1 Mathematical Research Branch, National Institutes of Health, Bethesda 20892-2690; and 2 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We utilized immunofluorescent immunolabeling of renal tissue sections to identify and count tubules at specified depths of the rat renal inner medulla. We used primary antibodies to aquaporin-1 (AQP1; labeling thin descending limbs), aquaporin-2 (AQP2; labeling inner medullary collecting ducts), the rat kidney-specific chloride channel (ClC-K1; labeling thin ascending limbs), and von Willebrand factor (labeling descending vasa recta). Secondary antibodies conjugated to different fluorophores were used, giving up to a three-color display. Labeled structures were then identified and counted. At each level sampled in the inner medulla, many more thin limbs were labeled by ClC-K1 than AQP1. In addition, thin limbs were found to label with antibodies to ClC-K1 on both sides of their hairpin turns. We conclude that the descending thin limbs shift from expressing AQP1 to expressing ClC-K1 some distance before the point where they turn and begin to ascend. Mathematical models can use our quantitative data to explore implications for the urine-concentrating mechanism.
loop of Henle; urine-concentrating mechanism; inner medulla; aquaporin; chloride channel; urea channel
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
WATER ABSORPTION FROM THE descending limbs of Henle's loop is of primary importance for the concentrating ability of the mammalian kidney, because ~90% of filtered water is reabsorbed before it reaches the cortical collecting duct. Reports in the literature describe water- and solute-permeable descending limbs in hamsters (8, 19), rats (1, 3, 9), chinchillas (1, 2, 6), and rabbits (10, 13, 21). The descending limb of Henle's loop has been found in all species to be water permeable and moderately permeable to NaCl and urea. The adjacent ascending thin limb (ATL) of Henle's loop is characterized by extremely low water permeability and very high chloride permeability (7).
Chou et al. (1) showed that the osmotic water permeability of the descending limb in chinchillas is not uniformly high along its length. Instead, the distal 20% of the long-loop descending thin limb (DTL) was shown to have a relatively low water permeability (50 µm/s). Chou et al. (2) also showed the NaCl and urea permeabilities to be nonuniform in the chinchilla descending limb, attaining a maximum in the innermost segment of 98.4 and 47.6 µm/s, respectively.
Osvaldo and Latta (24) used electron microscopy to examine different levels of the inner medulla to characterize the structure of thin limbs of the rat kidney. Kriz and colleagues (16, 17) and Dieterich et al. (5) expanded these observations in their characterizations of thin limbs in mouse and other species. Osvaldo and Latta observed that, in a cross section from the upper layer of the inner medulla, there are "complex" (identified as DTL) and "simple" (identified as ATL) thin limbs and that the structure of the complex type becomes more simplified as they descend. They further suggested that for the longest loops a transition from complex to simple epithelium might occur at variable levels within the descending segment. This would explain the predominance of simple thin limbs found toward the papillary tip.
Dieterich et al. (5) described four types of epithelia. Type 1 epithelia, characteristic of short loops, were the smallest in diameter and had the thinnest cells. Types 2, 3, and 4 were found in the long loops. Type 2 epithelia occur in the inner stripe of the outer medulla. Type 3 epithelia, distal to type 2, were smaller than type 2, with a smaller diameter than type 2 but one greater than that of type 1. Type 3 cells appeared to be less specialized than those of type 2. The ascending thin limbs form type 4, with the epithelium appearing to be simple relative to the other types. Furthermore, the transition from type 3 to type 4 was found to occur some distance before the bend of the loop of Henle (5). Early morphological studies in rats, mice, and rabbits (15) lacked the markers needed to evaluate the distribution of transporters in the different regions described.
Recently, Pannabecker et al. (25) have shown that, for multiple species, many inner medullary thin limbs exhibit structural characteristics of DTL immediately adjacent to segments with structural characteristics of ATL at sites above the bend. They have shown that AQP1 is expressed in the DTL-type but not in ATL-type regions of these mixed-type thin limbs, whereas the chloride channel ClC-K1 is expressed in ATL-type but not in DTL-type regions of mixed-type thin limbs.
AQP1 is a constitutively active water channel found in DTLs and in descending vasa recta (DVR) in rat kidney, but not found in ATLs (18, 23). ClC-K1 is found in ATL cells (29, 30) and in ATL-type DTL segments (2, 5). On the other hand, AQP2 is a water channel found in collecting duct principal cells and in inner medullary collecting duct cells (22).
Here, we used immunofluorescent immunolabeling of tissue sections to identify and count AQP1, AQP2, and ClC-K1-positive tubules in rat inner medulla. The data obtained give an estimate of the fraction of descending limbs that are water permeable and the fraction that are solute permeable at a given distance from the papillary tip. The data also serve to estimate the number and diameter of tubules that extend to various depths of the inner medulla, which are important for mathematical models of the urine-concentrating mechanism (26, 20) that use the number and dimensions of loops of Henle and their transport properties as parameters.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental animals. In view of evidence that ClC-K1 expression may be regulated by vasopressin (30), all tissue for immunocytochemistry was taken from animals exposed to high levels of vasopressin. Three male Brattleboro rats (210-260 g, Harlan Sprague Dawley, Indianapolis, IN) were implanted with osmotic minipumps (Alzet model 2001, Alza, Palo Alto, CA) to administer 20 ng/h 1-desamino-8-D-arginine vasopressin, the V2 receptor-selective agonist of vasopressin. In addition, tissue from three male Sprague-Dawley rats (200-225 g) was also examined. These animals were maintained on a water-restricted diet for 5 days as previously described by Kim et al. (11).
Antibodies. To perform immunolocalization studies, we used peptide-directed polyclonal antibodies to 1) AQP1, raised in rabbits (L266) and chickens (LC18), to the same peptide (28); 2) ClC-K (COOH-terminal peptide of ClC-K2, KKAISTLINPARK), kindly provided by Klaus Steinmeyer, that recognizes both ClC-K1 and ClC-K2 (30) or ClC-K antibody (AB5392; Chemicon, Temecula, CA); 3) von Willebrand factor (vWF; Cedarlane Labs, Ontario, PQ); and 4) AQP2 raised in guinea pigs [GP7; COOH-terminal peptide of AQP2 to the same peptide previously used to produce antibodies in rabbits (22)].
Fixation of tissue and immunocytochemistry. Kidneys of ketamine-pentobarbital-anesthetized animals were fixed for immunolocalization by retrograde perfusion through the abdominal aorta, and antibodies were immunolocalized on frozen sections as previously described by Wade et al. (31). Sections, usually 12 µm thick and beginning at the papillary tip, were counted to obtain an estimate of the distance of each section from the papilla. The sections were incubated overnight at 4°C with primary antibodies diluted to 10 µg/ml. Secondary antibodies were species-specific donkey anti-chicken, donkey anti-rabbit, and donkey anti-guinea pig antibodies (Jackson ImmunoResearch Labs, West Grove, PA) coupled to Alexa 488, Alexa 568 (Molecular Probes, Eugene, OR), and Cy5, respectively.
Data analysis. Immunofluorescent-labeled tissue cross sections were used to identify and count the number of tubules labeled by each fluorophore. The number of AQP1-labeled plus the number of ClC-K1-labeled tubules minus the number of AQP1- and vWF-labeled vasa recta serves to estimate the number of descending and ascending limbs of the loop of Henle in a cross section at each distance from the papillary tip. Because one-half of the limbs of Henle's loop at each medullary depth are descending and one-half ascending, we approximated the number of descending limbs in each cross section as one-half of the total number of limbs present in the cross section.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunofluorescent localization in rat inner medulla.
We used triple immunofluorescent immunolabeling of cross sections from
the tip and from the base of the inner medulla. Figure 1 shows immunofluorescent labeling of a
section of inner medulla from a 1-desamino-8-D-arginine
vasopressin-infused Brattleboro rat. Secondary antibodies are
conjugated to different fluorophores, giving a three color display. The
0.25-mm2 section is ~840 µm from the papillary tip.
ClC-K1 is labeled red and is localized in ATL-type limbs of Henle's
loop; AQP1-sensitive structures are labeled green, and AQP2-sensitive
collecting duct cells are labeled blue.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Data summarized in Table 1 show that ClC-positive limbs of Henle's loop exceed AQP1-positive limbs of Henle's loop at all medullary depths and cross sections studied. On average, one expects that one-half of the limbs of Henle's loop will be descending, and Table 1 shows 62-11% of descending limbs to be AQP1 positive for Brattleboro rats, whereas the fraction that are AQP1 positive decrease and the fraction that are ClC positive increase toward the papillary tip. Pannabecker et al. (25) reported that ~55% of thin limbs isolated from distinctly above the bend of the loop were of the mixed type, meaning that they expressed both AQP1 and ClC-K1 somewhere along their length. Our findings suggest that the incidence of regions expressing AQP1 decreases substantially near the papillary tip, and we estimate that ~70-90% of DTLs are ClC positive near the tip of the papilla.
Data for 0.25-mm2 sections show a maximum of 110 limbs of Henle's loop turning/section within 900 µm of the tip and 30 limbs turning/0.25-mm2 section nearest the tip. This is comparable to the 400 and 90/mm2, respectively, reported by Knepper et al. (12) (Fig. 4). Table 1 also shows the number of inner medullary collecting ducts and ATLs to approach asymptotically near the papillary tip, which was shown previously (20), and data for Sprague-Dawley rats that were water-restricted are shown to be consistent with that for Brattleboro rats.
Implications for mathematical models. The mechanism utilized by the mammalian kidney to generate a concentration gradient in the inner medulla is still unclear after decades of study (see review in Ref. 4). One possible mechanism is water absorption from thin descending limbs in conjunction with a preferential solute reabsorption from thin ascending limbs and countercurrent multiplication (14, 26).
In vitro perfusion studies of thin descending limbs in hamsters (8), rats (1), and chinchillas (1, 2) have demonstrated a water-permeable descending limb. Low-to-moderate solute permeabilities have been measured for several species in DTL, e.g., sodium permeability in hamsters (8) and NaCl permeability in chinchillas (1, 2). In addition, little or no Na+,K+-ATPase activity has been recorded in thin descending limbs in the inner medulla, e.g., in rats (27). The measurements from perfusion studies and the data from immunolocalization suggest that mathematical models of the urine-concentrating mechanism might investigate a distribution of water- and solute-transporting segments of DTL on the basis of the distribution of AQP1 and ClC channels observed. Our observations, as well as those of others (25), show transitions from AQP1-positive to ClC-positive segments with no significant colocalization of solute and water channels. Hence, mathematical models might test the effect of water-permeable segments of DTL juxtaposed and in series with solute-permeable segments of DTL on solute concentration gradients in the inner medulla and on the kidney's ability to concentrate urine.Conclusion. We conclude that, at distinct axial distances within the inner medulla, a variable fraction of DTL cells express AQP1 compared with those expressing ClC-K1. Our observations suggest that a smaller fraction of the DTL at each depth are water permeable than are carrying out solute transport. The finding that the proportion labeled by AQP1 falls axially suggests that water reabsorption from DTL may diminish near the tip of the inner medulla. Mathematical models of the urine-concentrating mechanism can use this data to explore possible implications for the urine-concentrating mechanism.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors gratefully acknowledge the encouragement and very helpful advice of Dr. Mark Knepper of the National Institutes of Health. We also appreciate the expert technical assistance of Jie Liu. Images were processed with IDL version 5.2, Research Systems, Inc., on the Helix System of the Center for Information Technology at the National Institutes of Health.
![]() |
FOOTNOTES |
---|
This study was supported by National Institutes of Health Grant DK-32839 (J. B. Wade). Brattleboro rats with 1-desamino-8-D-arginine vasopressin minipumps were provided by Dr. James Terris of the Uniformed Services University. Polyclonal antibody was kindly provided by Dr. Klaus Steinmeyer of the University of Hamburg.
Address for reprint requests and other correspondence: R. Mejia, BSA Bldg., Suite 350, National Institutes of Health, Bethesda, MD 20892-2690 (E-mail: ray{at}helix.nih.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00340.2000
Received 14 December 2000; accepted in final form 31 October 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Chou, C-L,
and
Knepper MA.
In vitro perfusion of chinchilla thin limb segments: segmentation of osmotic water permeability.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F471-F476,
1992.
2.
Chou, C-L,
and
Knepper MA.
In vitro perfusion of chinchilla thin limb segments: urea and NaCl permeabilities.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F337-F343,
1993
3.
Chou, C-L,
Knepper MA,
Hoek AN,
Brown D,
Yang B,
Ma T,
and
Verkman AS.
Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.
J Clin Invest
103:
491-496,
1999
4.
Chou, C-L,
Knepper MA,
and
Layton HE.
Urinary concentrating mechanism: the role of the inner medulla.
Semin Nephrol
13:
168-181,
1993[ISI][Medline].
5.
Dietrich, HJ,
Barrett JM,
Kriz W,
and
Bulhoff JP.
The ultrastructure of the thin loop limbs of the mouse kidney.
Anat Embryol
147:
1-18,
1975[ISI][Medline].
6.
Flessner, MF,
Mejia R,
and
Knepper MA.
Ammonium and bicarbonate transport in isolated perfused rodent long-loop thin descending limbs.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F388-F396,
1993
7.
Imai, M,
and
Kokko JP.
Sodium chloride, urea, and water transport in the thin ascending limb of Henle. Generation of osmotic gradients by passive diffusion of solutes.
J Clin Invest
53:
393-402,
1974[ISI][Medline].
8.
Imai, M,
Taniguchi J,
and
Yoshitomi K.
Transition of permeability properties along the descending limb of long-loop nephron.
Am J Physiol Renal Fluid Electrolyte Physiol
254:
F323-F328,
1988
9.
Jamison, RL,
Buerkert J,
and
Lacy F.
A micropuncture study of Henle's thin loop in Brattleboro rats.
Am J Physiol
224:
180-185,
1973[ISI][Medline].
10.
Johnston, PA,
Battlilana CA,
Lacy FB,
and
Jamison RL.
Evidence for a concentration gradient favoring outward movement of sodium from the thin loop of Henle.
J Clin Invest
59:
234-240,
1977[ISI][Medline].
11.
Kim, GH,
Ecelbarger C,
Mitchell C,
Packer RK,
Wade JB,
and
Knepper MA.
Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop.
Am J Physiol Renal Physiol
276:
F96-F103,
1999
12.
Knepper, MA,
Danielson RA,
Saidel GM,
and
Post RS.
Quantitative analysis of renal medullary anatomy in rats and rabbits.
Kidney Int
12:
313-323,
1977[ISI][Medline].
13.
Kokko, JP.
Sodium chloride and water transport in the descending limb of Henle.
J Clin Invest
49:
1838-1846,
1970[ISI][Medline].
14.
Kokko, JP,
and
Rector FC, Jr.
Countercurrent multiplication system without active transport in inner medulla.
Kidney Int
2:
214-223,
1972[ISI][Medline].
15.
Kriz, W.
Differences in the cytological organization of the epithelia of loops of Henle.
Curr Probl Clin Biochem
6:
320-327,
1976[Medline].
16.
Kriz, W,
and
Koepsell H.
The structural organization of the mouse kidney.
Z Anat Entwicklungsgesch
144:
137-163,
1974[ISI][Medline].
17.
Koepsell, H,
Kriz W,
and
Schnermann J.
Pattern of luminal diameter changes along the descending and ascending thin limbs of the loop of Henle in the inner medullary zone of the rat kidney.
Z Anat Entwicklungsgesch
138:
321-328,
1972[ISI][Medline].
18.
Maeda, Y,
Smith BL,
Agre P,
and
Knepper MA.
Quantification of aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA.
J Clin Invest
95:
422-428,
1995[ISI][Medline].
19.
Marsh, DJ.
Solute and water flows in the thin limbs of Henle's loop in the hamster kidney.
Am J Physiol
218:
824-831,
1970[Medline].
20.
Mejia, R,
Sands JM,
Stephenson JL,
and
Knepper MA.
Renal actions of atrial natriuretic factor: a mathematical modeling study.
Am J Physiol Renal Fluid Electrolyte Physiol
257:
F1146-F1157,
1989
21.
Miwa, T,
and
Imai M.
Flow-dependent water permeability of the rabbit descending limb of Henle's loop.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F743-F754,
1983[ISI][Medline].
22.
Nielsen, S,
DiGiovanni SR,
Christensen EI,
Knepper MA,
and
Harris HW.
Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.
Proc Natl Acad Sci USA
90:
11663-11667,
1993[Abstract].
23.
Nielsen, S,
Pallone T,
Smith BL,
Christensen EI,
Agre P,
and
Maunsbach AB.
Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F1023-F1037,
1995
24.
Osvaldo, L,
and
Latta H.
The thin limbs of the loop of Henle.
J Ultrastructure Res
15:
144-168,
1966[ISI][Medline].
25.
Pannabecker, TL,
Dahlmann A,
Brokl OH,
and
Dantzler WH.
Mixed descending- and ascending-type thin limbs of Henle's loop in mammalian renal inner medulla.
Am J Physiol Renal Physiol
278:
F202-F208,
2000
26.
Stephenson, JL.
Concentration of urine in a central core model of the renal counterflow system.
Kidney Int
2:
85-94,
1972[ISI][Medline].
27.
Terada, Y,
and
Knepper MA.
Na+-K+-ATPase activities in renal tubule segments of rat inner medulla.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F218-F223,
1989
28.
Terris, J,
Ecelbarger CA,
Nielsen S,
and
Knepper MA.
Long-term regulation of four renal aquaporins in rats.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F414-F422,
1996
29.
Uchida, S,
Sasaki S,
Nitta K,
Uchida K,
Horita S,
Nihei H,
and
Marumo F.
Localization and functional characterization of rat kidney-specific chloride channel, ClC-K1.
J Clin Invest
95:
104-113,
1995[ISI][Medline].
30.
Vandewalle, A,
Cluzeaud F,
Bens M,
Kieferle S,
Steinmeyer K,
and
Jentsch TJ.
Localization and induction by dehydration of ClC-K1 chloride channels in the rat kidney.
Am J Physiol Renal Physiol
272:
F678-F688,
1997
31.
Wade, JB,
Lee AJ,
Liu J,
Ecelbarger CA,
Mitchell C,
Bradford AD,
Terris J,
Kim GH,
and
Knepper MA.
UT-A2: a 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopression.
Am J Physiol Renal Physiol
278:
F52-F62,
2000