force of potassium transport. When the eleetronegative value of the distal tubule lumen is reduced it m a y lead to a decrease in K diffusion into the nephron, and like,rise to a drop in potassium excretion. I t seems that a lack of significant potassium excretion in our studies and a drop in K excretion, together with a significant natrinresis caused by mercurial dmreties in the works [2,9], may partly be due to a decrease in the transtnbular potential of the distal nephron. The reduction of potassium excretion in the work of Duarte et al. [4] by amiloride was aecompained by a marked decrease in the transtubular distal potential difference. The experiments with ethaerynie acid and euphylline have shown that an increase in potassium excretion may partly result from an increased value of the tubular potential, thus enhancing ion diffusion into the nephron. We must emphasize that the study of the transtubular potential of the distal tubule has been carried out under dynamic recording eondi. tions. In one set of experiments we made studies of the tubule potentiaI in the distal segment of the nephron by discrete mieropuneture of several distal tubules. The experiments revealed a drop of potential in the distal tubule under the action of norm, ire from 24.5 _% 1.5 my to 17.5 ~ 1.4mv (P < 0.05) and only a slight increase of transtubular potential under
Fig. 2. Effect of novurite infused i.v. (25 mg/kg) on transt.ubular potential difference (mV) of distal renal tubule. Arrow indicates infusion of diuretic
88
V. A. Kantariya and A. A. Lebedev
Fig. 3. Effect of eulchylline infused i.v. (12.5 mg/kg) on transtubular potential difference (mV) of distal tubule
the action of ethacrynic acid and euphylline from 24.5 ~ 1.5 m y to 26.5 =~ 1.5 mv and --27.6 =[: 2.3 mv, respectively (the difference is not statmticaly valid). The data obtained from dynamic and discrete recordings of the transtnbular potential of the distal tubule may not be in complete agreement because of the variable electrical gradient along the distal nephron [18]. As a result, depending on the mieroelectrode position along the tubule length, potentials of different amplitudes are recorded. Wright [18] proves that a low potential i s present at the beginning of the distal tubule, which rises to -- 60 mv toward the end of the tubule. Dynamic recordings of the distal tubule potentials together with a permanent localization of the microelectrode along the nephron helps carry out an adequate study of the transtubular potential in this segment of the nephron (Figs. 2, 3).
References 1. Boulpaep, E. L.: Permeability changes of the proximal tubule of Necturus during saline loading. Amer. J. Physiol. 221~, 517--531 (1972) 2. Bowman, F.J., Landon, E.J.: Organic mercurials and net movement of potassium in rat kidney slices. Amer. J. Physiol. 218, 1209--1217 (1967) 3. Deetjen, P. : A study of the effectiveness and localisation of the action of furosemide. Cardiology 9, 12--17 (1969) 4. Duarte, C. G., Chonuti, F., Giebiseh, G.: Effect of amiloride, ouabain and furosemide on distal tubular function in the rat. Amer. J. Physiol. 221,632-- 639 (I971) 5. Giebisch, G.: Measurements of electrical potentials and Ion fluxes on single renal tubules. Circulation 221, 879--891 (1960)
Diuretics and Electrophysiology of the Nephron
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6. Heller, J. : The influence of Lissamine green on tubular reabsorption of electro. lytes and water in rats. Pflfigers Arch. ~23, 27--33 (1971) 7. Karger, W., Eigler, F. W., Hampel, A. : t)ber eine Mel~ordnung zur Bestimmung elektrischer Gr6~en an biologischen Membranen mit aktiven Ionentransport. Pflfigers Arch. ges. Physiol. 272, 187--190 (1960) 8. Klimmenkoff, A. P. : The effect of diuretic on sodium transport across the cellular membranes of the frog urinary bladder. In: The mechanism of diuretic action, Symposium, pp. 43--44. Kuybishev, 1970 9. Landon, E. J., Forte, L. R. : Cellular mechanism in renal pharmacology. Ann. Rev. Pharmacol. 11, 171--188 (1971) 10. Lebedev, A. A. : The mechanism of action of mercuric diuretics on the sodium tubular transport. Cardiology 11, 89--94 (1971) 11. Maeknight, A. D. : The effect of ethacrynic acid on the electrolyte and water contents of rat renal cortical slices. Biochim. biophys. Acta (Amst.) 173, 223--233 (1969) 12. Salaco, L. A., Smith, A. J.: Effects of amiloride on active sodium transport by the isolated frog skin: evidence concerning site action. Brit. J. Pharmaeol. 38, 702--718 (1970) 13. Shelepov, V. A. : The mechanism of diuretic action. In: The kidney and electrolytes. Proc. of Kuybishev Medical Institute, Vol. 43, pp. 100--107, Kuybishev 1967 14. Steinhausen, M.: Eine Methode zur Differenzierung proximaler und distaler Tubuli der Nierenrinde yon Ratten in vivo und ihre Anwendung zur Bestimmung tubulgrer StrSmungsgeschwindigkeiten. Pflfigers Arch. ges. Physiol. 277, 23--35 (1963) 15. Tasaki, K., Tsukahara, Y., Ito, I.: A simple, direct and rapid method for filling microelectrodes. Physiol. Behav. 3, 1009--1010 (1968) 16. Volynsky, B. G., Freidman, S. L. : On the mechanism of purins diuretic action. In: The mechanism of diuretic action, Symposium, pp. 23--24. Kuybishev 1970 17. White, H. Z., Role, D., Bisno, A. L.: Water and sodium exchange in renal tubule fluid. Amer. J. Physiol. 200, 595--600 (1961) 18. Wright, F. S. : Increasing magnitude of electrical potential along the renal distal tubule. Amer. J. Physiol. 220, 624--638 (1971) V. A. Kantariya A. A. Lebedev Xuybishev Medical Institute Chair of Pharmacology Chpaev St. 89 Kuybishev, USSR
Effects of Certain Diuretics on the Electrophysiological Characteristics of the Nephron in the Rat Kidney V. A. K a n t a r i y a a n d A. A. L e b e d e v The Ulyanov Memorial Kuibyshev Medical Inst.itu~, Chair of Pharmacology, Kuibyshev, USSR Received April 12, 1975
Summary. 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 potentiM and transepithelial resistance. Ethacrynic acid decreased the shortcircuit cttrrent in the proximal tubule, without changing the permeability characteristies 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 distal tubule under the action of novurite may serve to explain a lack of significant 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. Key words: Micropuncture -- Active Transport -- Sodium Transport -- Potassium Transport -- Diuretics. A l t h o u g h a g r e a t n u m b e r of e x p e r i m e n t s to d e t e r m i n e the a c t i o n of m o d e r n diuretics have b e e n carried out, the cellular m e c h a n i s m of m o s t of said drugs is n o t clear. The m e t h o d of the eleetrophysiological s t u d y of tile n e p h r o n plays a definite role i n the u n d e r s t a n d i n g of the cellular a n d m e m b r a n e aspects of the action of diuretics. The p r e s e n t paper deals with the cellular m e c h a n i s m of the action of diuretics d e t e r m i n e d b y m e a n s of the electrophysiological s t u d y of the n e p h r o n i n the r a t kidney.
Methods Experiments were performed on both male and female albino rats weighing 180 to 250 g, anesthetized by intraperitoneal injection of Thiopentalum (50 mg/kg body weight). The left kidney was placed into a plastic holder-cup. The Kidney surface was con~stantly washed by warm physiological solution. A Traeheotomy 6 PfltigersArch., Vol. 360
82
V. A. K a n t a r i y a and A. A. Lebedev
was performed and catheters were inserted into the external jugular veins for infusion of saline and diuretics solution. Polyethilene catheter was inserted into the left kidney ureter for free urine flow and sampling. Urine samples were collected in glass mieroeylinders and analyzed for electrolyte concentration b y flame photometer FPL-1. The eleetrophysiological characteristics of nephron were studied in eontrols and at peak diuresis during diuretic action. Diuretics were injected i.v. a t the rate of 12.5 mg/kg of euphylline, 25 mg/kg of novurite and 50 mg/kg of ethaerynic acid. A solution of sodium chloride was injected i.v. during the experiment at the rate of 0.05 ml/min. Mieropuneture of superfieial cortical tubules was performed under the stereoscopie microscope MBS-2 (90--120 magnification). Microeleetrodes 2 o. d. filled with 3 M KCL according to the method of Tasaki et al. [15] with the use of fiberoptics were used in our experiment. The use of such microeleetrodes facilitates visual control of microcleetrode position in the tubule lumen and eliminates recording of the electro-physiological cell activities, which lead to errors in the study of t r a n s t u b u l a r electrical properties. The diffusion potential of such microelectrodes is less t h a n 5 mv, the resistance--no more t h a n 500 K ohms. A V 2--11 high-resistance D.C. amplifier was used to measure t r a n s t u b u l a r potential. Stable potential in proximal tubule for at least 1 rain, on condition t h a t change in the diffusion potential of the microelectrodes did not exceed 1 m y was considered favorable for adequate reeording of the process under study. Several supeifieial tubules were punctured during the experiment. I n the distal tubule it is possible to take dynamic recordings of the stable potential for at least 20 to 30 rain, whereas the same t r a n s t u b u l a r potential in the proximal tubule tends to decrease spontaneously with time. EPP-09 D.C. electronic potentiometer with pen recorder was used to take dynamic recordings of the t r a n s t u b u l a r potential in the distal nephron. The early parts of the distal tubules were punetured, evidenced b y the lissamine green t h a t appeared after passing the loop of I-Ienle [14]. The use of low-resistance microeleetrodes 2 ~zo.d. helps to measure the effective electrical resistance of the nephron wall aeeording to the method of Karger et al. [7] and carry out a study of the short-circuit e u r r e n t - - a criterion of the active sodium transport. This method was ruled out in the distal tubule because of the high concentration gradients across the tubule wall which prevented the use of this method in the distal p a r t of the nephron. The experimental segment of the nephron was localized visually by a distinct bright b a n d inside the proximal tubule which is absent in the distal segment [3]; i.v. injection of lissamine green or indigocarmine helps to identify the tubule. Because of the inhibitory effect of lissamine green on the proximal tubule, reabsorption dye was injected either at the very end of the experiment or a t half-hour intervals [6]. I n a n u m b e r of experiments we used microelectrodes filled with 3 ~ solution of E v a n s blue, which was injeeted into the lumen b y iontophoresis, identified the segment of the nephron, and localized the tip of the mieroeleetrode in the tubule lumen (Fig. 1). Itistologieal slices were prepared to check localization of the dye. I t is very convenient to use microeleetrodes filled with Evans blue to record tubule potential. However, they are not of much use when the study of the effective electrieal resistance of the nephron wall and the short-eireuit current is carried out because they are. high-resistant and their eleetrieal characteristics are less stable over a period of time. We usually used mieroelectrodes filled with 3 M KCL solution. A very careful selection of microeleetrodes is imperative. They should be low-resistant, their diffusion potential at the tip should be small, their electrical characteristics should be maintained stable t h r o u g h o u t micropuneture and when current is passed.
Diuretics and Electrophysiology of the Nephron
83
Fig. 1. E v a n s blue into the tubule lumen identify microelectrode position inside the nephron. Photomicrograph of a histological section of superficial tubules of the r a t kidney. Sections were fixed in Bowin fixation and stained with hematoxylin a n d eosin. Magnification 400
Results
In the present study, ethacrynic acid, euphylline and novurite produced a marked diuretic and saluretic reaction (Table l). When ethacrynic acid and euphylline were administrated they produced a Table 1. Effect of i.v. injected diuretics on the urine flow and electrolyte excretion in the r a t kidney. Means • S. E. Group
l~enal excretion V ~zliter/min
Control Ethaerynic acid Control Novurite Control Euphylline
6*
U~AV [zEq/min
UKV ~zEq/min
2.7 • 0.9
0.83 ~ 0.23
0.16 ~ 0.05
65.5 i 16.0 P < 0.001
11.52 • 2.90 P < 0.001
1.08 • 0.25 P < 0.001
4.5 :j: 0.8 13.5 :[: 1.9 P < 0.01
0.50 i
0.10
0.49 • 0.10
2.81 ~ 0.70 P < 0.001
0.70 ~ 0.13 P > 0.05
3.6 • 0.3
0.74 • 0.15
0.28 • 0.01
19.0 :t: 2.8 P < 0.001
8.00 ~_ 1.70 P < 0.01
0.85 ~ 0.18 P < 0.01
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V.A. Kantariy~ and A. A. Lebedev
pharmacological effect which was evidenced directly after the infusion of diuretics. Xanthine diuretic euphylline and ethacrynic acid significantly increased potassium exretion; mercurial diuretic novurite did not produce the same effect in our studies. The diuretic effect of euphylline as a rule persisted only 30 to 40 rain, then diminished considerably, and finally dropped to its original level. Conversely, the diuretic and saluretie effect of novurite developed only by the end of the 30 to 40-rain test period after i.v. inihsion. The study of etectrophysiologieM characteristics of the nephron at peak diuresis revealed discrepancies in the changes produced by different drugs. Novnrite decreased the transtubular potential and transepithelial resistance of the proximal tubule and reduced the short-circuit current (Table 2). Ethacrynic acid, on the other hand, caused a sharp drop in the short-circuit current without affecting the transepithelial resistance or transtubular potential of the proximal tubule. Euphylline caused the tubule wall to become hyperpolarized and a definite rise in the transepithelial resistance was recorded. The difference in the effect of diuretics on electrophysi01ogicaI characteristics of the nephron has been revealed in the distal tubules as well. Dynamic recording has shown that novurite decreased transtubular potential from 19.8 m y to 12.0 mv (A = 6.6 :~ 1.6 my, P < 0.05) and reduced the effective transepithelial resistance from 0.84 d: 0.07 M ohms to 0.60 ~ 0.05 M ohms (P < 0.01) ~. Ethacrynie acid has increased transtubutar potential from 17.6 my to 26.3 mv (A == 8.6 ~ 1.1 my, P < 0.02); resistance of the tubule wall in presence of diuretic --0.74 ~ 0.07 M ohms (N.S.). Euphylline increased tubular potential from 22.4 my to 30.0 my (A -- 7.6 ~= 1.1 my, P < 0.01), transepithelial resistance -- 0.90 =j= 0.09M ohms (N. S.) and under the action of diuretics has been done to study transepithelial resistance in the distal tubule. 0.84 =j= 0.07 M ohms-common controls.
Discussion We may assume from the data obtained that different diuretics tend to block the tubular reabsorption of electrolytes affecting the work of certain stages of transtubular ion transport. Mercurial diuretic novurite, on the one hand, inhibits the active sodium transport,, reducing the short-circuit current in the proximal tubule, on the other hand, it changes permeability characteristics of the nephron wall. I t is well known that transtubular potential and transepithelial resistance are values that in general reflect the degree of renal epithelium permeability to ions [1,5, t8]. Transepithelial resistance in the proximal segment of the nephron is due to extracelln!ar shunts for fluid and electrolyte transDiscrete mierol0uneture of several tubules in controls.
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85
Table 2. Effect of i.v. injected diuretics on the eIectrophysiological characteristics of the proximal tubule in rat kidney Group
Transtubular potential (my)
Transepithelial resistance (~ ohms)
Short-circuit current (10 --9 A)
Control
--9.4 4- 1.0 (27) 0.24 4- 0.02 (25)
8.0 4- 0.7 (t4)
Novurite
-- 6.9 ~: 0.5 (32) 0.12 4- 0.02 (24) P < 0.05 P < 0.001
4.4 ~ 0.5 (22) P < 0.001
Control
--8.5 ~ 0.7 (37) 0.24 • 0.04 (36)
6.8 ~ 0.9 (15)
Ethaerynic acid
--9.3 ~_ 0.8 (31) 0.22 4- 0.03 (23) P > 0.05 P > 0.05
2.7 ~ 0.2 (t9) P < 0.001
Control
--5.7 ~ 0.4 (54) 0.31 4- 0.03 (25)
4.0 • 0.3 (13)
Euphylline
-- 8.7 4- 0.4 (52) 0.40 • 0.04 (29) P < 0.05 P > 0.05
4.0 ~ 0.2 (15) N.S.
Figures in brackets denote number of punctured tubules. port (I). In our studies a decrease of transepithelial resistance and transtubular potential under the action of novurite is a result of high-tubulewall ion permeability to electrolyte diffusion. Increase of the passive ion permeability of the skin, bladders, cellular membranes of the mammalian kidney preparation under lbhe action of mercurial substances is noted in a nmnber of studies [8, i0,13]. White et al. [17] have revealed an increase of radioactive sodium diffusion into the proximal tubule lumen of kidney in the presence of mercurhydrine. We m a y assume that sodium diffusion into the nephron lumen along with the high permeability of paraeellular pathway to ions is responsible to a certain extent for an increase in sodium excretion caused by mercurial diuretics. We cannot rule out the inhibitory effect of mercurial drugs on the functioning of the "sodium pump". The electrophysiological study of Necturus kidney has revealed a drop in the transtubular potential under the action of 5Iereurhydrine and a marked decrease of peritubular-membrane potential due to the action on sodium pump "built in" the basal membrane of the renal epithelium [5]. Thus, we m a y assume that sodium-pump blockade and an increase of ion diffusion into the nephron lumen play a certain role in effecting sodium excretion by mercurial drugs. Ethaerynie acid in our studies did not affect permeability characteristics of the renal tubules, nor did it change the transtubular potential and transepitheliM resistance of the nephron wall. The pharmacological effect of ethacrynic acid was manifest in a definite increase of the width of tubule lumen as a result of high intratubular pressure due to an
86
V. A. Kantariy~ and A. A. Lebedev
increased amount of nonreabsorbed fluid which lead to the danger of a "leak" around the mieroelectrode tip and a drop in transtubular potential and transepithclial resistance. Nontheless, we did not find any changes in permeability characteristics of the nephron under the action of ethacrynic acid. An increase in the width of the tubule lumen, on the one hand, hampered localization of the microelectrode position in the nephron lumen; on the other hand, however, it helped perform micropuncture of dilatated renal tubules and reduce possible recordings of eleetrophysiologieal cell activities. Blockade of active sodium transport is imperative for the pharmacological effect of ethacrynie acid. Ethaerynie acid was mainly responsible for a sharp drop in short-circuit current of the proximal tubule. Macknight [11] revealed a depression of cell metabolism under the action of ethaerynic acid. Landon and Forte [9] emphasize the blocking action of diuretic on the energy supply system of the active sodium transport in renal cells. Thus, the inhibition of active ion transport and energy supply is mainly responsible for the development of the ethaerynic acid effect. Contrary to cell phenomena of novurite and ethacrynie acid, xanthine diuretic euphylline did not inhibit active sodium transport in the proximal tubule nor did it increase ion "leakage" via the extraeel]ular pathway. Diuretic hyperpolarized the tubule wall and caused a slight increase in the renal epithelial resistance. The hyperpolarization of the renal tubule may be due to an increased permeability of the luminal membrane to sodium. When sodium enters the cells, it depolarizes the luminal membrane and increases transtubular potential. This is evidenced by an increased radioactive sodium uptake across the mucosal membrane of the amphibian bladder under the action of theophylline [8], and also by the action of amiloride which blocks this membrane effect of theophylline [12]. Amiloride is known to reduce the permeability of the luminal membrane to sodium. Xanthinc does not seem in any way to block biochemical processes of sodium transport. There is an evident increase of the activities of the certain encymes: suceinate dehydrogenase, Na-K stimulated ATPase in the renal epithelium, a high rate of oxidative phosphorylation, and 02 consumption of the kidney in the presence of xanthine derivates [16]. The changes of electrophysiological characteristics of the nephron wall under the euphylline action evidenced in the present study are in no way proof of the inhibitory effect of diuretic on the functioning of the active ion transport system. We understand that main points of the pharmacological effect of euphyllinc include the action of this drug on intrarenal hemodynamies, which is evidenced on the level of renal microcirculation and hydrodynamics of the nephron and may not necessarily be connected with an increase of the total glomerular filtration and general renal blood flow.
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87
Euphylline and ethacrynie acid were remarkable for producing an increase of potassium excretion by the kidney. Novurite, however, did not increase potassium excretion. It is well known, that potassium excretion depends on the level of ion nltrafiltration, reabsorption along the nephron length, and secretion in the distal tubule. The electrical gradient across the nephron wall is the main characteristics of the distal potassium secretion ae~ing as an electromotive force of potassium transport. When the eleetronegative value of the distal tubule lumen is reduced it m a y lead to a decrease in K diffusion into the nephron, and like,rise to a drop in potassium excretion. I t seems that a lack of significant potassium excretion in our studies and a drop in K excretion, together with a significant natrinresis caused by mercurial dmreties in the works [2,9], may partly be due to a decrease in the transtnbular potential of the distal nephron. The reduction of potassium excretion in the work of Duarte et al. [4] by amiloride was aecompained by a marked decrease in the transtubular distal potential difference. The experiments with ethaerynie acid and euphylline have shown that an increase in potassium excretion may partly result from an increased value of the tubular potential, thus enhancing ion diffusion into the nephron. We must emphasize that the study of the transtubular potential of the distal tubule has been carried out under dynamic recording eondi. tions. In one set of experiments we made studies of the tubule potentiaI in the distal segment of the nephron by discrete mieropuneture of several distal tubules. The experiments revealed a drop of potential in the distal tubule under the action of norm, ire from 24.5 _% 1.5 my to 17.5 ~ 1.4mv (P < 0.05) and only a slight increase of transtubular potential under
Fig. 2. Effect of novurite infused i.v. (25 mg/kg) on transt.ubular potential difference (mV) of distal renal tubule. Arrow indicates infusion of diuretic
88
V. A. Kantariya and A. A. Lebedev
Fig. 3. Effect of eulchylline infused i.v. (12.5 mg/kg) on transtubular potential difference (mV) of distal tubule
the action of ethacrynic acid and euphylline from 24.5 ~ 1.5 m y to 26.5 =~ 1.5 mv and --27.6 =[: 2.3 mv, respectively (the difference is not statmticaly valid). The data obtained from dynamic and discrete recordings of the transtnbular potential of the distal tubule may not be in complete agreement because of the variable electrical gradient along the distal nephron [18]. As a result, depending on the mieroelectrode position along the tubule length, potentials of different amplitudes are recorded. Wright [18] proves that a low potential i s present at the beginning of the distal tubule, which rises to -- 60 mv toward the end of the tubule. Dynamic recordings of the distal tubule potentials together with a permanent localization of the microelectrode along the nephron helps carry out an adequate study of the transtubular potential in this segment of the nephron (Figs. 2, 3).
References 1. Boulpaep, E. L.: Permeability changes of the proximal tubule of Necturus during saline loading. Amer. J. Physiol. 221~, 517--531 (1972) 2. Bowman, F.J., Landon, E.J.: Organic mercurials and net movement of potassium in rat kidney slices. Amer. J. Physiol. 218, 1209--1217 (1967) 3. Deetjen, P. : A study of the effectiveness and localisation of the action of furosemide. Cardiology 9, 12--17 (1969) 4. Duarte, C. G., Chonuti, F., Giebiseh, G.: Effect of amiloride, ouabain and furosemide on distal tubular function in the rat. Amer. J. Physiol. 221,632-- 639 (I971) 5. Giebisch, G.: Measurements of electrical potentials and Ion fluxes on single renal tubules. Circulation 221, 879--891 (1960)
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6. Heller, J. : The influence of Lissamine green on tubular reabsorption of electro. lytes and water in rats. Pflfigers Arch. ~23, 27--33 (1971) 7. Karger, W., Eigler, F. W., Hampel, A. : t)ber eine Mel~ordnung zur Bestimmung elektrischer Gr6~en an biologischen Membranen mit aktiven Ionentransport. Pflfigers Arch. ges. Physiol. 272, 187--190 (1960) 8. Klimmenkoff, A. P. : The effect of diuretic on sodium transport across the cellular membranes of the frog urinary bladder. In: The mechanism of diuretic action, Symposium, pp. 43--44. Kuybishev, 1970 9. Landon, E. J., Forte, L. R. : Cellular mechanism in renal pharmacology. Ann. Rev. Pharmacol. 11, 171--188 (1971) 10. Lebedev, A. A. : The mechanism of action of mercuric diuretics on the sodium tubular transport. Cardiology 11, 89--94 (1971) 11. Maeknight, A. D. : The effect of ethacrynic acid on the electrolyte and water contents of rat renal cortical slices. Biochim. biophys. Acta (Amst.) 173, 223--233 (1969) 12. Salaco, L. A., Smith, A. J.: Effects of amiloride on active sodium transport by the isolated frog skin: evidence concerning site action. Brit. J. Pharmaeol. 38, 702--718 (1970) 13. Shelepov, V. A. : The mechanism of diuretic action. In: The kidney and electrolytes. Proc. of Kuybishev Medical Institute, Vol. 43, pp. 100--107, Kuybishev 1967 14. Steinhausen, M.: Eine Methode zur Differenzierung proximaler und distaler Tubuli der Nierenrinde yon Ratten in vivo und ihre Anwendung zur Bestimmung tubulgrer StrSmungsgeschwindigkeiten. Pflfigers Arch. ges. Physiol. 277, 23--35 (1963) 15. Tasaki, K., Tsukahara, Y., Ito, I.: A simple, direct and rapid method for filling microelectrodes. Physiol. Behav. 3, 1009--1010 (1968) 16. Volynsky, B. G., Freidman, S. L. : On the mechanism of purins diuretic action. In: The mechanism of diuretic action, Symposium, pp. 23--24. Kuybishev 1970 17. White, H. Z., Role, D., Bisno, A. L.: Water and sodium exchange in renal tubule fluid. Amer. J. Physiol. 200, 595--600 (1961) 18. Wright, F. S. : Increasing magnitude of electrical potential along the renal distal tubule. Amer. J. Physiol. 220, 624--638 (1971) V. A. Kantariya A. A. Lebedev Xuybishev Medical Institute Chair of Pharmacology Chpaev St. 89 Kuybishev, USSR
