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Isolated nephron segments as pharmacological tools
FI P S - J a n u a r y i q S I
determines the health, capacity and lilt:span of man and Ihat this inflow is dual (energy ,'rod structural information), this p h c n o m c m m may be examined in the lisht of N. Bohr's contplcmcntarily principle, developed by him for the corpuscular-~ ave dualism in physics. AI one time physics encountered the problem of elements of set determining the quantum slate of the macroscopic syslem. Flowe~er. for a substance consist mg of I ()= electrons and nuclei, the literal picture could be oblained b} solution of Ihe v,a~e equation with 3 × I (t=s unknm~ n member~. This phenomenological thermodynamics was opposed by Gibbs" statktical thermodynamics, which abstracts ilself from all coml~ments of the system and la:.es account ol the net resull of the ensemble. Thc individual substances of coml~site natural complexes certainly have their specific actions; but however different were the fimdstuffs of different peoples. they achieved the same objective, i.e. the provision of man with eveD thimz needed for life and propagation of the species. The preservation of health and treatment of illnesses is achieved by different sets of herbs in different parts of the world. It is imp, wtam and necessary to dcfine and com-
prehend deepl} the ,peciticll} ol acllon ,4 individual ~,ubst ante,.. This purel} phenomemflogieal approach ha,, great significance, llul it b, praclieally im~s,qbl¢ to ev;duate the imlm~rtanec and c, mtribulion of e ~ e r y sub,,lance. ,,I all p,~ssible eooperali~,e e[fecls. Moreover. indP, idual [acts musl nol "obscure Ihe ~.iev+ ol lhe w ~ d for the tree'.'.Therefore lhe ,4atNt,cal approach dctcrmined b~ the +.iguificance O[ Mructurill information is no less iml~+r[anl. For lhe normal grov.lh and development <+f capacil}, non-ntorbidit,, and finall} hcahh, the organ,sm ol man musl get a definite amount (~olumc. critical mass) of slruclural inlormatitm [rom Nature.
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Attempts to learn the sites and nlechanisnts of action of a variety of agents in an organ as complex as the kidney arc often flustrated by the limitations of the available techniques. Many drutm appear to have complex effects and multiple sites of action; they may change not only transtx+rt function but also alter such factors as renal blood flow or glomerular filtration rate. Physiologists have long been interested in techniques which permit direct stud}' o[ the transport of solutes and water by specific segments of the nephron. This interest can be satisfied by micropuneture studies of those structures which occur on the surface
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Isolated nephron segments as pharmacological tools New York 13210, U.5.A+
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port rate. ot ~atcr and ~an,,u..olut~. In addition It+ gix ing the im, e,,tigator aeee-', to tubular ,egmcm~, + hich ca:mot bc -.tudicd directlx %+ith t,ther technique,,, thl', methodok+g} offer,, other atd'.antagc-. .Xmt,ng tht.,,e are the preel,e and IndeD:ndent control ot the content,, o[ the bathing mcdiurn and perfu,a:te. :,nd the at ailabilit.,, of the tubule lr,lgmelli it-~cIt for analx,,l', ot eel!.llar coml~',itlon. I he,,e adxantage, dl,,~ not onl~ ph},,iologic:d ,,tudJe, but ,ll,,~, ,,t udic,, ,limed ;.tt ¢ l t l C I t L l l i f l ~ t h+~."",lte'~Ol lr:An.+porland Ili~.'ch,tni',m', O[ ;.l,.'tion ol x ariOtlS il~CIltS, some o[ '.%hich ++re knm%n h+r their tberal',,:utt+ salue I he detail- ot 1h¢
of the kidnex. The lack ot techniques t~ stud} the properties of nephron segment, found deep in the renal muss led Burg and t e c h n i q u e ~, f o r p e r t t l , , i n g t r a g n t e l l t ' ~ o [ t h e his eo-x~orkers ill the National In,,titute', o! nlanlnl;.tli;.m nephrtm in +,lro. Including He:IItlP to develop methods to dissect and •,omc o[ the di[ficultic, encountered .rod o | t h e method. l~rfu.~ in vitro various segments of the the major c , o n l r i b u t l o n , , rubhit nephron. [n this technique short hate recently Ix'en :cuexsed a a Here. l limit nlxs¢ll to txso ,treat ot disfragmentst;f the nephron (usually I-2 mm in length) are suspended bctx~een pare, of cusslon: the impact of this methodok+g', +n concentric glass pipets. Fluid of knm~n our understanding O [ t h e t r a n s p o r t o f ,A e d k comlx~sition is forced into the lumen of one organic acids acro,,x proximal nephron end of the tubule and collected for anal) sis ,egments. and the site,, and m e e h a n i M l l s O t in the pipets at the other end. Changes in action of x.:trious diuretics. In Iqb,-a Tune, Burg ;rod Patlak 4 r u b the chemical or radioisotopie contents o[ the perfusate arc umd to as.~s.,, the tr.'ms- lished ~+.hat now must IX."regarded a,, a I t~,+~t %,.lth H,' .rod ,tl:,,m:d+. ,+ Pr,-. , , , ;
T I P S - J a n u a r y 1981
classic study on para-aminohippurate (PAH) transport acro~,s the epithelium of rabbit tubulesin vitro. By utilizing a variety of experimental prott~cols (including analysis of cellular contents), they determined fact¢~rswhich led to a detailed model of the transport process. More recently, a very similar model for the transport of organic acids across proximal tubule fragments from garter snakes has been developed by Dantzler and associates 5, In rabbit and snake the late portions of the prt~ximal nephrons appear to ~crete PAH at much higher rates than the early portions. As depicted by the first two bars of Fig. i. when either the pars recta of the rabb~t or the late proximal tubule of the snake are placed in a bathing medium containing low concentrations (approximately 2 × IO--SM) of radioactive PAH. the concentration of PAH in the pcrfused fluid is substantially higher than that in the medium. This demonstration that net PAH movement fro.. the peritubular to the lumen compartments occurs against a concentration gradient led to the conclusion that PAH secretion is an active process. Because of the very. large concentration difference of PAH between the cytosol and bathing medium (right side. Fig. i) it was po~,tulated that the entry of PAH into the cell across the peritubular membrane must be an active process. Since the conceutration of PAH in the cytosol exceeded that of lumen (center. Fig. I) the organic acid moved to the lumen from the cell down its chemical gradient. Both groups concluded that this mt~vement could be passive diffusion. The observation that the apparent permeability of the lumen membrane appeared t o b~ unaffected by probenecid added to the bathing medium was seen by lrune et aL as further evidence supporting this interpretation. While the major features ol the model stood the test of time, two aspects are not universally accepted. Recent evidence. utilizmt+lumen membrane vesicles isolated from dog kidney cortex, suggests that PAH movement through the luminal membrane w.ay t.e a facilitated process s. Furthermore. Dant~ler and Bentley ~ have shown that ,-hen ,:old PAH. phenol red. probenecid or SITZ (an inhibitor of an ion exchange in red cells) are added to the perfusate of snake tubules, secretion of I"C]PAH is inhibited. They too suggest that the "downhill" movement of PAH into the tubular lumen may not be simple diffusion. Unfortunately comparable information has not been published for other animals. Sh/ili and Roch-Ramel (personal corn-
munication) h;~verecently been able to dissect and stuEy PAH uptake iato nonperfused tubules of two other mz mmalian species, the pi~,and monkey. In the pig the pars convoluta takes up radioactive PAH at a higher rate than the later Ix:,rtions of the proximal nephron. The investigators do not find marked regiomd differences between the rate,,; of PAH uptake of proximal convoluted tubule and the first part of the pars recta in ~he monkey. Thus it seems that species differences exist. The area of renal pharmacology in which this methodology has had the greatest impact is that concerning the mechanisms of action of diuretics m various distal nephron segments. Diuretics inhibit .~dute reabsorption and. secondarily, reduce water r c a b ~ r p t i o n by the nephron. Perhaps t h e most notable of such studies are those by Dr M. B, Burg of the xlational Institutes of Health in which he ~ie~ribes not only the ,miqueness of NaCI transport by the thick a~ending limb but a l ~ the effects of various "loop diuretics' on this transport process. In 1973 t~o laboratories reIx~ed that this tubule -~egment in the rabbit exhibits transport characteristics quite different from those of other nephron ~gments. They showed the segment to be impermeable to water and to transport NaCI vigorously from the lumen to the peritubular surface. This phenomenon of diluting the urine ,~as predicted by studies with other mcthodologies. The surprising feature of their data was that the spontaneous transepithelial voltage w'as lumen positive v, hen identical solutions bathed both sides of the epithelium a. it was concluded that since chloride ion moved against both an electri-
cal and a chemical gradient it must be an active process. Curiously, NaCI rcabsorplion was fl)und I~) t)4."~nsitive to otLabain, a cardiac glycoside known to be a specific inhibitor of active sodium transpo;l. While not unequivocal, Burg acquired evidence that sodium ab.~twption was probably a passive process. Using the Ncrnsl equation he showed that the lumen positive voltage was of sufficient magnitude to explain the sodium gradient dcvelopeti at very slow perfusion rates, It was concluded that these data were consistent with the h)pothesis that sodium rcabsorption could be passive, down the elect rical gradient. Variability in the data and the comparatively high sodium pe rlncability of this tubule segment make it difficult to discriminate between a mechanism in which chloride true,sport is independent of sodium and one ~n which both sodium and chloride are transported. Despite our inability to describe a detailed model of the events at the various membrane surfaces for NaCI absorption by the thick a~'ending limb, a major conclusion arises from the data. Chloride moves against an ch:ctro-che mical gradient during reubsorption. This process appe~rs to be dramatically different from the active proccsses which extrude sodium and chloride from other cells of the body. Dr Burg has suggested that this difference may be an imlx~rtant as[~ct of the therapeutic efficacy of the loop diuretics, Thus, a ¢irug such as fumsemidc- may elicit relatively few side effects bccau~ it acts to inhibit t~ process unique to the thick ascending liml'.. The effect of several diuretics ,m NaCI absorption in -the thick ascending :imb and the associated transepithelial voltage are presented in Table I. Furosemide, mersalyl
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Fig. I. Tubular fluM an d O'to',ol to n ediurn m tif~~oJ PA H .~r rabbit par~ recta and snake distal proximal nt'ph ton segments. ()pen bar~ represent data lot the rabbit and were modi~ed from Tune. Burg and Patlak 4. Solid bars rep. re~entfiJr the snake, from Damzler =.
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(a mercurial diuretic) and cthacryniccysteine all reduce substantially both the rate o f NaCI absorption and the spontaneous lumen positive voltage when placed in the perfu.~lte. The effect of each was readily reversible '° when the drug was removed from the lumen. None of these drugs had any major effects when placed in the bath at concentrations which are effective from the luminal surface. Dr Burg concluded that these diuretics act to inhibit active chloride transport at the lumen surface. MK196 is an experimental diuretic developed by Merck, Sharp and Oohme; its diuretic and natriuretic effects are similar to those of furosemide. As shown in Table I M K I 9 6 reduces NaCI ab,~rption and voltage in this tubule segment. Conversely, amiloride, a diuretic known for its weak natriuretic and potassium "sparing" qualities, has no effect on the thick ascending limb of Henle even at concentrations 5-10 times that o f furosemide. These data are consistent with effects of these drugs in intact animals. Relatively little information is available for the rabbit distal convoluted tubule hi vitro. Unfortunately, the distal convoluted tubule is short in the rabbit (0.5-1.0 ram) and difficult to dissect without cau~,ing visible damage. The spontaneous ~oltage across various sites of the distal convoluted tubule remains controversial to both the micropuneturist and tho~' who ti~: pert'usion in vitro techniques. In our laborator~ we find the early distal tubule ha¢ lumen I~silive voltage which is inhibiled by furosenlide'. O n the other hand, Ihe hire distal tubule has a lumen negative voltage which is sensitive to amiloride. T:,'ansport data presented in Table I1 for the effects of diuretics on Na + and K + transport ::ire consistent with these electrical data. The loop diuretic, f u r o ~ m i d e , reduces sodium transport to about 50% of its ~xmtrol value while having no significant effect on K +
secrt.iOll. Amilorldc reducc,, .,odium abslllption hi about .ill% ill its cllnlrill ~,alue and nearl~ climinate~ K" secrctitm Because ill the regional e[fecls ol the%" diuretics on vollag¢ of the distal tubule to. it seems rea~mable to .,f~'eulate thai fumsemide inhibits Na('l reab,,orption m the early distal tubule and amiloride in the late distal tubule. The apparent heterogeneity o f thin tubule segment nlakes preci~ '.tatcments al~mt the mechanisms of ion Iraw, p~rt difficult The rabbit cortical collecting tubule ha, been shoran t(~ reab,,orb Na" and ~.¢retc K" ~. Both processes arc thoughl to bc active. This segment is sensitile hi anlidiuretic hormone and aldnsteronc, lt~ high electrical resistance and low catitmic I~rmeability are consisteni with its abilit~ t,~ maintain hlrgc ionic gradients. A,, short n in Table III. the K-sparing diuretic amiloridc profl)undly inhibits the abilit~ of this segment to reabm)rb Na" and secrete K ' . In addition, the tran.sepithelial voltage as~t~ciated with active sodium transport is abolished. The specific resistance of the collecting tubule is increased b~ about 30% (from 266 to 358 ohms cm -~) iil the presence of amiloride ~-'°. These data are con-i~tent w'ith a simple model. Reduction of the permeability of the lumen membrane to seaiium b~ amiloride pre~ents the cntr~ of sodium into the cytoplasm of the cell. Thi~ leads to a reduction of the c.~io~olic ,,odium available for transptnt at the pcritubular membrane. In essence, the s~dium pump i~ deprived of substrate. The effects of amiloridc on the active r e a b ~ r p t i o n of sodium in the collecting tubule appear to be limited to the lumen surface since this drug has no effect on NaCI reabsorption ~ h e n phiced on the bhx~l surface :it high concentration,. Data fi~r t he effects of two "loop diuretics" on collecting tubule arc a l ~ presented in Table III. Neither furosemid¢ nor MKIUe, has any significant effect on the tran,,cpithchal 1.-%1tl l: 11 [ { f e e d ~*f d t u r c l l t x o n . o d i u m . i b ~ r p i l , , n .intt p O l . l ~ l u m ~ . t r t . l l O l l . l i ' i ' t l ~ ) f i t r,il~l~lI Jl~l.il < o n t o . luted iubule [ I,In~|%li't ,Is ~> ~ o n l l ~ q x.iiti~'
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~,ollagc t~r lra.q,,r~rl atl thl'. ,,cgmcnl ]he techniqu.: ~q pcrlu,,lon in i ztro ~,i renal lubulc" f l a . ' m r nt, ha', pr-~en to bc , i ' ~ e r l u l t~)l in thc ~t,.ld~ ot the ,*I¢', ,lnd mcchani~m~ o f ict,+n ~t a ht+q ~,1 pharmacological agcnt~. All o! thc,.c ;lgcnl., ahcr Ihc" tran~pt~rt pr,l~.rtic, ,ff lh¢ tubular epithchum. Man~ i~f the,q: agent', .ire kno~n h~ alter other a,t~ct~ of rcn;d function a', ~cll. A,ah~at~. the cffcum,,tance/rr wtro se~Cs to qmplit.~ the e ~ . p r , ' t l ' n c f l t a ! proce,,,. In ~.uch ,t ~a) that ¢%trapolatlon o! the data to intact anlm,d functam mu-t bc done v, ffh care. The need to integrate etfe¢ti~ek ,,uch data in +ttr+. x~flh p~..~inent eM-icrimcntal method,, ~, l i t , , ]-. cxcrprc~'nt.
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determines the health, capacity and lilt:span of man and Ihat this inflow is dual (energy ,'rod structural information), this p h c n o m c m m may be examined in the lisht of N. Bohr's contplcmcntarily principle, developed by him for the corpuscular-~ ave dualism in physics. AI one time physics encountered the problem of elements of set determining the quantum slate of the macroscopic syslem. Flowe~er. for a substance consist mg of I ()= electrons and nuclei, the literal picture could be oblained b} solution of Ihe v,a~e equation with 3 × I (t=s unknm~ n member~. This phenomenological thermodynamics was opposed by Gibbs" statktical thermodynamics, which abstracts ilself from all coml~ments of the system and la:.es account ol the net resull of the ensemble. Thc individual substances of coml~site natural complexes certainly have their specific actions; but however different were the fimdstuffs of different peoples. they achieved the same objective, i.e. the provision of man with eveD thimz needed for life and propagation of the species. The preservation of health and treatment of illnesses is achieved by different sets of herbs in different parts of the world. It is imp, wtam and necessary to dcfine and com-
prehend deepl} the ,peciticll} ol acllon ,4 individual ~,ubst ante,.. This purel} phenomemflogieal approach ha,, great significance, llul it b, praclieally im~s,qbl¢ to ev;duate the imlm~rtanec and c, mtribulion of e ~ e r y sub,,lance. ,,I all p,~ssible eooperali~,e e[fecls. Moreover. indP, idual [acts musl nol "obscure Ihe ~.iev+ ol lhe w ~ d for the tree'.'.Therefore lhe ,4atNt,cal approach dctcrmined b~ the +.iguificance O[ Mructurill information is no less iml~+r[anl. For lhe normal grov.lh and development <+f capacil}, non-ntorbidit,, and finall} hcahh, the organ,sm ol man musl get a definite amount (~olumc. critical mass) of slruclural inlormatitm [rom Nature.
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342-352. Mo.,cov. 4 "~irlcr. N . l l q 6 ~ l ( y b e r n e t i t ~ e ~ r f ' + , ~ l t r + ~ i , t n d B t m d and .~ath#ne. 2nd ed,L PuN
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Larry C. Stoner S t u t e Univer.sity o f N e w Y o r k U p s t a t e . $ h ' d i c a l i e t u e r , l)el,,arttll ' n t ,>l l+h+ sl,,h+q+. " e ~ / r x / r l l , '
Attempts to learn the sites and nlechanisnts of action of a variety of agents in an organ as complex as the kidney arc often flustrated by the limitations of the available techniques. Many drutm appear to have complex effects and multiple sites of action; they may change not only transtx+rt function but also alter such factors as renal blood flow or glomerular filtration rate. Physiologists have long been interested in techniques which permit direct stud}' o[ the transport of solutes and water by specific segments of the nephron. This interest can be satisfied by micropuneture studies of those structures which occur on the surface
h I).+m,,n. •
Reading lisl
Isolated nephron segments as pharmacological tools New York 13210, U.5.A+
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port rate. ot ~atcr and ~an,,u..olut~. In addition It+ gix ing the im, e,,tigator aeee-', to tubular ,egmcm~, + hich ca:mot bc -.tudicd directlx %+ith t,ther technique,,, thl', methodok+g} offer,, other atd'.antagc-. .Xmt,ng tht.,,e are the preel,e and IndeD:ndent control ot the content,, o[ the bathing mcdiurn and perfu,a:te. :,nd the at ailabilit.,, of the tubule lr,lgmelli it-~cIt for analx,,l', ot eel!.llar coml~',itlon. I he,,e adxantage, dl,,~ not onl~ ph},,iologic:d ,,tudJe, but ,ll,,~, ,,t udic,, ,limed ;.tt ¢ l t l C I t L l l i f l ~ t h+~."",lte'~Ol lr:An.+porland Ili~.'ch,tni',m', O[ ;.l,.'tion ol x ariOtlS il~CIltS, some o[ '.%hich ++re knm%n h+r their tberal',,:utt+ salue I he detail- ot 1h¢
of the kidnex. The lack ot techniques t~ stud} the properties of nephron segment, found deep in the renal muss led Burg and t e c h n i q u e ~, f o r p e r t t l , , i n g t r a g n t e l l t ' ~ o [ t h e his eo-x~orkers ill the National In,,titute', o! nlanlnl;.tli;.m nephrtm in +,lro. Including He:IItlP to develop methods to dissect and •,omc o[ the di[ficultic, encountered .rod o | t h e method. l~rfu.~ in vitro various segments of the the major c , o n l r i b u t l o n , , rubhit nephron. [n this technique short hate recently Ix'en :cuexsed a a Here. l limit nlxs¢ll to txso ,treat ot disfragmentst;f the nephron (usually I-2 mm in length) are suspended bctx~een pare, of cusslon: the impact of this methodok+g', +n concentric glass pipets. Fluid of knm~n our understanding O [ t h e t r a n s p o r t o f ,A e d k comlx~sition is forced into the lumen of one organic acids acro,,x proximal nephron end of the tubule and collected for anal) sis ,egments. and the site,, and m e e h a n i M l l s O t in the pipets at the other end. Changes in action of x.:trious diuretics. In Iqb,-a Tune, Burg ;rod Patlak 4 r u b the chemical or radioisotopie contents o[ the perfusate arc umd to as.~s.,, the tr.'ms- lished ~+.hat now must IX."regarded a,, a I t~,+~t %,.lth H,' .rod ,tl:,,m:d+. ,+ Pr,-. , , , ;
T I P S - J a n u a r y 1981
classic study on para-aminohippurate (PAH) transport acro~,s the epithelium of rabbit tubulesin vitro. By utilizing a variety of experimental prott~cols (including analysis of cellular contents), they determined fact¢~rswhich led to a detailed model of the transport process. More recently, a very similar model for the transport of organic acids across proximal tubule fragments from garter snakes has been developed by Dantzler and associates 5, In rabbit and snake the late portions of the prt~ximal nephrons appear to ~crete PAH at much higher rates than the early portions. As depicted by the first two bars of Fig. i. when either the pars recta of the rabb~t or the late proximal tubule of the snake are placed in a bathing medium containing low concentrations (approximately 2 × IO--SM) of radioactive PAH. the concentration of PAH in the pcrfused fluid is substantially higher than that in the medium. This demonstration that net PAH movement fro.. the peritubular to the lumen compartments occurs against a concentration gradient led to the conclusion that PAH secretion is an active process. Because of the very. large concentration difference of PAH between the cytosol and bathing medium (right side. Fig. i) it was po~,tulated that the entry of PAH into the cell across the peritubular membrane must be an active process. Since the conceutration of PAH in the cytosol exceeded that of lumen (center. Fig. I) the organic acid moved to the lumen from the cell down its chemical gradient. Both groups concluded that this mt~vement could be passive diffusion. The observation that the apparent permeability of the lumen membrane appeared t o b~ unaffected by probenecid added to the bathing medium was seen by lrune et aL as further evidence supporting this interpretation. While the major features ol the model stood the test of time, two aspects are not universally accepted. Recent evidence. utilizmt+lumen membrane vesicles isolated from dog kidney cortex, suggests that PAH movement through the luminal membrane w.ay t.e a facilitated process s. Furthermore. Dant~ler and Bentley ~ have shown that ,-hen ,:old PAH. phenol red. probenecid or SITZ (an inhibitor of an ion exchange in red cells) are added to the perfusate of snake tubules, secretion of I"C]PAH is inhibited. They too suggest that the "downhill" movement of PAH into the tubular lumen may not be simple diffusion. Unfortunately comparable information has not been published for other animals. Sh/ili and Roch-Ramel (personal corn-
munication) h;~verecently been able to dissect and stuEy PAH uptake iato nonperfused tubules of two other mz mmalian species, the pi~,and monkey. In the pig the pars convoluta takes up radioactive PAH at a higher rate than the later Ix:,rtions of the proximal nephron. The investigators do not find marked regiomd differences between the rate,,; of PAH uptake of proximal convoluted tubule and the first part of the pars recta in ~he monkey. Thus it seems that species differences exist. The area of renal pharmacology in which this methodology has had the greatest impact is that concerning the mechanisms of action of diuretics m various distal nephron segments. Diuretics inhibit .~dute reabsorption and. secondarily, reduce water r c a b ~ r p t i o n by the nephron. Perhaps t h e most notable of such studies are those by Dr M. B, Burg of the xlational Institutes of Health in which he ~ie~ribes not only the ,miqueness of NaCI transport by the thick a~ending limb but a l ~ the effects of various "loop diuretics' on this transport process. In 1973 t~o laboratories reIx~ed that this tubule -~egment in the rabbit exhibits transport characteristics quite different from those of other nephron ~gments. They showed the segment to be impermeable to water and to transport NaCI vigorously from the lumen to the peritubular surface. This phenomenon of diluting the urine ,~as predicted by studies with other mcthodologies. The surprising feature of their data was that the spontaneous transepithelial voltage w'as lumen positive v, hen identical solutions bathed both sides of the epithelium a. it was concluded that since chloride ion moved against both an electri-
cal and a chemical gradient it must be an active process. Curiously, NaCI rcabsorplion was fl)und I~) t)4."~nsitive to otLabain, a cardiac glycoside known to be a specific inhibitor of active sodium transpo;l. While not unequivocal, Burg acquired evidence that sodium ab.~twption was probably a passive process. Using the Ncrnsl equation he showed that the lumen positive voltage was of sufficient magnitude to explain the sodium gradient dcvelopeti at very slow perfusion rates, It was concluded that these data were consistent with the h)pothesis that sodium rcabsorption could be passive, down the elect rical gradient. Variability in the data and the comparatively high sodium pe rlncability of this tubule segment make it difficult to discriminate between a mechanism in which chloride true,sport is independent of sodium and one ~n which both sodium and chloride are transported. Despite our inability to describe a detailed model of the events at the various membrane surfaces for NaCI absorption by the thick a~'ending limb, a major conclusion arises from the data. Chloride moves against an ch:ctro-che mical gradient during reubsorption. This process appe~rs to be dramatically different from the active proccsses which extrude sodium and chloride from other cells of the body. Dr Burg has suggested that this difference may be an imlx~rtant as[~ct of the therapeutic efficacy of the loop diuretics, Thus, a ¢irug such as fumsemidc- may elicit relatively few side effects bccau~ it acts to inhibit t~ process unique to the thick ascending liml'.. The effect of several diuretics ,m NaCI absorption in -the thick ascending :imb and the associated transepithelial voltage are presented in Table I. Furosemide, mersalyl
25
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Fig. I. Tubular fluM an d O'to',ol to n ediurn m tif~~oJ PA H .~r rabbit par~ recta and snake distal proximal nt'ph ton segments. ()pen bar~ represent data lot the rabbit and were modi~ed from Tune. Burg and Patlak 4. Solid bars rep. re~entfiJr the snake, from Damzler =.
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(a mercurial diuretic) and cthacryniccysteine all reduce substantially both the rate o f NaCI absorption and the spontaneous lumen positive voltage when placed in the perfu.~lte. The effect of each was readily reversible '° when the drug was removed from the lumen. None of these drugs had any major effects when placed in the bath at concentrations which are effective from the luminal surface. Dr Burg concluded that these diuretics act to inhibit active chloride transport at the lumen surface. MK196 is an experimental diuretic developed by Merck, Sharp and Oohme; its diuretic and natriuretic effects are similar to those of furosemide. As shown in Table I M K I 9 6 reduces NaCI ab,~rption and voltage in this tubule segment. Conversely, amiloride, a diuretic known for its weak natriuretic and potassium "sparing" qualities, has no effect on the thick ascending limb of Henle even at concentrations 5-10 times that o f furosemide. These data are consistent with effects of these drugs in intact animals. Relatively little information is available for the rabbit distal convoluted tubule hi vitro. Unfortunately, the distal convoluted tubule is short in the rabbit (0.5-1.0 ram) and difficult to dissect without cau~,ing visible damage. The spontaneous ~oltage across various sites of the distal convoluted tubule remains controversial to both the micropuneturist and tho~' who ti~: pert'usion in vitro techniques. In our laborator~ we find the early distal tubule ha¢ lumen I~silive voltage which is inhibiled by furosenlide'. O n the other hand, Ihe hire distal tubule has a lumen negative voltage which is sensitive to amiloride. T:,'ansport data presented in Table I1 for the effects of diuretics on Na + and K + transport ::ire consistent with these electrical data. The loop diuretic, f u r o ~ m i d e , reduces sodium transport to about 50% of its ~xmtrol value while having no significant effect on K +
secrt.iOll. Amilorldc reducc,, .,odium abslllption hi about .ill% ill its cllnlrill ~,alue and nearl~ climinate~ K" secrctitm Because ill the regional e[fecls ol the%" diuretics on vollag¢ of the distal tubule to. it seems rea~mable to .,f~'eulate thai fumsemide inhibits Na('l reab,,orption m the early distal tubule and amiloride in the late distal tubule. The apparent heterogeneity o f thin tubule segment nlakes preci~ '.tatcments al~mt the mechanisms of ion Iraw, p~rt difficult The rabbit cortical collecting tubule ha, been shoran t(~ reab,,orb Na" and ~.¢retc K" ~. Both processes arc thoughl to bc active. This segment is sensitile hi anlidiuretic hormone and aldnsteronc, lt~ high electrical resistance and low catitmic I~rmeability are consisteni with its abilit~ t,~ maintain hlrgc ionic gradients. A,, short n in Table III. the K-sparing diuretic amiloridc profl)undly inhibits the abilit~ of this segment to reabm)rb Na" and secrete K ' . In addition, the tran.sepithelial voltage as~t~ciated with active sodium transport is abolished. The specific resistance of the collecting tubule is increased b~ about 30% (from 266 to 358 ohms cm -~) iil the presence of amiloride ~-'°. These data are con-i~tent w'ith a simple model. Reduction of the permeability of the lumen membrane to seaiium b~ amiloride pre~ents the cntr~ of sodium into the cytoplasm of the cell. Thi~ leads to a reduction of the c.~io~olic ,,odium available for transptnt at the pcritubular membrane. In essence, the s~dium pump i~ deprived of substrate. The effects of amiloridc on the active r e a b ~ r p t i o n of sodium in the collecting tubule appear to be limited to the lumen surface since this drug has no effect on NaCI reabsorption ~ h e n phiced on the bhx~l surface :it high concentration,. Data fi~r t he effects of two "loop diuretics" on collecting tubule arc a l ~ presented in Table III. Neither furosemid¢ nor MKIUe, has any significant effect on the tran,,cpithchal 1.-%1tl l: 11 [ { f e e d ~*f d t u r c l l t x o n . o d i u m . i b ~ r p i l , , n .intt p O l . l ~ l u m ~ . t r t . l l O l l . l i ' i ' t l ~ ) f i t r,il~l~lI Jl~l.il < o n t o . luted iubule [ I,In~|%li't ,Is ~> ~ o n l l ~ q x.iiti~'
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~,ollagc t~r lra.q,,r~rl atl thl'. ,,cgmcnl ]he techniqu.: ~q pcrlu,,lon in i ztro ~,i renal lubulc" f l a . ' m r nt, ha', pr-~en to bc , i ' ~ e r l u l t~)l in thc ~t,.ld~ ot the ,*I¢', ,lnd mcchani~m~ o f ict,+n ~t a ht+q ~,1 pharmacological agcnt~. All o! thc,.c ;lgcnl., ahcr Ihc" tran~pt~rt pr,l~.rtic, ,ff lh¢ tubular epithchum. Man~ i~f the,q: agent', .ire kno~n h~ alter other a,t~ct~ of rcn;d function a', ~cll. A,ah~at~. the cffcum,,tance/rr wtro se~Cs to qmplit.~ the e ~ . p r , ' t l ' n c f l t a ! proce,,,. In ~.uch ,t ~a) that ¢%trapolatlon o! the data to intact anlm,d functam mu-t bc done v, ffh care. The need to integrate etfe¢ti~ek ,,uch data in +ttr+. x~flh p~..~inent eM-icrimcntal method,, ~, l i t , , ]-. cxcrprc~'nt.
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