Response of the Distal Tubule and Cortical Collecting Duct to Vasopressin in the Rat

Philip B. Woodhall, C. Craig Tisher

J Clin Invest. 1973;52(12):3095-3108. https://doi.org/10.1172/JCI107509.

Research Article

Renal micropuncture observations in the rat suggest that the entire “distal tubule” (defined by the micropuncturist as that portion of the renal tubule extending between the macula densa and its first junction with another (renal tubule) may be responsive to vasopressin. However, this portion of the renal tubule contains two segments that are morphologically dissimilar. The “early” distal tubule is lined by epithelium characteristic of the distal convoluted tubule, while the “late” distal tubule is lined by epithelium characteristic of the cortical collecting duct. Thus, the present study was initiated to identify the most proximal site of action of vasopressin in the distal renal tubule. A water diuresis was established in rats with hereditary hypothalamic diabetes insipidus. In one-half of the animals the diuresis was interupted by an i.v. infusion of exogenous vasopressin. Morphological preservation of the kidneys was initiated after induction of vasopressin-induced antidiuresis or during maximum water diuresis. Cell swelling and dilatation of intercellular spaces, morphological findings indicative of vasopressin responsiveness, were observed in the cortical collecting duct including the late segment of the distal tubule, a segment that has also been described by morphologists as the initial collecting tubule. Morphological evidence of vasopressin-responsiveness was not observed in the early distal tubule (distal convoluted tubule). Additional morphological studies in Wistar, Long-Evans, and Sprague-Dawley rats demonstrated a marked difference […]

Find the latest version: https://jci.me/107509/pdf Response of the Distal Tubule and Cortical Collecting Duct to Vasopressin in the Rat

PHILIP B. WOODHALL and C. CRAIG TISHER From the Division of Nephrology, Department of Medicine and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

A B S T R A C T Renal micropuncture observations in the late distal tubule (initial collecting tubule) and that rat suggest that the entire "distal tubule" (defined by the vasopressin-induced osmotic equilibration is a function micropuncturist as that portion of the renal tubule ex- of the latter segment alone. The findings emphasize the tending between the macula densa and its first junction importance of morphological characterization of those with another (renal tubule) may be responsive to vaso- segments of the renal tubule that are subjected to physio- pressin. However, this portion of the renal tubule contains logical investigation. two segments that are morphologically dissimilar. The "early" distal tubule is lined by epithelium characteristic of the distal convoluted tubule, while the "late" distal INTRODUCTION tubule is lined by epithelium characteristic of the corti- The distal tubule of the rat kidney, as defined by the cal collecting duct. Thus, the present study was initiated morphologist, is composed of three segments that are to identify the most proximal site of action of vasopressin histologically distinct. These include the ascending thick in the distal renal tubule. A water diuresis was estab- limb of Henle (pars recta), the macula densa (pars lished in rats with hereditary hypothalamic diabetes in- maculata), and the distal convoluted tubule (pars coni- sipidus. In one-half of the animals the diuresis was in- voluta) (1, 2). The remaining portion of the renal terupted by an i.v. infusion of exogenous vasopressin. tubule, the collecting duct, is lined by epithelium morpho- Morphological preservation of the kidneys was initiated logically distinct from that lining the convoluted portion after induction of vasopressin-induced antidiuresis or of the distal tubule (1, 2). The first portion of the cor- during maximum water diuresis. Cell swelling and tical collecting duct has been variously termed the con- dilatation of intercellular spaces, morphological findings necting portion (2) or the initial collecting tubule (3). indicative of vasopressin responsiveness, were observed The latter term will be used in this paper. In most mam- in the cortical collecting duct including the late segment malian kidneys the transition from the distal convoluted of the distal tubule, a segment that has also been de- tubule to the cortical collecting duct (i.e., the initial scribed by morphologists as the initial collecting tubule. collecting tubule) occurs well before the first junction of Mbrphological evidence of vasopressin-responsiveness one renal tubule with another; thus, the first junction of was not observed in the early distal tubule (distal con- two renal tubules is formed by two so-called initial col- voluted tubule). Additional morphological studies in lecting tubules. Wistar, Long-Evans, and Sprague-Dawley rats demon- Despite the fact that the convoluted segment of the strated a marked difference in the random availability of distal tubule (as defined morphologically) ends well be- distal convoluted tubules versus initial collecting tubules fore its first junction with another tubule, it has been potentially available for micropuncture just beneath the common practice at the micropuncture table to define renal capsule. The results suggest that hypotonic tubu- the "distal tubule" or "distal convolution" as that seg- lar fluid entering the early distal tubule (distal convoluted ment of the renal tubule that extends between the macula tubule) remains hypotonic to plasma until it enters the densa and the site of the first junction with another distal tubule or distal convolution (4-7). When defined in Dr. Tisher is the recipient of Research Career Develop- this manner, it can be seen that this segment of the re- ment Award HE 440074 from the U. S. Public Health in character. Service. nal tubule is structurally heterogeneous Received for publication 30 May 197.? and in revised form Tlhtus, the "early" distal tubule of the renal micropunc- 3 August 1973. twrist is lined by epithelium characteristic of the distal The Journal of Clinical Investigation Volume 52 December 1973 3095-3108 3 09.o convoluted tubule (pars convoluta), whereas the "late" opened and the left kidney was isolated and prepared for distal tubule is lined by epithelium characteristic of the micropuncture as described previously (11). The temperature of the exposed kidney was maintained by a constant intial collecting tubule. surface drip of warmed (370C) isotonic saline. Timed col- The present investigation was initiated to determine lections of urine were obtained from each separate kidney whether the structural heterogeneity of the so-called via the placement of polyethylene catheters in the left distal tubule (as defined at the micropuncture table) was ureter (P.E. 50) and the bladder (P.E. 60). Urine osmolality was measured cryoscopically with an Advanced associated with morphological evidence of functional Instruments osmometer (Advanced Instruments, Inc., Need- heterogeneity in response to the administration of exoge- ham Heights, Mass.). Experimental manipulation and/or nous vasopressin. Previous morphological studies have morphological observations were initiated when a sustained demonstrated that certain segments of the collecting water diuresis, equivalent to at least 35 p1/min/kidney, had duct of the mammalian kidney are responsive to vaso- been established. In five animals renal morphological examination was carried out during water diuresis. In six pressin. In the presence of a favorable osmotic gradient, other DI rats renal tissue was fixed and prepared for that is, from the lumen to the interstitium, vasopressin- morphological examination between 6 and 60 min after induced bulk water flow across the tubular epithelium is the rapid conversion of water diuresis to antidiuresis via the associated with cell swelling and institution of a constant i.v. infusion of aqueous vasopressin dilatation of the lateral (Pitressin, Parke, Davis & Co., Detroit, Mich.) at a rate and basilar intercellular spaces. These morphological of 0.15 mU/min. changes have been observed in vivo in the medullary col- Two additional DI rats weighing 206 and 163 g, respec- lecting duct of the rat (8) and in vitro in isolated seg- tively, were studied during vasopressin administration after ments of the rabbit cortical tubule (9). Thus, these same the institution of varying degrees of water deprivation. Both of these animals were starved overnight and then morphological features were employed in the present prepared surgically as described above. One animal was study as a morphological marker to determine the most allowed free access to water before study but additional proximal site of action of vasopressin in the renal tubule fluid was not administered after the induction of anes- of the rat. The results of this investigation revealed that thesia (except for an infusion of isotonic saline at 0.02 ml/min for 40 min during surgical preparation to replace the entire cortical collecting duct (beginning with the ini- estimated surgical blood loss). The second animal was de- ial collecting tubule) was responsive to vasopressin. On prived of water for 9 h before surgery but i.v. fluids were the other hand, no evidence was found to suggest that the not given either during or after surgical preparation. distal convoluted tubule, as defined morphologically, was After surgical preparation was completed, both animals received an i.v. infusion of vasopressin (0.15 mU/min) for responsive to vasopressin. 10 min. Fixation of renal tissue was then initiated in preparation for morphological examination. METHODS Morphological observations on that segment of the renal Selected morphological features of the distal convoluted tubule between the macula densa and the first junction with tubule and the cortical collecting duct were documented in another renal tubule were made in 7 Wistar rats and 34 13 animals with hereditary hypothalamic diabetes insipidus Sprague-Dawley rats. All animals were studied during (DI) 1 (10) during water diuresis, vasopressin-induced anti- normally spontaneous antidiuresis, and surgical preparation diuresis without hydropenia, or after vasopressin adminis- was carried out in a manner identical to that outlined above tration after the antecedent institution of water deprivation. for DI rats. Additional observations on the morphology of the distal Morphological studies in DI rats. Renal tissue was convoluted tubule and cortical collecting duct were recorded preserved for morphological investigation by dripping either during antidiuresis in 7 Wistar rats and 34 Sprague-Dawley 2%o OsO4 buffered in potassium phosphate (osmolality: rats. 225 mosmol/kg H20; pH 7.4-7.6) or a mixture of glutar- In the initial phases of the investigation, 11 adult DI aldehyde and formaldehyde (12) diluted to half-strength rats weighing 183-257 g were studied after an overnight and buffered in potassium phosphate (osmolality: 960 fast during which free access to water was permitted. On mosmol/kg H20; pH 7.4-7.6) over the surface of the the morning of the experimental day, water diuresis was undisturbed left kidney for 30 min. Thereupon the kidney initiated by the administration of two separate water loads was excised quickly and the well-preserved outer cortex via gastric gavage at 30-min intervals (each water load was was removed and divided into several blocks approxi- equivalent to 3% of the body weight). Immediately there- mately 1 mm' in size. Aldehyde-preserved tissue was fixed after the rats were anesthetized via the intraperitoneal for an additional 4 h at room temperature in the same administration of sodium pentobarbital (35 mg/kg body fixative solution; it was then rinsed overnight in a potas- weight), tracheostomy was performed, and polyethylene sium phosphate buffer with added sucrose and postfixed catheters were inserted into a carotid artery and one for 1 h in 2% OsO4. Tissue initially fixed in OSO4 was jugular vein. Water diuresis was sustained by the constant fixed for 1 additional h in OS04 and then rinsed briefly i.v. infusion of a hypotonic sodium chloride solution (110 in the potassium phosphate buffer. All tissues were de- mM Na Cl; 25 mM Na HCO3; osmolality=260 mosmol/ hydrated in a graded series of alcohols and embedded in kg H20) at 0.2 ml/min. The mean arterial blood pressure Epon epoxy resin (Epon Adhesive Synthetic Resin, Shell was monitored constantly with a P23D pressure transducer Chemical Co., New York (13). connected to a Sanborn recorder (Hewlett-Packard Co., Tissue sections for light microscopy were cut 1 Am in Waltham Div., Waltham, Mass.). The abdomen was then thickness from the Epon-embedded material and stained with toluidine blue (14). Sections for electron microscopy "Abbreviation used in this paper: DI, diabetes insipidus. were cut with diamond knives, doubly stained with uranyl 3096 P. B. Woodhall and C. C. Tisher acetate (15) and lead citrate (16), and examined and photographed with an AEI 6B electron microscope (AEI Scientific Apparatus Inc., Elmsford, N. Y.). The morphology of the distal convoluted tubule and the cortical segment of the collecting duct in the diuretic and antidiuretic DI animals was first evaluated qualitatively by both light and electron microscopy. The material was then subj ected to quantitative morphometric techniques in an attempt to document the presence or absence of vasopressin- induced transcellular or intercellular osmotic water flow or both in the two segments of the renal tubule under investigation. Since transcellular water flow causes an increase in cell volume that should be reflected by an increase in cell height, the height of individual cells lining the cortical segment of the collecting duct and the distal convoluted tubule was measured. By planimetric techniques, the total cross-sectional area of the epithelium lining the cortical segments of the collecting duct was also deter- mined as another indication of cell volume change in the two physiological conditions. These measurements included the area occupied by the intercellular space and intra- cellular vacuoles, since the two compartments could not be distinguished at the light microscopic level. For these measurements distal convoluted tubules and cortical collecting ducts appearing in the cross-section in the 1-,um tissue sections were photographed with a Zeiss Photomicro- scope II (Carl Zeiss, Inc., New York) at an initial magnification of 160, with Kodak Panatomic Photographic Film (Eastman Kodak Co., Rochester, N. Y.). All prints were enlarged by a factor of 12 (final magnification: 1,920 X). To insure proper orientation, only those sections with identifiable renal capsule were used. In addition to the light microscopic measurements of cell height in distal convoluted tubules, electron micrographs were also taken of the distal convoluted tubules of DI rats in water diuresis and vasopressin-induced antidiuresis to compare the width of the lateral intercellular spaces in the two physiological FIGURE 1 Photomicrograph depicting the typical appear- TABLE I ance of a cortical collecting duct from an untreated DI Urine Flow Rate and Urine Osmolality in DI Rats before rat during water diuresis. Magnification X 1,400. Tissue Preservation FIGURE 2 Photomicrograph demonstrating cell swelling and dilatation of lateral and basilar intercellular spaces Animal (arrows) in a cortical collecting duct from DI rat in Study group no. Urine flow Urine osmolality which the water diuresis was converted to antidiuresis by the administration of exogenous vasopressin. Magnification j4/min/kidney mosmoll'kg H20 X 1,400. Water diuresis 008 74 153 200 91 241 190 63 245 conditions. For these measurements three separate tubules 202 69 197 were examined in each animal. Six measurements of width 17 69 176 were taken randomly from two different cells within each tubule examined. Width of the intercellular spaces was Mean 73 202 418* measured with an Optical Comparator, Model P-1523, Antidiuresis 009 3.2 (37.7)$ 530 (201)§ (Scherr-Tumico, Inc., St. James, Minn.) directly from 010 4.0 (52) 357 (145) negatives taken at an original magnification of 5,000 X. 210 28.7 (37) 483 (227) 2 5.0 (38) 1,000 (165) These measurements were made without prior knowledge 58 22 (42) 784 (132) of the physiological status of the animal, i.e., water diuresis 129 56 (76) 369 (102) versus antidiuresis. Morphological studies in Wistar and Sprague-Dawley Mean 10.2 592 4141* rats. These observations were made for two primary pur- poses: (a) to characterize the type of epithelium that lines * SEM. those from macula Value in parenthesis indicates maximum urine flow before administration so-called distal tubules (extending the of vasopressin. densa to the first junction with another renal tubule) that § Value in parenthesis indicates urine osmolality at the time of maximum are potentially accessible to renal micropuncture, and (b) urine flow. to define the type of epithelium present at the point of 11 The mean value does not include animals 58 and 129, fixed 1 h after junction between two distal tubules. l-,um tissue sections administration of vasopressin. were examined by light microscopy and compared with Vasopressin Responsiveness of Distal Tubule and Collecting Duct 3097 -s'wEv~W.'; cceeiZs g::p;\ , !i. d

FIGURE 3 Low-magnification electron micrograph depicting a short segment of cortical collecting duct from an untreated DI rat in water diuresis. No enlargement of intercellular spaces (arrows) or evidence of cell swelling is present in either the light or principal cells on the right and left, or the dark or intercalated cell in the center. Magnification X 12,000. 3098 P. B. Woodhall and C. C. Tisher sections obtained from DI rats. Only those sections that initial collecting tubules) differed markedly in the included the renal capsule were used and only those six DI rats treated with vasopressin before fixation tubules (excluding proximal tubules) that lay directly beneath the capsule and which would normally have been (Fig. 2). Light microscopic examination revealed that accessible to micropuncture were evaluated. The tubules the outer diameter of the collecting ducts, when viewed were classified on the basis of their cellular composition in cross-section, was somewhat reduced in comparison to as distal convoluted tubule, transition region, or cortical that observed in animals undergoing water diuresis; collecting duct. The transition region was defined as that segment of the renal tubule in which cells resembling both furthermore, the cells lining the tubules were greater in the distal convoluted tubule and the cortical collecting duct height. These two events led to a reduction in luminal could be seen in the same cross-section. In these same ani- diameter and they suggested that cell swelling had oc- mals (as well as in DI rats), serial 1-Am tissue sections curred. Most striking, however, was the presence of were also cut to trace the formation of the first junction of one renal tubule with another. Junctional observations markedly dilated lateral and basilar intercellular spaces were not limited to sections with visible renal capsule; separating the individual cells lining the collecting ducts. junctions throughout the outer one-third of the cortex were Differences in the morphologic appearance of collect- examined. ing duct epithelium in the two physiological conditions Numerical data were analyzed by Student's t test. Data were even more obvious with electron microscopy. In are expressed as mean ± standard deviation unless indi- cated otherwise and P values less than 0.05 were considered untreated DI rats undergoing water diuresis, enlargement significant. of the intercellular spaces was not evident and the lateral cell membranes and the interlocking lateral interdigita- RESULTS tions of adjacent cells were immediately juxtaposed (Fig. Physiological observations (Table I). The average 3). On the other hand, the exposure of collecting duct urine flow at the time of fixation in the five untreated DI epithelium to vasopressin during maximum water diuresis rats (water diuresis alone) was 73 Al/min/kidney; the was accompanied by the appearance of the large lateral average urine osmolality averaged 202±SE 18 mos- and basilar intercellular spaces noted previously with mol/kg H20. In the six animals in which water diuresis light microscopy (Fig. 4). The majority of the lateral was interrupted by the administration of exogenous vaso- interlocking processes of adjacent cells were no longer in pressin, the urine osmolality immediately before fixation direct apposition to each other. There was marked rare- had increased to an average value of 592±SE 141 mos- faction of the cytoplasm of the principal or light cells, mol/kg H20 and urine flow had fallen to 10.2 Al/min/ suggesting that the cells were swollen and had increased kidney. An amount of urine sufficient for analysis could in volume. The most marked enlargement of the inter- not be obtained from the two animals subjected to water cellular spaces was noted in kidney tissue fixed 6-10 min deprivation before the administration of exogenous vaso- after the establishment of antidiuresis. The collecting duct pressin. Physiological observations were not obtained in epithelium from kidneys fixed at 60 min continued to healthy and presumably antidiuretic non-DI rats. exhibit evidence of cellular swelling, but the frequency Morphological observations. The general Morphologi- of lateral intercellular space dilatation was sharply re- cal appearance of the collecting duct epithelium and that duced. In many animals the intercellular spaces were lining the distal convoluted tubule in DI rats was identi- dilated most markedly within the initial collecting tu- cal to that described previously in other strains of labora- bule at a point near its transition with the distal convo- tory rats and it will not be detailed further. The terminal luted tubule. Hydropenic animals receiving exogenous portion of the distal tubule of the micropuncturist was vasopressin did not exhibit detectable morphological lined by epithelium that was collecting-duct in type, i.e., evidence of cellular swelling or intercellular space like that of the initial collecting tubule of the morpholo- dilatation. gist. In contrast to the above observations on epithelium of In the five untreated DI rats in which the kidney was the collecting duct, water diuresis and vasopressin-in- fixed in situ during water diuresis, the lumen of the cor- duced antidiuresis were not associated with discernible tical collecting ducts (including the initial collecting tu- differences in the morphological appearance of the bule) was widely patent. Both the light or principal cells distal convoluted tubule on light microscopy (Figs. 5 and and the dark or intercalated cells were cuboidal in shape 6). The luminal and outside diameters of the distal con- and enlargement of the lateral or basilar intercellular voluted tubules were similar in the two physiological spaces was not observed (Fig. 1). In contrast, the ap- conditions as was the height of individual cells lining pearance of the cortical collecting ducts (including the the tubules. These findings were confirmed with electron FIGURE 4 Low-magnification electron micrograph depicting a segment of a cortical collecting duct from a DI rat in which the water diuresis was converted to antidiuresis with exogenous vasopressin. Lateral and basilar intercellular spaces are widely dilated and the light or principal cells on the right and left are swollen. A dark or intercalated cell is present just to the right of center. Magnification X 10,000. Vasopressin Responsiveness of Distal Tubule and Collecting Duct 3099 I

FIGURE 5 Photomicrograph of distal convoluted tubule from a DI rat in water diuresis. Magnification X 1,050. FIGURE 6 Photomicrograph of a distal convoluted tubule from a DI rat converted from water diuresis to antidiuresis by the administration of exogenous vasopressin. Magnification x 1,075. microscopy (Figs. 7 and 8). Neither cell swelling nor cells in the four untreated animals undergoing water dilatation of the lateral and basilar intercellular spaces diuresis was 5.7±SD 1.0 jum (n= 71) vs. 6.7±SD 1.3 was observed in the distal convoluted tubules from vaso- itm (n = 71 ) in the three vasopressin-treated animals pressin-treated antidiuretic animals. (P <0.001). The average height of the dark or inter- The qualitative morphological observations described calated cells in the untreated diuretic animals was 5.3+ above were confirmed by semiquantitative morphometric SD 1.1 jum (n = 61) vs. 6.8+SD 1.0 atm (n = 59) in measurements. These measurements were performed on the treated animals (P < 0.001). These results sug- cortical collecting ducts (including the initial collecting gest that the cells of the collecting ducts in vasopressin- tubules) and distal convoluted tubules from 7 of the 13 treated animals were swollen, and cellular volume had animals in which tissue fixation was initiated at the increased. They confirm the qualitative morphological height of water diuresis (four rats) or up to 20 min after observations and they are consistent with an increased the beginning of vasopressin-induced antidiuresis (three cellular entry of wvater through the apical or lutminal rats). Aldehyde fixation was used in each of these seven membrane in the presence of vasol)ressin and a favorable animals. The results of these measurements are depicted osmotic gradient. The results of planimetry support such in Tables II and III. In the cortical collecting duct, in- a conclusion as well, despite the fact that statistical sig- cluding the initial collecting tubule, the outside diameter nificance was not quite achieved; the average cross-sec- of the tubule did not differ significantly in the two groups tional area of the epithelium lining the cortical collecting of animals. However, a significant reduction in luminal duct of untreated diuretic animals was 601±SD 150 tm-n diameter was observed in the vasopressin-treated anti- (n = 33) vs. 668±SD giM2 (n = 33) in the animals re- diuretic group (26.1±+SD 5.1 /lm vs. 22.7+SD 4.7 uam, ceiving vasopressin (P < 0.06). P < 0.01). The average height of the light or principal The results of the morphometric measurements of 3100 P. B. Woodhall and C. C. Tisher rse~~~~~~l:-S~~~~*4' ( .4- Sot

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FIGURE 8 Electron micrograph of distal convoluted tubule from a DI rat converted from water diuresis to antidiuresis by the administration of exogenous vasopressin. There is no evidence of cell swelling or intercellular space enlargement. Magnification X 12,600. 3102 P. B. Woodhall and C. C. Tisher TABLE I I Morphometric Measurements in Cortical Collecting Ducts of DI rats*

Tubule diameter Cell height

Study group Outside Inside Light Dark Cell area

pm Am pm2 \Water diuresis 38+5.8 26.1±5.1 5.7±1.3 5.3±1.11 601±155 (n = 4) (n = 34) (n = 35) (n = 71) (n = 61) (n = 33) Antidiuresis 36.1±5.8 22.7±4.7 6.7±1.0 6.8± 1.0 668±180 (n = 3) (n = 34) (n = 34) (n = 71) (n = 59) (n = 33) P > 0.10 P < 0.01 P < 0.001 P < 0.001 P < 0.06 * Values represent the mean4the standard deviation.

distal convoluted tubules are depicted in Table III. The collecting tubules (the first portion of the cortical col- average height of the cells in the untreated diuretic ani- lecting duct before the junction) are readily accessible to mals was 10.5+SD 1.4 Am (n = 100) vs. 10.7+SD 1.6 micropuncture in this particular strain of Wistar rat, Am (n = 100, P > 0.3) in vasopressin-treated rats. and that the micropuncturist may well be apt to obtain Measurements of the average width of the lateral inter- samples of distal fluid from regions of differing cell type cellular spaces of the distal convoluted tubules in the two with equal frequency. However, in both Sprague-Dawley physiological conditions equalled 178±+SD 70 A (n = and DI rats, there appears to be a greater likelihood of 144) in untreated diuretic rats and 182+SD 82 A (n = entering the distal convoluted tubule during micropunc- 108) in vasopressin-treated animals (P > 0.5; Table ture studies of the so-called distal tubule. III). These morphological observations are in agreement Finally, in all three strains of rats, the first junction with those obtained qualitatively and they suggest that of one renal tubule with another was always formed by vasopressin-induced osmotic water flow does not oc- tubules lined with epithelium characteristic of the col- cur in the convoluted portion of the distal tubule. lecting duct (Fig. 10). In no instance was the junction of When the surface of the kidney is viewed in vivo one renal tubule with another formed by one or more within the range of magnification usually employed for distal convoluted tubules. A total of 1,628 sections were micropuncture (60-10OX), it is impossible to distinguish examined in 34 Sprague-Dawley rats. Nine junctions (at least on the basis of histological appearance alone) were observed and all were formed by tubules lined with the distal convoluted tubule (early distal tubule) from collecting duct epithelium. Identical findings were noted the initial collecting tubule (late distal tubule); either in DI rats (2, 405 sections, 16 junctions) and Wistar rats, segment is potentially accessible to renal micropuncture (944 sections, 11 junctions). Lastly, when a subcapsular (Figs. 9a and b). Table IV depicts the results of an tubule was identified as collecting-duct in type and fol- analysis of 829 subcapsular tissue sections in which the lowed by serial sections until it had formed a junction cellular composition of the so-called distal tubule of the with another renal tubule, that segment of the tubule micropuncturist was assessed in three different strains beyond the first junction was never observed to return to of rats. Distal convoluted tubules, initial collecting tu- the surface before descending into the medulla. bules, and tubules representing regions of transition between these two morphologically distinct segments could all be identified just beneath the renal capsule. The cel- TABLE III lular composition of subcapsular distal tubules was simi- MIorphometric Measurements in Distal Convoluted Tubules lar in Sprague-Dawley and DI rats; of the three types of DI Rats* tu- of tubules identified and counted, distal convoluted Width of lateral bules comprised 75.2 and 77.3% of the total subcapsular Study group Cell height intercellular spaces tubules in the two species, respectively, while subcapsular tubules lined by collecting duct epithelium repre- pm A 178± 78 sented 19.1 and of the total number, re- \Water diuresis 10.5±1.4 only 10.9% (n = 4) (n = 100) (n = 144) spectively. However, in the Wistar rat, 48.3% of 60 Antidiuresis 10.7± 1.6 182 ±82 subcapsular tubules were lined by epithelium character- (n = 3) (n = 100) (n = 108) istic of the distal convoluted tubule while 45% were P > 0.3 P > 0.5 lined by collecting duct epithelium. The latter findings suggest that both distal convoluted tubules and initial * Values represent the mean-the standard deviation. Vasopressin Responsiveness of Distal Tubule and Collecting Duct 3103 FIGURE 9 (a) Located just beneath the renal capsule shown at the top of this photomicrograph is a distal convoluted tubule from a Wistar rat, which is thus accessible to micropuncture. Magnification X 1,000. (b) Located just beneath the renal capsule shown at the top of this photomicrograph is an initial collecting tubule from a Wistar rat, which is thus accessible to micropuncture. Magnification X 865.

DISCUSSION of the mammalian renal tubule, although similar morpho- The present observations in DI rats provide morphologi- logical findings have been observed in vitro in isolated cal evidence (cell swelling, increased cell height, and perfused cortical segments of the rabbit collecting tu- dilatation of the lateral and basilar intercellular spaces) bule. Earlier, Tisher, Bulger, and Valtin (8) described that the cortical segment of the collecting duct, including a similar morphological response to exogenous vaso- the initial collecting tubule, is responsive to vasopressin pressin in medullary segments of the collecting duct in vivo. These results represent the first in vivo docu- from DI rats. Thus, the present results, when coupled mentation of vasopressin responsiveness by this segment with those of earlier studies (8), provide morphological

TABLE IV Cellular Composition of Subcapsular Distal Tubules Potentially Available for Micropuncture

Strain Lined by epithelium characteristic of

Distal No. of convoluted Collecting Transition tubules tubule tubule region Sprague-Dawley 141 106 (75.2%o') 27 (19.1%7c) 8 (5.7%) Wistar 60 29 (48.3%6) 27 (45%) 4 (6.7%) Long-Evans (Brattleboro) 119 92 (77.3%) 13 (10.9%0) 14 (11.8%)

3104 P. B. Woodhall and C. C. Tisher FIGURE 10 Photomicrograph of a junction at the upper right formed by two renal tubules lined by epithelium characteristic of the initial collecting tubule from a \Vistar rat. PT, proximal tubule; DCT, distal convoluted tubule; RC, renal corpuscle. Magnification X 620.

evidence that the entire length of the collecting duct of tubule, like those of the proximal convoluted tubule, DI rats is responsive to vasopressin, and that vaso- are highly complex in structure, possessing elaborate pressin-induced osmotic water flow presumably occurs invaginations of the basal plasmalemma. This configu- along its entire length as well. ration greatly increases the cell surface-to-volume ratio In contrast to the implications of certain physiologi- and may permit greater bulk fluid flow in the absence cal studies (5, 6, 17, 18), no morphological evidence of obvious structural changes such as cell swelling and was found to suggest that the convoluted segment of intercellular space dilatation. Thus, possible limitations the distal tubule in the rat is responsive to vasopressin. in the applicability of the morphometric techniques em- In the presence of vasopressin there was no increase ployed to detect transepithelial water flow must be in the height of the individual cells, there was no in- acknowledged. In recent studies by Tisher and Kokko crease in the width of the lateral and basilar inter- the same morphometric techniques were employed to cellular spaces between individual cells, and there was relate changes in intercellular space width to varying no change in the general appearance of the cells that rates of net transepithelial water flow in isolated proxi- might reflect the presence of increased transepithelial nmal convoluted tubules exposed to various oncotic osmotic water flow in this segment of the nephron. It pressure gradients. Changes in net fluid reabsorption is recognized, however, that the failure to demonstrate as small as 0.40-0.50 nl mm1 min-' were associated with such changes in the distal convoluted tubule does not significant differences in intercellular space width (19, totally exclude the possibility that some water reab- unpublished observations). Calculations based on data sorption may occur in this segment of the nephron. In derived from earlier micropuncture and microdissection contrast to the epithelium of the collecting duct and studies in the rat suggest that rates of net transepi- the toad bladder, the cells of the distal convoluted thelial water reabsorption well in excess of 0.50 nl

Vasopressin Responsiveness of Distal Tubule and Collecting Duct 33105 mm-'min-' could be anticipated if the entire distal samples of fluid obtained from the late distal tubule tubule (from the macula densa to the first junction with (initial collecting tubule) would be more likely to another renal tubule) is responsive to vasopressin. approach isotonicity. However, in contrast, micropunc- From the work of Schnermann et al. (20) it can be ture studies in either the Sprague-Dawley rat or the calculated that tubular fluid is delivered to the early Brattleboro strain of the Long-Evans hooded rat would distal tubule at a rate of approximately 9 nl/min in appear to be accompanied by a reduced likelihood of en- a water-loaded DI rat. The average length of the distal tering the initial collecting tubule and a greater likeli- tubule from macula densa to the first junction with hood of obtaining samples of tubular fluid from the distal another renal tubule is approximately 2.4-2.5 mm (21, convoluted tubule. Thus, in the latter two strains of rats, and Gottschalk, personal communication). In hydro- one might anticipate that the fluid would appear to re- penic rats the average TF/P inulin ratio in the early main hypotonic to plasma throughout the accessible distal tubule is approximately 5 and increases to an length of the so-called distal tubule (as defined by the average value of 20 in the late distal tubule (Gottschalk, micropuncturist). personal communication). Calculation of the rate of net Morphological differences such as those described in fluid reabsorption from these data yields a value of the present study may help to explain the described approximately 2.7 nl mm-' min', five times that asso- differences between the renal micropuncture observa- ciated with detectable intercellular space dilatation in tions of Gottschalk and Mylle (5) and Lassiter, Gott- the isolated proximal convoluted tubule. Thus, since schalk, and Mylle (6) and those of Lechene, Morel, smaller increments of transepithelial water flow have Guinnebault, and de Ruffignac (22). The first two been detected in the proximal convoluted tubule by the papers suggested that distal fluid approached isotonicity same morphological techniques employed in the present beyond the mid-portion of the distal tubule whereas study, we believe it quite likely that if there is sig- the last paper suggested that distal fluid remained hypo- nificant osmotic water flow across the early distal osmotic to plasma throughout the accessible portion of tubule (distal convoluted tubule) in the presence of the distal tubule under a similar array of experimental vasopressin, it should also be detectable. conditions (hydropenia, saline diuresis, urea diuresis, The results of the present study suggest that the mannitol diuresis, etc.). Although the studies by both hypotonic tubular fluid that enters the early distal con- groups were performed in Wistar rats, the strains used voluted tubule from the ascending thick limb of Henle were not identical (23). In later studies (23) it was remains hypotonic to plasma throughout the distal con- found that more samples of fluid collected from the voluted tubule until it enters the initial collecting late distal tubule in Saclay Wistar rats (strain used tubule. Thereafter, one might expect that osmotic by Colindres and Lechene [23]) remained hypo-osmotic equilibration with plasma would be achieved or ap- than from the Wistar rats commonly used by Gott- proached as tubular fluid progresses further along the schalk and his co-workers under the same experimental length of the initial collecting tubule. The present conditions, and that the mean TF/P osmolality in the morphological demonstration of dissimilar responsive- second half of the distal convolution was significantly ness to vasopressin along the distal tubule of the micro- lower in the Saclay Wistar animals. It was suggested puncturist also provides a possible explanation for the that hereditary or environmentally induced strain differ- results of earlier micropuncture studies in Wistar rats, ences might account for the differences observed in where the osmolality of distal tubule fluid was found the latter study. Based on observations in the present to approach isotonicity at a point approximately 50- study, we believe it quite likely that hereditary differ- 60% along the accessible length of the distal tubule (5, ences in distal tubule morphology exist in these two 6). As shown in Table IV, both the distal convoluted strains of animals. We would predict that the Saclay tubule (early distal tubule) and the initial collecting rats resemble the Sprague-Dawley and Long-Evans tubule (late distal tubule) are accessible to micro- strains of animals, having a greater percentage of true puncture in approximately equal numbers over the distal convoluted tubules among those distal tubules surface of the kidney of the Wistar rat.2 Therefore, available for micropuncture. Of course, despite the fact according to the present morphological results in Wis- that the present results do provide further insight into tar rats, it might be anticipated that samples of tubular the random availability of distal convoluted tubules fluid from the so-called early distal tubule (distal con- versus initial collecting tubules on the kidney surface voluted tubule) would be hypotonic to plasma, while of differing strains of rats, it must also be acknowledged that they do not establish the average percent a Two of the seven WVistar rats in this study were ob- length of the distal tubule of the micropuncturist that tained f rom the laboratory of Dr. C. W. Gottschalk. The might be lined by the differing cell types. The demon- remaining five animals were obtained from the same sup- plier (Hilltop Lab Animals, Scottdale, Pa. 15683. stration of a close relationship between such a figure 3106 P. B. Woodhall and C. C. Tisher and measured osmolalities of tubular fluid at various Greater future reliance should be placed on studies points along the distal tubule (as defined by the micro- employing both structural and functional techniques puncturist after the dissection of latex casts) would to identify the precise site of micropuncture along the provide further support for the thesis. nephron. A technique has recently been described that Similar morphological dissimilarities among animal permits the fixation of renal tubules for electron species could also account for the difference in the microscopy immediately after samples of fluid have been osmolality of distal tubule fluid observed in the dog obtained via micropuncture (25); the parallel injection (7) and the rhesus monkey (24) as compared with of latex into the lumen of the fixed tubule and the sur- the rat. Micropuncture studies in the dog have demon- rounding interstitium at the site of puncture facilitates strated that the tubular fluid remains hypo-osmotic precise histological identification of underlying cell throughout the accessible portion of the distal tubule, types at the site of micropuncture. again defined as "that segment of the distal nephron The present demonstration of cellular responsiveness between the macula densa and the point at which it to vasopressin can probably be attributed to the rapid joins another distal tubule to form a cortical collecting induction of antidiuresis in diuretic animals where the duct," (7). Combined morphological and micropuncture rate of fluid flow within the tubule presumably was studies in the dog have revealed that samples of hypo- high. The findings are consistent with the concept that tonic tubular fluid are obtained from segments of the a large volume of hypotonic fluid was entering the distal nephron that are morphologically characteristic cortical collecting duct at a time when the luminal of the distal convoluted tubule (C. C. Tisher and J. R. cell membrane was suddenly rendered more permeable Clapp, unpublished observations). to water by the action of vasopressin. The sudden in- In 1964 Ullrich, Rumrich, and Fuchs (78) reported crease in permeability to water permitted an initially an increase in the water permeability of the rat distal high rate of transtubular fluid flow, which effected the convolution during antidiuresis. By an in vivo micro- appearance of cell swelling and intercellular space en- perfusion technique with the Stamm FW 49 strain of largement, at least transiently. Three lines of evidence albino rat, the water permeability of the distal con- support this view: first, the cortical collecting ducts of volution during water diuresis was found to be 2.6 X animals whose kidneys were fixed -within 6-10 min 10' cm3/cm'/s/cm H20. In the same strain of rat the after the establishment of antidiuresis exhibited larger water permeability during antidiuresis was 7.6 X 10' and more numerous dilated intercellular spaces than cm'/cm2/s/cm H20. Based on our own morphological did the collecting ducts of those animals whose kidneys observations, it is possible that the authors were per- were fixed at 30-60 min; second, enlarged intercellular fusing, at least in part, the initial collecting tubule, spaces were observed infrequently in the cortical col- a segment that would correspond to the late distal lecting ducts of the DI rats made severely hydropenic tubule or late distal convolution. The authors' de- before the administration of vasopressin; finally, the scription is unclear as to just how much of the distal most marked intercellular space enlargement was ob- tubule was actually perfused, and histological identifica- served just beyond the region of transition from the tion of the perfused tubule segments was not under- distal convoluted tubule to the initial collecting tubule, taken. i.e., that point in which the osmotic gradient between The presence of functional (vasopressin responsive- the hypotonic fluid leaving the distal convoluted tubule ness) and structural heterogeneity within the rat distal and the isotonic interstitial fluid surrounding the tubule tubule (as defined at the micropuncture table) should should be greatest. serve to sound a note of caution to investigators at- tempting to establish the precise site of various physio- ACKNOWLEDGMENTS logical events in this segment of the renal tubule. It The authors express their appreciation to Dr. Roscoe R. appears likely, at least in some instances, that reported Robinson for many helpful discussions during the course functional differences between the early and late distal of this investigation and for his assistance during the functional differences between the preparation of this manuscript. The authors also express tubule actually reflect their appreciation to Mrs. Kathy Blake, Mrs. Helen Parks, distal convoluted tubule and the initial collecting tubule. and Mrs. Betty Waller for technical assistance and to Mrs. Such a circumstance should not be unexpected in view Jessie Calder for preparation of the illustrative material. of these two nmor- Supported in part by grants AM 13845 and AM 10844 of differing embryological origins from the U. S. Public Health Service. phologically distinct segments of the mammalian renal tubule. 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3108 P. B. Woodhall and C. C. Tisher