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Function of Distal Convoluted and Connecting Tubules Studied by Isolated Nephron Fragments MASASHI IMA! and Ryuichi NAKAMURA

Function of Distal Convoluted and Connecting Tubules Studied by Isolated Nephron Fragments MASASHI IMA! and Ryuichi NAKAMURA

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Kidney International, Vol. 22 (1982), pp. 465—4 72

Function of distal convoluted and connecting studied by isolated fragments MASASHI IMA! and RYuIcHI NAKAMURA

Department of Pharmacology, Jichi Medical School, Tochigi, Japan

Conventional micropuncture studies designate the distal tu-are packed with numerous elongated microvilli distributed bules as that portion of the distal nephron extending from thealong the basal infolding [2, 3, 7] (Fig. 1). to the first branch point of the collecting . The CNT segment appears granular in its position between Recent studies [1—4] define considerable epithelial heterogene-DCT and CCT. The transition from true DCT to CNT is also ity within this portion of the nephron, including discrete mor-abrupt (Fig. 2). Despite the early recognition of a distinct CNT phologic characteristics of: (1) cortical thick ascending limb ofsegment [8], functional distinction awaited the intranephron Henle (CAL); (2) true (DCT); (3)localization by Morel, Chabardès, and Imbert of connecting tubule (CNT); and (4) cortical collecting tubulesensitive adenylate cyclase activity [11. (CCT). These segments are heterogeneous in function as well as Morphologic studies [2, 3, 7] have demonstrated the presence morphology. of two cell types within this segment, connecting tubule cells Perfusion of isolated renal tubules, as developed by Burg et aland intercalated (dark) cells. The latter cell type is also found in [5], allows characterization of the functional heterogeneity ofCT (Fig. 1) and is further subdivided into black and gray cells nephron segments. Detailed information regarding the specific[2, 3]. Connecting tubules draining long looped (deep) properties of ion transport in these distal nephron segments isare arranged in arcades as several connecting tubules fuse. Both fragmented because isolation of these short segments is diffi-the granular appearance and confluent arcades aid in CNT cult. While a comprehensive review of the transport propertiesidentification during dissection. of the DCT and the CNT is impossible, this study addresses the Embryological studies in human [9] suggest that the responses of these segments to stimulation by several hor-CNT originates from nephrogenic blastema, differing from the mones. Where available, relevant anatomical and biochemicalcollecting tubule which develops from the ureteral bud. Howev- information is included. er, as cell type intermingling exists between CNT and CCT [2, Anatomical aspects. Practical requirements of interpuncture3, 7, 10] as described, the CNT should be regarded as an standardization have prompted micropuncturists to designateintermediate segment. distal tubule puncture sites with reference to the macula densa, Recent morphometric studies [11—14] suggest the existence of located at the vascular pole of the , and the initialfunctional heterogeneity among these cell types. Wade et al [II] branch point of the collecting tubule. In locating these sites andfound an increase in basolateral membrane surface area limited expressing puncture sites with reference to these landmarks,to the principal cells of rabbit cortical collecting tubule, follow- distal convoluted tubule function has been described; however,ing chronic DOCA treatment. In -adapted rats, Raste- as represented in Figure 1, this definition does not represent agar et al [12] found similar changes in only principal cells of rat homogenous epithelium. medullary collecting tubule. Stanton et al [13] further demon- The tubule segment including and immediately distal to thestrated in potassium-adapted rats that basolateral membrane macula densa was thought previously to include epithelium ofsurface area was increased in the connecting tubule cell type, as distal convoluted tubule, with isolated macula densa specializa-well as in principal cells of CCT. In the aggregate, these findings tion. Morel, Chabardès, and Imbert [1] demonstrated, in rabbitsuggest that both connecting tubule cell and principal cell renal tubules isolated from collagenase-treated kidneys, thatmediate potassium in the distal nephron. CAL extends beyond macula densa. Precise morphological In contrast, Stetson, Wade, and Giebisch [14] showed that studies [6, 7] in rat kidney confirm this finding. Abrupt transi-potassium depletion in the rat was accompanied by both an tion from the CAL to true distal convoluted tubule (DCT)increase in luminal membrane surface area of intercalated cells occurs at a site approximately 0.1 to 0.2 mm beyond the maculaand increased density of rod-shaped particles in this membrane, densa. True DCT in the rabbit nephron is quite short, averagingdemonstrated by the freeze-fracture technique. These changes only 0.5 mm, with the longest segments less than 0.8 mm [1, 4].were limited to the intercalated cell, suggesting the possibility Although DCT isolated from collagenase-treated rabbit kidneythat the intercalated cell is responsible for increased potassium appears bright under a phase contrast microscope (Fig. 2), this is not always the case for untreated nephrons. A single cell type comprises this segment, characterized by deeply invaginated Received for publication June 15, 1982 basolateral membrane interdigitated with neighboring cells. The 0085—2538/82/0022—0465 $01.60 luminal surface is covered with short, blunt microvilli and cells © 1982 by the International Society of

465 466 Imai and Nakamura

DCT CCT activity was observed in proximal convoluted tubules during chronic acidosis. This study suggests that the site of enhanced ammonia production in response to acidosis is not the distal convoluted tubule. The origin of urinary kallikrein from distal nephron was first suggested by a stop-flow study in the dog [301. Histochemical studies [31, 32] also suggest distal tubule localization of kalli- Distal tubule cell krein. Tomita, Endow, and Sakai [331 found in rabbit isolated renal tubules that the aprotinin inhibitable kallikrein-like activi- ty was demonstrable exclusively in CNT. Proud, Knepper, and Pisano [34] recently used direct radioimmunoassay to quantify kallikrein location in distinct tubular segments. They found the highest kallikrein content in the connecting tubule, with signifi- Connecting tubule cell cant content also present in DCT and CCT segments. Further, Carone et al [35] have shown that labelled bradykinin injected into the was hydrolyzed almost completely, while labelled peptide added in the distal tubule appeared almost completely intact in the . Another stop-flow study by Scicli, Gandolfi, and Carretero [361 supports the impression Intercalated cell that kinins are formed in the distal nephron. The exact role, however, of kinins in regulation of renal flow and transport of salt and awaits further definition. Recognition that the kidney is the major target organ of action has long antedated recognition of precise nephron distribution of aldosterone receptors. Recent work by Farman, Vandewalle, and Bonvalet [371 using autoradiographic Principal cell techniques to define specificity and distribution of rabbit kidney Fig. 1.Schematicillustration of the distal nephron segments. aldosterone binding demonstrated high affinity aldosterone binding in DCT, CNT, and CCT; however, dexamethasone competition for binding sites was considerable in DCT and CNT. Doucet and Katz [38] characterized high affinity aldoster- in potassium depletion states. While some investi-one specific binding found exclusively in cortical and outer gators have reported an increase in the intercalated cell numbermedullary collecting tubule, suggesting these segments are in potassium depletion [15, 16], a recent study by Hansen,target sites for mineralocorticoid action in the rabbit kidney. Tisher, and Robinson [17] demonstrated no change in the Chronic administration of mineralocorticoids was shown to intercalated cell number accompanying conditions of variedincrease Na-K ATPase activity in the CNT and CCT of rabbit renal tubular potassium or hydrogen ion transport. kidney [28]. Recent work by Petty, Kokko, and Marver [39] Biochemical aspects. The kidney's great capacity for glucosedelineated the acute (3-hr) stimulation of rabbit CCT Na-K generation is well recognized [18]. However, intrarenal utiliza-ATPase postadrenalectomy as owing to a specific effect of tion of limits net glucose production considerably. Justmineralocorticoids and not to glucocorticoid effects. Work by as proximal tubules play the major role in intrarenal gluconeo-Doucet and Katz [401 previously failed in mice to demonstrate genesis [19—21], distal nephron segments are apparently majorDCT and CCT Na-K ATPase stimulation following acute in sites of glucose utilization. In support of this notion, enzymesvivo and in vitro aldosterone treatment. Recent data from responsible for glycolysis are predominantly distributed inMernissi and Doucet [411 confirm the acute effect of in vivo distal nephron segments [22—25]. It is, however, unclear wheth-aldosterone treatment in stimulating Na-K ATPase in their er or not glucose utilization is related directly to the transportstudies of CCT and outer medullary collecting duct segments. It capacity of the distal nephron. remains to be determined whether true physiologic interspecies Na-K ATPase plays a crucial role in transepithelial sodiumdifferences account for the above discrepancy or anatomical transport, as well as in maintenance of intracellular ion compo-variations are being reflected. sition. Through methods developed to measure Na-K ATPase The distal nephron is also the site of in distinct nephron segments [26—281, the DCT has been demon-(PTH) and action, although the discrete sites of strated to have the highest activity of this enzyme [27—28],action differ. Calcitonin stimulates adenylate cyclase activity in suggesting a high capacity for sodium transport in this segment.the DCT [42, 43], whereas PTH increases the enzyme activity in Phosphate-dependent glutaminase (PDG) is important in am-the CNT [1, 43, 44]. sensitive adenylate cyclase moniagenesis through its deamination of glutamine. Curthoysactivity was also demonstrated in the CNT as well as in the and Lowry [29] examined nephron segment distribution of PDGCCT [1, 451. Arginine vasopressin is more potent in CCT than in activity in rat kidney under various conditions including chronicCNT in stimulating adenylate cyclase activity [1, 45]. acidosis and alkalosis. Although the activity of PDG was Isoproterenol sensitive adenylate cyclase was also demon- greater in distal convoluted tubules than in proximal tubulesstrated in CNT and CCT. In contrast to the findings with under control conditions, a marked stimulation of enzymevasopressin, isoproterenol is more potent in CNT than in CCT Distal tubules 467

Fig. 2. Light microscopic appearance of the distal convoluted tubule (DCT, upper panel) and the connecting tubule (CNT, lower panel). The arrow indicates the transition from the DCT to the CNT.

[1, 461. The stimulation of adenylate cyclase with isoproterenol Physiological aspects in these nephron segments could be abolished with propranolol [46], implicating beta adrenergic receptors in this effect. Using Transepithelial voltage. Wright [521 first demonstrated that [3H]-dihydroalpranolol (DHA) in binding experiments with iso-the magnitude of the transepithelial voltage across the distal lated nephron fragments, Nakamura and Imai [47] looked atconvoluted tubule of rat increases from about —8 to —45 mV "specific" beta adrenergic receptor binding as defined by DHAbetween early and late distal tubule. This was confirmed by displacement with i04 M (±) propranolol. Binding distributionobservations from other laboratories [53—55]. DeBermudez and throughout DCT, CNT, CCT, and the outer medullary collect-Windhager [54] demonstrated that electrical resistance also ing tubule (Fig. 3) differed from the adenylate cyclase stimula-changes as a function of the length of the distal convoluted tion data in the inclusion of DCT. Specifically, CCT DHAtubule. They suggested that the changes in electrical resistance binding was characterized by low affinity (Kd 4.3 X ioM)andare caused by changes in osmolality of the luminal fluid and are high capacity (maximum binding 34.3 fmoles/cm). However,responsible for changes in the transepithelial voltage along the DHA displacement by (+) propranolol, a stereoisomer withoutdistal convoluted tubule. affinity for beta adrenergic receptors, as well as by other beta Although it is accepted generally that late or randomly blockers with membrane stabilizing action, suggests that mem-punctured distal convoluted tubules give negative poten- brane stabilizing effects of certain beta adrenergic antagoniststials ranging about —40 to —60 mY [56], the orientation of the affect ligand binding in tubule segments. Although high affinityvoltage in the early distal tubule is controversial [55—57]. Using beta adrenergic binding sites have been demonstrated in mem-electrodes of large diameter (3 to 5 sm), Baratt et al [571 found brane preparations from whole kidneys [48—511, discriminatingan average voltage of +3.7 mY in early distal convoluted detection of beta adrenergic receptors in single nephron seg-tubules of Munich-Wistar rats. They claimed that the use of ments must await development of more sensitive methods. Ling-Gerard microelectrodes introduces an artifact. Hayslett et 468 Imaiand Nakamura

tested the hypothesis that changes in osmolality of perfusate may modulate the transepithelial voltage. The CNT may correspond to the late distal convoluted tubule defined by micropuncture studies. The transepithelial voltage of the rabbit CNT was found to be about —30 mV, a value comparable to that observed in the DCT in the same series of Total binding experiments [4]. Thus, intrinsic differences in transepithelial C Nonspecific binding voltage could not be demonstrated between the DCT and CNT. In both the DCT [59] and the CNT [4], the transepithelial 0.4 P<0.01 voltage was shown to be dependent on either hydrostatic pressure gradient or perfusion rate or both. When the height of 2 * * 4 the reservoir was lowered under 10 cm H20, the transepithelial I voltage increased sharply. In the CNT it was observed that 0.2 obstruction of tubular outflow under constant perfusion pres- I sure caused a gradual increase in the voltage, suggesting that the decrease in flow rate is also responsible for the increase in voltage when perfusion pressure was reduced [4]. The lumen negative voltage in both the DCT and CNT is Glom irIIPCT PST CAL DCT CNT CCT MCT inhibitable with l0 M ouabain added to the bath [4, 59], N 6) (U) (6) (6) (6) (6) (9) (6) supporting the role of Na-K ATPase at the basolateral mem- Fig. 3. The ncphron distribution of[3H1-dihydroalprenolol (DHA) in the brane in generating the transepithelial voltage. This view is rabbit. Renal tubular fragments isolated from collagenase-treated rabbit compatible with the observations that Na-K ATPase activity is kidneys were incubated at 37°C for 15 mm with 10—8 moles/liter [°H]- high in these segments [26—28]. sharply decreased DHA in the presence (nonspecific binding) or absence of iO moles! the transepithelial voltage of the DCT [59] when it was added to liter of (±)-propranolol (total binding). the perfusate, suggesting that an amiloride sensitive sodium channel exists in the luminal membrane of this segment as well as in the CCT. Transport of sodium and potassium. Shareghi and Stoner [60] a! [55],onthe other hand, found lumen negative voltage in themeasured net transport rates of sodium and potassium in small early distal convoluted tubules of Sprague-Dawley rats andnumbers of DCT and CNT segments. In five DCT experiments Munich Wistar rats when simultaneous measurements were(portions of CNT may have been included) they found an made with both macro- and Ling-Gerard microelectrodes. Theyaverage net sodium absorption of 82.3 and net potassium suggested that macroelectrode measurements are confoundedsecretion of 7.0 pEq mm' min'. In three CNT segments, the by an important artifact caused by rapid inflow or outflow ofmean net sodium absorption was 62.2 and mean net potassium fluids from the tip of pipettes. More recently, Allen and Barrattsecretion 7.0 pEq mm' min. Summarizing other data in light [58]confirmedthe earlier observation of Barratt et a! [571 andof these, the net sodium absorption in the DCT [601 is about suggested that macroelectrodes cause lumen negative diffusiontwice that in CCT [61], and net potassium is about potential by an artifact due to leakage of concentrated potassi-threefold greater in DCT than CCT. In recent work, Lombard, um chloride from the electrode into the . At thisKokko, and Jacobson [62] reported comparable DCT sodium time, it is difficult to decide which observations are correct. reabsorption (107.8 pEq mm' min) but higher DCT potassi- The in vitro perfusion of isolated renal tubules might beum secretion (51.1 pEq mm' min). expected to provide a useful tool to solve these problems. Effect of mineralocorticoids. It has been confirmed by sever- However, discrepant results have also been reported on theal groups of investigators [4, 59, 63—65] that the transepithelial transepithelial voltage of the rabbit DCT perfused in vitro [4,voltage of the CCT is dependent on mineralocorticoids. The 59, 60]. Gross, Imai, and Kokko [59]foundthe lumen negativemagnitude of the negative transepithelial voltage of the CCT voltage of about —40 mV in the rabbit DCT when the tubulesobtained from rabbits pretreated with deoxycorticosterone ace- were perfused with a solution of identical composition to that oftate (DOCA) is greater than that obtained from untreated the bathing medium. Although a subsequent report of Imai [41rabbits [4, 59, 63—65]. On the other hand, both the DCT and confirmed this observation, Shareghi and Stoner [60] reportedCNT consistently show lumen negative voltage of about 30 to that the lumen positive potential was observed in the early—40 mV irrespective of presence or absence of pretreatment DCT, with the lumen negative voltage in the late DCT. Thewith DOCA [4, 591. Gross and Kokko [63] reported that the reason for this discrepancy is unknown at this time. Difficulty intransepithelial voltage of the DCT obtained from adrenalecto- identification of the early part of the DCT might be responsiblemized rabbits was identical to that obtained from DOCA- for part of this discrepancy. Although in earlier studies [4, 59]treated ones, whereas the average transepithelial voltage of the the DCT was obtained from the portion of the renal tubuleCCT obtained from adrenalectomized animals was +1.6 mV. distributed close to the glomeruli, the transition from theFurthermore, addition of 2 x 10 M aldosterone to the cortical thick ascending limb of Henle's loop was not alwaysperfusate generated a lumen negative voltage in the CCT but did confirmed. It is possible that the presence of the cortical thicknot affect the voltage of the DCT. However, it is perplexing that ascending limb of Henle's loop in the preparation of early DCTa higher concentration of aldosterone (10 M) was required to could cause lumen positive voltage. None of these studiesobtain the effect on the voltage of CCT when the hormone was Distal tubules 469

ECT, 0.1 pg/mI cause significant change in transepithelial voltage or net trans- port rates of sodium and potassium in CNT. Imai [66] also confirmed that PTH had no effect on the voltage of the CNT. Thus it appears unlikely that PTH mediates natriuresis through its action on CNT. Effect of vasopressin. Osmotic water permeability of the 0 DCT and CNT was shown to be very low [4, 591 and was unaffected by arginine vasopressin (AVP). In the CNT, addition a, of AVP to the bath caused a transient increase and a subsequent 0 decrease in lumen negative voltage [4]. Analysis of the dose- response relationship for suppression of voltage revealed that F AVP is more potent in the CCT than in the CNT. This is compatible with the order of adenylate cyclase responses of these segments to AVP [45]. Recently Holt and Lechene [67] provided evidence that endogeneous are responsible for the suppression C E R C E R C E R of lumen negative voltage of the CCT by AVP. It is unknown DCT CNT CCT whether or not prostaglandins also mediate the effect of AVP in Fig. 4. Effect of synthetic eel calcitonin on transepithelial voltage of the the CNT. rabbit distal nephron segments. Abbreviations: DCT, distal convoluted Effect of isoproterenol. Isoproterenol was shown to decrease tubule; CNT, connecting tubule; CCT, cortical collecting tubule. the transepithelial voltage of the CNT and CCT, but not of the DCT [4]. Analysis of the dose-response relationship showed that isoproterenol is more potent in the CNT than in the CCT. added to the bath. The affect of or inThis is again in keeping with the order of adenylate cyclase the bath to block the voltage effect of aldosterone suggestsresponses of these segments to isoproterenol [461. Recently, aldosterone acts via the basolateral membrane. lino, Troy and Brenner [68] also confirmed that isoproterenol Although the transepithelial voltage of the CNT was alsodecreases the lumen negative voltage of the CCT. They showed independent of DOCA pretreatment, this dose does not excludethat the effect of isoproterenol was inhibitable with (—)-pro- CNT as a target segment of aldosterone action. These studiespranolol but not with phenoxybenzamine, indicating that the do not exclude the possibility that aldosterone modulatesstimulation of adrenergic beta-receptors is responsible for this electrically silent sodium transport. However, the binding andeffect. They further showed that administration of both isopro- ATPase data cited earlier [38, 391 speak against CNT targetterenol and ouabain induced lumen positive voltage and sug- primacy. More precise studies are necessary to draw finalgested that isoproterenol stimulates electrogenic chloride trans- conclusions. port across the CCT. Effect of calcitonin. Although the highest activity of calcito- Effect of beta-adrenergic blocking agents. In an attempt to nm sensitive adenylate cyclase was observed in the rabbit DCTinhibit the isoproterenol effect with (± )-propranolol, we found [42, 431, physiological significance for this response remains tothat (±)-propranolol, by itself, sharply suppressed the lumen be determined. To examine this issue, we observed the effect ofnegative voltage of the CNT and CCT [69, 70] at concentrations a synthetic analog of eel calcitonin (ASU1'7-ECT, Toyo Jyozoranging from l0— to i03 M. (+)-Propranolol, which has little Tokyo, Japan) on the transepithelial voltage of the distalor no affinity for beta-adrenergic receptors, also has a similar nephron segments including the DCT, CNT, and CCT (unpub-effect. To examine the possibility that the membrane stabilizing lished observation). ASU"7-ECT was selected because it isaction common to both agents participates in the voltage more stable than natural eel or salmon calcitonin. It wassuppressing effect, we tested various beta-blocking agents with confirmed that ASU"7-ECT stimulates adenylate cyclase activ-or without membrane stabilizing action. As summarized in ity in the CNT and the thick ascending limb as salmon calcito-Table 1, it is apparent that the voltage suppressing effect of beta nm does [431. Preliminary results are summarized in Figure 4.adrenergic blocking agents is related closely to their membrane In the DCT, 0.1 ig/ml (0.1 M.R.C. U/mI) ASU"7-ECT caused astabilizing action. These observations are in good agreement sharp decrease in lumen negative voltage from —12.33.0 towith the results of the [3H]-DHA binding study previously —4.7 1.5 when it was added to the bath. By contrast, ASU"7-mentioned [47]. Since such an effect is not found in the proximal ECT did not affect voltage of the CNT and the CCT. Thesetubule and the cortical thick ascending limb of Henle's loop, the observations strongly suggest that the DCT is the site at whichresponsive sites seem to be localized to the distal nephron calcitonin exerts its natriuretic effect. It is unknown whether orsegments, suggesting that physicochemical properties of the not changes in the voltage are responsible for the anticalciuricbasolateral membrane in the distal nephron segments are dis- effect of calcitonin. tinct from those in the more proximal segments. Since iO- M Effect of parathyroid hormone. The CNT is the major site of(±)-propranolol also inhibited sodium effiux in the CCT (unpub- action of parathyroid hormone (PTH) with respect to adenylatelished observation), direct tubular action of propranolol may, at cyclase stimulation [1, 43, 44]. It has been confirmed that PTHleast in part, be responsible for natriuresis caused by a larger stimulates absorption in this segment [60, 66]. Thisdose of this agent. However, the mechanism by which mem- topic will be discussed in greater depth elsewhere in thisbrane stabilizing action modulates sodium transport remains symposium. Shareghi and Stoner [60] reported that PTH did notundefined. 470 Imaiand Nakainura

Table1.Effects of various beta-adrenergic blocking agents on [3H1-DHA binding and transepithelial voltage of the rabbit cortical collecting tubule (±)-PPL (+)-PPL ATL BNT PND STL

Membrane stabilizing action (++) (++) (-) (+) (4-) (—) Receptor selectivity 131,132 () 13i 131,132 13,132 13,132 inhibition of [3H]-DHA binding5 (++) (++) (-) Inhibition of voltagec (++) (++) (—) (+) (+) (—) Abbreviations: PPL, propranolol; ATL, altenolol; BNT, bunitrolol; PND, pindrol; STL, sotalol; DHA, dihydroalprenolol. aSymbols:(++) strong action, (+) moderate to weak action, (—)noaction. bSymbols:(+-4-) inhibition more than 80%, (+) inhibition 50 to 70%, (—)inhibition less than 20%. Data are based on studies of Nakamura and Imai [43]. Symbols: (+ +) 50%inhibitionat 3 x 10 5_3 Xl0moles/liter, (+) 50%inhibitionat 3 x iO—lO moles/liter, (—)noinhibition at 10 moles/ liter. (Data are based on studies of Imai [69].)

Summary and conclusion. Functions of the DCT and the 3. KRIZ W,KAI5SLING B,PSZALLAM:Morphological characteriza- CNT were reviewed briefly with respect to anatomical, bio- tion of the cells in Henle's loop and the distal tubule, in New Aspects of Renal Function, edited by VOGEL HG, Ui I RICH KJ. chemical, and physiological aspects. Although anatomical and Amsterdam, Excerpta Medica, 1978, pp. 67—78 biochemical aspects may be beyond the scope of this sympo- 4.IMAIM: The connecting tubule: A functional subdivision of the sium, they are mentioned briefly to provide information rele- rabbit distal nephron segments. Kidney mt 15:346—356, 1979 vant to understanding the physiological aspects. It is clear that 5.BURG M, GRANTHAM J, ABRAMOW M, ORLOFF J: Preparation and the DCT and the CNT, at least in rabbit kidneys, are distinct study of fragments of single rabbit nephron. Am J Physiol 210:1293— segments with respect to these three features. The functional 1298,1966 6. KAISLINGB,PETER5,KRIZW: The transition of the thick distinctions are especially evident when hormonal responses ascendinglimb of Henle's loop into thedistal convoluted tubule in are considered. The DCT is responsive to calcitonin, whereas the nephron of the rat kidney. ('eli Tissue Res182:111—118, 1977 the CNT responds to PTH, AVP, and isoproterenol. The 7.CRAYEN M, THOENES W: Architektur und cytologische Charakteri- multiplicity of the responses of the CNT may reflect the sierung des distalen Tubulus der Rattenniere. FortschrZool 23:270—288, 1975 anatomical multiplicity. Although it is possible that different 8.PETER K: Untersuchungen uber den Bau der Niere. Jena, G. types of cells may be effecting different functions, definite Fischer, 1909 evidence for such intrasegmental heterogeneity is still lacking. 9. OsATHANDONH V, POTTER EL: Development of human kidney by There are a few common characteristics between the DCT microdissection. III. Formation and interrelationship of collecting tubules and nephrons. Arch Pathol Lab Med 756:290—302, 1963 and the CNT. Both segments are impermeable to water and 10. YOUNG D, WIs5IG SL: A histologic description of certain epithelial probably have high capacity for active sodium transport. The and vascular structure in the kidney of the normal rat. Am J Anat basolateral membrane of both segments may have a common 115:43—70, 1964 physicochemical property allowing specific binding of agents 11. WADE JB, O'NEIL RG, PRYOR JL, BOULPAEP EL: Modulation of with membrane stabilizing action. area in renal collecting tubules by . J Cell Biol 81:439—445,1979 The rabbit is thus far the only mammalian species in which 12.RASTEGAR A, BIEMESDERFER D, KASUGARIAN M,HAYSLETTJP: we can examine the function of the DCT and CNT by the in Changes in membrane surfaces of collecting duct cells in potassium vitro microperlusion technique. The possibility of interspecies adaptation. Kidney mt 18:293—301, 1980 differences must be taken into consideration before generalizing 13. STANTON BA, BIEMESDERFER D, WADE JB, GIEBI5cH G: Structur- al and functional study of the rat distal nephron: Effects of the findings from the rabbit nephron. potassium adaptation and depletion. Kidney mt 19:36—48, 1981 As mentioned in the introduction, knowledge of the DCT and 14. STETSON D, WADE JB, GIEBISCH G: Morphological alterations in the CNT is still fragmentary. Many important questions remain rat medullary collecting duct following potassium depletion. Kidney unanswered. mt 17:45—56,1980 15.OLIVERJ,MACDOWELLM,WELTLG, HOLLIDAY MA,HOLLAND- Acknowledgments ER W JR, WINTERS RW, WILLIAMS TF, SEGAR WE: The renal legions of electrolyte imbalance. I. The structural alterations in A portion of the work cited in this review has been supported by a grant-in-aid for scientific research from the Japanese Ministry of potassium-depletedrats. J Exp Med 106:563—574,1957 Education, Science, and Culture. We thank the Toyo Jyozo Co. for 16.RICHET G, HAGEGE J: Dark cells of the distal convoluted tubules and collecting ducts. II. Physiological significance. FortschrZool supplying synthetic eel calcitonin (ASU1 '7-eel calcitonin). 23:229—306. 1975 17.HANSEN GP, TISHER CC, ROBINSON RR: Responseof collecting Reprint requests to Dr. M. Imai, Department of Pharmacology, Jichi ductsto disturbance of acid-base and potassium balance. Kidney Medical School, Minamikawachi, Tochigi, 329—04 Japan mt 17:326—337, 1980 18. COHEN JJ, KAMMDE:Renal metabolism: Relation to renal func- References tion,in The Kidney, (2nd ed), edited byBRENNER BM, RECTOR FC I. MOREL F, CHABARDES D, IMBERT M: Functional segmentation of JR. Philadelphia, W. B. Saunders, 1981, pp. 126—214 the rabbit distal tubule by microdetermination of hormone-depen- 19. GUDER WG, SCHMIDT U:Thelocalization of gluconeogenesis in rat dent adenylate cyclase activity. 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