AJP - Renal Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 274: F374-F383, 1998;
0363-6127/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elger, M.
Right arrow Articles by Hentschel, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elger, M.
Right arrow Articles by Hentschel, H.
Vol. 274, Issue 2, F374-F383, February 1998

Na-Pi cotransport sites in proximal tubule and collecting tubule of winter flounder (Pleuronectes americanus)

M. Elger, A. Werner, P. Herter, B. Kohl, R. K. H. Kinne, and H. Hentschel

Institut für Anatomie und Zellbiologie I, Universität Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg; Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, D-44139 Dortmund, Germany; and Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Localization of a recently described and cloned Na-Pi cotransport system from flounder was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of microdissected tubules and by immunocytochemistry of kidney of winter flounder. Histological examination showed a small glomerulus, an extremely short proximal tubule PI with a selective affinity to Lens culinaris agglutinin from lentils, and an extensive second proximal tubule segment PII (>90% of proximal tubules), consisting of cells with numerous apical clear vesicles and extensive amplification of basolateral cell membranes. PII merged with the collecting tubule/collecting duct (CT/CD) system without a distal segment. By RT-PCR, PII cells revealed high levels of NaPi-II related RNA; low levels were also observed in CTs. Previously characterized antisera against different epitopes of flounder NaPi-II specifically labeled the basolateral regions of PII and the apical cell portion of CT/CD cells and of some PII cells. These results suggest that tubular secretion of Pi occurs in PII of teleost fish with modulation of urinary Pi content in the subsequent CT/CD system.

proximal tubule; immunocytochemistry; reverse transcription-polymerase chain reaction; sodium-phosphate cotransporter; collecting duct; flounder kidney

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PHOSPHATE BALANCE and utilization in adult vertebrates depend on dietary uptake, storage, and renal excretion of phosphate (Pi). In the mammalian kidney, phosphate excretion is regulated by variation of phosphate reabsorption in the proximal tubule. Na-dependent transport of phosphate through the apical membrane of proximal tubule cells by a well-defined transport system (NaPi-II) is thought to be the rate-limiting step in this homeostatic process (4, 27, 29, 30, 34).

In addition to renal phosphate reabsorption, net secretion of phosphate was observed in piscine and avian kidneys (for review, see Ref. 39). Moreover, it was shown that freshwater carp respond to an intravenous infusion of Pi by a considerable increase in tubular secretion of phosphate (26). Studies using micropuncture in elasmobranch fish (43), transport by isolated tubules (7), or cell culture (17) of teleost kidney tubules indicated that the proximal tubule is the site of net phosphate secretion. However, the nature of the transepithelial transport was unknown.

At variance to the mammalian proximal tubule, which consists of three mainly reabsorptive segments, S1-S3, a variable number of proximal tubule segments has been described in different fish species (25). Based on their morphology and functional characteristics, the proximal tubule portions in fish fall into two major categories, namely the (early) proximal tubule PI and the (late) proximal tubule PII (21). The first segment displays the morphological features of an apical endocytic apparatus indicative of an absorptive epithelium. The uptake of markers such as ferritin in plaice (36) or fluid phase markers in dogfish (23) is restricted to this segment. The second segment is clearly distinguished in many teleosts and in the elasmobranch kidney, because the apical endocytic apparatus is absent and uptake of fluid phase markers from the lumen is not observed (13, 14, 19, 21).

Recently, a Na-Pi cotransport system was cloned from the flounder Pleuronectes americanus, which shows a high homology to the mammalian renal Na-Pi (type II) transport system (46). The flounder Na-Pi transport system also displays transport kinetics similar to those in the mammalian kidney when expressed in Xenopus oocytes. In view of the morphological bipartition of the proximal tubule in fish (21), we aimed to study the distribution of NaPi-II in flounder kidney. We used reverse transcription (RT)-polymerase chain reaction (PCR) on microdissected renal tubule segments for detection of NaPi-II-related mRNA in nephron segments and immunocytochemistry on tissue sections for cellular localization of NaPi-II-related protein.

Our results indicate that, in the flounder, NaPi-II message and related protein are present in PII and collecting tubule and collecting duct (CT/CD) cells. The intracellular distribution differs, however. In PII, immunohistochemical labeling is found mainly in the basal portion of the cells, whereas in the CT/CD system, an apical localization predominates. The results suggest that phosphate secretion by the proximal tubular segment PII can be subsequently modulated by the CT/CD system.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals

Male and female winter flounder, Pleuronectus (formerly Pseudopleuronectes) americanus (total body length 15-25 cm) were captured by local fishermen in Frenchman Bay (osmolality of the sea water approximately 850 osmol/kgH2O) for Mount Desert Island Biological Laboratory during July and August. The fish were maintained for no longer than 2 wk before use in 2,000-liter tanks with running aerated seawater (average temperature 15°C). In addition, several animals were provided by AquaBios Product Sciences, Salsbury Cove, ME.

RT-PCR of Microdissected Renal Tubules

Microdissection of renal tubules. After decapitation of the animals, the kidneys were excised and transferred into ice-cold flounder Ringer solution (in mM: 140 NaCl, 2.5 KCl, 1 CaCl2, 1 MgCl2, and 20 NaHCO3, adjusted to approximately pH 7.3 by 2% CO2-98% O2). All chemicals were purchased from Sigma Chemical (St. Louis, MO; molecular biology grade). The kidney was cut into small pieces, and microdissection of tubule segments was performed in ice-cold flounder Ringer solution (15).

PI was identified by its connection to the glomerulus; the small glomeruli were searched for by looking for intrarenal arteries or by tracking the CD system back to the smaller CTs, to which the glomeruli have a close spatial association. The transition of PI to PII was recognized by an abrupt increase in the tubular diameter. PII of developing nephrons was extensively coiled, and PII of mature nephrons frequently showed wider bends, which by their curvature were generally differing from the straight rami of the furcating CD system. CTs and CDs could be rapidly dissected out of the caudal part of the kidney. For RT-PCR, only tubules that could be easily freed from the interstitial tissue were used. PI was either separated from the neck segment and the glomerulus, or all three were pooled, without a difference in results. Pieces (0.5-2 mm long) of single defined tubule segments were transferred to small Eppendorf tubes containing 10 ml of flounder Ringer solution plus 1 U/ml of ribonuclease (RNase) inhibitor (RNasin; Promega), using a Pasteur pipette coated by briefly dipping into a 0.1% bovine serum albumin (BSA) solution, frozen, and stored in liquid nitrogen or at -80°C until use for RT-PCR.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Lectin binding to renal structures in histological sections of flounder kidney

RT-PCR. RT-PCR of microdissected tubules was performed according to the protocol described for RT-PCR, using total RNA of flounder kidney (28) and the RT system from Promega. The frozen tubules were kept on ice, and the prepared RT mix was added. The microdissected pieces were thawed during short mixing and spinning. The reaction was performed for 30 s at 42°C. The 20-µl RT sample contained the tubules, 5 mM MgCl2, 10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 1 mM of each nucleotide, 20 units RNasin, 0.25 mg oligo(dT) primer, and 15 units avian myeloblastosis virus reverse transcriptase. The tubes were chilled on ice, and the cDNA was used immediately for PCR [10 ml RT mix, and, in addition, 50 mM KCl, 10 mM Tris · HCl (pH 9.0), 0.1% Triton-X 100, 1.5 mM MgCl2, 40 mM of each primer, and 2.5 units of Taq DNA polymerase (Promega or Perkin-Elmer Cetus)]. The flounder NaPi-II specific primers (22 mers) are located at positions 1312 and 2126. The actin primers derive from the beta -actin genomic sequence from the common carp (Cyprinus carpio). Therefore, the length of the amplified fragments obtained with flounder tissues are close approximations. The 25-mer primers start at nucleotides 2352 (sense) and 3219 (antisense). The sequences of both NaPi-II and actin-specific primers are published (28). After an initial melt for 60 s at 94°C, 35 two-step cycles (10 s at 94°C, 55 s at 60°C, tube control mode) with a final polymerization step (5 min at 72°C) were performed in an Omnigene thermocycler (Hybaid).

Antibody Preparation

Polyclonal antibodies against a sequence from the NH2-terminal (AK33 and AK34) or against a sequence from a presumably extracellular hydrophilic loop (AK54 and AK55) of the type II Na-Pi cotransporter from flounder were raised and previously characterized (28). In the present study, we used immune sera against both portions.

Quantitative Histology

Three sexually mature females of flounder were anesthetized with tricaine (MS 222; Sigma), and renal tissue was fixed in situ by dripping ice-cold fixative [2% paraformaldehyde (PFA) and 0.5% picric acid in 80% ethanol; fixative I] on the exposed kidney surface for 5 min. Tissue blocks were fixed for an additional hour in the same fixative and embedded in paraffin. Serial sections (7 µm thickness, 100 µm apart) were prepared from three regions (cranial, middle, caudal) of the elongated kidney and stained with alcian blue-periodic acid- Schiff reagent for histochemical detection of neutral and acid glycoconjugates. This staining allowed clear identification of nephron segments in this species. For morphometry, 10 entire cross sections (~300 µm apart from each other) from the middle portion of the kidney were photographed and printed at a final magnification of ×250. Volumes of glomeruli, proximal tubule segments, and the CT/CD system in relation to total kidney volume as well as in relation to total volume of glomeruli and tubules combined were determined by the point-counting technique according to standard stereological formulas (45).

Immunohistochemistry

Renal tissue was preserved by one of the following protocols: 1) fixative I was used according to the procedure described above, and 2) fixative II consisting of 2.5% PFA, 0.1% glutaraldehyde, and 0.2% picric acid in Sorensen's buffer, pH 7.4, was perfused through the vasculature as described previously (18). In brief, a polyethylene cannula was introduced through the heart chamber into the bulbus, and the chilled fixative was perfused for 5 min at a pressure of 100-120 mmH2O. Small kidney pieces were washed in cold phosphate-buffered saline and frozen in isopentane cooled by liquid nitrogen. Seven to ten micrometer cryosections were prepared on a Frigocut E cryostate (Reichert-Jung) or Frigomat (Leica). In addition, cryosections of unfixed tissue were obtained after quick-freezing of tissue samples in melting isopentane cooled with liquid nitrogen.

Before immunolabeling, the sections were treated with 0.2% borohydride for 10 min. Blocking of unspecific binding sites was performed for 15 min either with 10% horse serum and 0.1% BSA in PBS or with 50 mM glycine in PBS followed by PBS containing 5% goat serum, 0.2% gelatin, and 0.5% BSA (24). Antisera against NaPi-II protein were diluted 1:300 to 1:500 in PBS containing 0.2% gelatin and 0.5% BSA and applied for 1-2 h at room temperature or overnight at 4°C. The reaction was visualized with goat anti-rabbit immunoglobulin G antibody conjugated to Cy3 (Dianova, Hamburg, Germany) using the same vehicle as for the primary antibody. In double and triple labeling experiments with Lens culinaris agglutinin (LCA; see below), the lectin was diluted 1:50 in the same solution. 4',6-Diamidino-2-phenylindole (Sigma) was used as a nuclear counterstain. Sections incubated with preimmune serum instead of primary antiserum or without incubation with primary antiserum served as controls. Further controls involved incubation with the antibody in the presence of the peptide antigen. The sections were examined by epifluorescence with the Axiophot microscope with Plan-Neofluar lenses (Zeiss) and by confocal laser scanning microscopy (Bio-Rad 600).

Lectin Histochemistry

For the identification of proximal tubule segments in immunohistochemical sections, 19 plant lectins (Table 1) were screened for their affinity to flounder kidney structures on deparaffinized sections of tissue preserved with fixative I. Incubation with the lectins conjugated with fluoresceine thiocyanate or tetramethylrhodamin isothiocyanate (Vector Laboratories) was performed for 1 h at room temperature in varying dilutions, from 1:40 to 1:120, corresponding to concentrations of 1-0.3 mg/ml, as described previously (22).


View larger version (155K):
[in this window]
[in a new window]
  		Fig. 1.   Apical zone of proximal tubule segment II (PII) cell. Smooth clear vesicles (a few are marked with asterisks) abound in the region beneath the brush border, which consists of long slender and loosely arranged, irregular microvilli. Single cilium is frequently revealed on micrographs (arrow). Mitochondria display only a few cristae in a dense matrix, thereby differing largely from mitochondria in PI cells. Endocytic organelles, such as dense tubules and large early endosomes, which are characteristically found in PI, are lacking in PII. Transmission electronmicrograph; magnification, ×32,000.

Electron Microscopy

Small blocks were dissected from kidneys fixed by perfusion with fixative II (see Immunohistochemistry, above), immersed for 2 h in fresh fixation fluid containing 2% PFA, 1.5% glutaraldehyde, and 0.5% picric acid in Sorensen's buffer pH 7.4, postfixed in 1% OsO4 in Sorensen's buffer, pH 7.4, dehydrated via a graded series of ethanol (20), and embedded in resin (Spurr's mixture). Thin sections were obtained with an ultramicrotome (Ultracut E; Reichert). Sections were stained with uranyl acetate and lead citrate and viewed with Zeiss EM 902 and Philips 301 electronmicroscopes.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Quantitative Histology

To get an overview on the general composition of the kidney, we first performed quantitative histology on paraffin sections, studied the carbohydrate moieties of membrane proteins by testing a variety of lectins, and examined the fine structure of renal tubular cell types.

The nephron proper was comprised of only two defined segments ("pauci-segmental nephron"; see Ref. 21). Glomeruli were very small, containing only a few capillary loops. An inconspicuous neck portion led to the proximal tubule consisting of two segments, PI and PII. PI was a proximal segment displaying an apical tubulovesicular apparatus of coated pits, coated vesicles, dense apical tubules as well as early endosomes, and a prominent lysosomal apparatus (results not shown), whereas, in PII cells, these features were lacking. Instead, the apical cytoplasm contained numerous small clear vesicles (Fig. 1). The brush border of PI cells was regular and rather dense, displaying many groups of two to three microvilli rooted in a common base. In contrast, at the apical cell membrane of PII cells, there were only few irregular long and slender microvilli, which were predominantly located near the lateral cell margin. The basolateral cell membrane of the PI and PII was amplified by an elaborate system of infoldings into the cytoplasm as is typical for the teleost kidney (Ref. 21 and Fig. 2); adjacent cells did not interdigitate at variance to most other vertebrate classes. In addition to single cilia projecting from the brush-border cells, multiciliated cells were present in both PI and PII. An intermediary segment and a distal segment were lacking. The PII tubules merged with the ramifying system of CTs and CDs. In the CT/CD cell system, the apical cytoplasm was rich in mucus granules (Fig. 3).


View larger version (177K):
[in this window]
[in a new window]
  		Fig. 2.   Basal region of PII cells. Numerous infoldings of the basolateral cell membrane are present. These folds have, close to the basolateral cell surface, a tubular appearance. Transmission electronmicrograph; magnification, ×29,000.


View larger version (158K):
[in this window]
[in a new window]
  		Fig. 3.   Apical zone of collecting tubule (CT) cell. Short stubby microvilli are occasionally present at the apical cell pole. Dense mucous vesicles (arrows) containing fibrous material are located at the apical cell membrane. Mitochondria are numerous and contain many cristae in a light matrix. Transmission electronmicrograph; magnification, ×40,000.

For the differentiation of PI and PII in immunohistochemical sections, we searched for a marker by screening a variety of lectins for their affinity to flounder kidney structures. The results are compiled in Table 1. The lectin LCA turned out to be suitable for this purpose, since in the renal tubule it reacted exclusively with the brush border only in the PI. This lectin (conjugated to a fluorochrome) thereafter served as a marker for PI in double-label experiments for immunohistochemistry (Fig. 6B).

Stereological examination revealed that glomeruli plus renal tubules (i.e., proximal tubule segments PI and PII and CT/CD) occupy less than one-third of the total kidney. Within this fraction of volume, glomeruli and PI were present with ~10% each and the CT/CD system with <10%. Thus roughly threequarters of renal tubules in the kidney of winter flounders were PII segments (Table 2). The renal tubules were embedded in an elaborate interstitial tissue, which occupied >65% of kidney tissue. The interstitial tissue contained mononuclear lymphoid-hematoblast cells, melanomacrophage centers, arterial vessels, and the sinusoid capillaries of the venous renal portal system.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Volume densities of renal structures in Pleuronectes americanus

RT-PCR of Microdissected Tubules

Several entire tubules were dissected out of fresh renal tissue (Fig. 4). Long nephrons extended from the dorsal glomeruli to the CT/CD system at the ventral side of the kidney. In addition, short, densely coiled nephrons were located in the vicinity of the CT/CD system. These tubules prevailed in the cranial portion of the kidney. The proximal segment PI was always short and coiled (1 to 2 bends).


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 4.   Microdissected renal tubules. Long nephron and a short coiled nephron (top, left) are shown. A small glomerulus (G) leads via a narrow neck segment to the short first brush-border segment PI, which has 2 to 4 bends in the vicinity of the glomerulus. The elaborate second proximal segment PII is distinctly thicker than PI and joins directly the CT/collecting duct (CD) system. Arrows mark the transition between PI and PII. Magnification, ×100.

RT-PCR starting from single microdissected tubule segments is very sensitive to contaminations with genomic DNA. To distinguish the amplicons derived from cDNA or genomic DNA, the primers were located on different exons of the genes. Whereas cDNA gave rise to fragments of 836 bp (NaPi-II) and 700 bp, the genomic DNA-derived amplicons were of 1245 and 900 bp, respectively.

RT-PCR revealed a distinct pattern of distribution of NaPi-II-related mRNA (Fig. 5, top). High levels of NaPi II-related mRNA were found in the second proximal segment PII, whereas the first segment PI and the glomeruli were negative. Segments of the CT/CD system expressed low levels of NaPi-II-related mRNA.


View larger version (68K):
[in this window]
[in a new window]
  		Fig. 5.   Expression of NaPi-II-related mRNA in microdissected renal tubule segments by polymerase chain reaction (PCR) after reverse transcription (RT) of mRNA. RT-PCR reaction was performed with single pieces of proximal tubule segments PI (lanes 1 and 2) and PII (lanes 3 and 4) and of the CT/CD system (CD; lanes 5 and 6). DNA fragments were separated on a 1% agarose gel and blotted. Samples in top were probed for NaPi-II, and bottom shows actin control. High levels of NaPi-II-related mRNA were revealed in PII, whereas PI was negative. Bottom represents the control PCR performed in parallel with primers for beta -actin. Lanes 7-9 show controls with purified RNA (lane 7 without RT, lane 8 with RT) or DNA (lane 9). To guarantee optimal sensitivity, a Southern blot is presented.

To control the reliability of the whole experiment, a NaPi-II-specific fragment and an actin fragment were amplified in parallel from the same RT sample. The presence of a cDNA-derived actin band (even a very faint one) demonstrates a functional RT. Therefore, the missing NaPi fragment in the tubular segment PI was not due to experimental problems. To increase sensitivity, the DNA was blotted and probed for NaPi-II and actin, respectively.

Immunohistochemistry

Because the RT-PCR results indicated a presence of NaPi-II mRNA in PII and CT/CD cells, the expression of the protein in the renal tubule was studied by immunohistochemistry.

Immunohistochemical localization of NaPi-II-related protein was performed with the two types of antisera, AK33/AK34 and AK54/AK55, directed against the NH2-terminal and a hydrophilic probably extracellular portion of this protein, respectively. Independent of the animals, of the fixative, or further treatment of the tissue, both types of antisera confirmed the results obtained by RT-PCR. The PII cells displayed specific reaction, whereas PI generally showed no labeling. The glomerulus and the neck segment were always negative. The CT/CD system in four animals of a total of seven animals that were investigated showed distinct reaction with AK55.

Labeling in PII profiles was in a basolateral and/or an apical location. A distinct basolateral localization with AK33 was obtained in paraffin sections with alcoholic PFA fixation (Fig. 6A), corresponding to previous results (28), as well as with formaldehyde glutaraldehyde mixture. AK55 in addition to labeling at the basal side showed binding in an apical cytoplasmic region (Fig. 7). AK34, when applied to cryostat sections of kidneys fixed with PFA, PFA-GA mixture, or of native tissue consistently displayed labeling in the basolateral region in most PII profiles (Fig. 8). Staining of the brush border was unspecific, as revealed by controls with preimmune serum. Control sections preincubated with antigenic peptide were negative.


View larger version (150K):
[in this window]
[in a new window]
  		Fig. 6.   Fluorescence microscopical localization of NaPi-II by double labeling of a histological section. Fixation with alcoholic formaldehyde solution and paraffin embedding. Magnification, ×200. A: binding of AK33 is very intense at the basolateral side of PII cells. Glomeruli (GL), PI, and CT display only faint signal by indirect immunofluorescence, respectively. Note autofluorescence of melanomacrophage centers (asterisks). B: Lens culinaris agglutinin-fluorescein isothiocyanate marks the brush border of PI cells, as well as endothelial cells of the glomeruli and of the sinusoid capillaries in the interstitium. Arrow points to a transition of PI to PII. Magnification, ×200.


View larger version (116K):
[in this window]
[in a new window]
  		Fig. 7.   Fluorescence microscopical localization of NaPi-II with antiserum AK55. Perfusion fixation with 2.5% formaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid in Sorensen's buffer, paraffin embedding. Pronounced immunostaining is seen at the apical region of CT cells, and less intense binding occurs at the base of many CT cells. Several PII profiles are well stained, and the antigen is present at the basolateral side of the cells as well as at the apex. Arrows mark the transition of PII to CT. Magnification, ×185.


View larger version (70K):
[in this window]
[in a new window]
  		Fig. 8.   Fluorescence microscopical localization of NaPi-II with antiserum AK34. Perfusion fixation with 2.5% formaldehyde, 0.1% glutaraldehyde, snd 0.2% picric acid in Sorensen's buffer, cryostat section. Antibody binds markedly in the region of basal infoldings of PII cells. Faint staining is present at the lateral cell borders and in the apical region of the cells. Magnification, ×480.

In the CT and CD, a clear reaction with AK54 and AK55 was observed in the apical zone, and irregular staining was observed at the basolateral side (Figs. 7, 9, and 10). Early stages of developing nephrons merging with the CTs did not bind the antibody (Fig. 9), and late stages of developing nephrons displayed only faint labeling (Fig. 10). Reaction intensities in these segments varied greatly between individual fish.


View larger version (68K):
[in this window]
[in a new window]
  		Fig. 9.   Confocal laser scanning micrograph of a cross section of CT. Perfusion fixation with 2.5% formaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid in Sorensen's buffer, paraffin embedding. Antiserum AK55 selectively binds to the apical region of epithelial cells. In addition, many cells display a low amount of binding at the basal and/or lateral cell membrane. Arrow points to a gap in the epithelium of the CT, indicating insertion of a nephron anlage, which has not yet aquired immunoreactivity to NaPi-II. Parallel sections revealed that a lumen was not present in the anlage. Magnification, ×570.


View larger version (93K):
[in this window]
[in a new window]
  		Fig. 10.   Confocal laser scanning micrograph of a longitudinal section of CT. Perfusion fixation with 2.5% formaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid in Sorensen's buffer and paraffin embedding. Apical region of all epithelial cells is distinctly stained. In addition, several cells show specific binding of the antiserum to the basal and/or lateral cell membrane region. Arrow indicates the location where a newly formed nephron with moderate immunoreactivity to NaPi-II merges with the CT. Magnification, ×560.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recently, a Na-Pi cotransport system was cloned from winter flounder (46). Here, we describe the localization of NaPi-II-related mRNA along the nephron performing RT-PCR with microdissected flounder tubules. In addition, because the nephron segmentation of the winter flounder kidney had not been studied systematically, we also performed qualitative and quantitative histology. Subcellular distribution of NaPi-II-related protein was investigated by light microscopic immunohistochemistry.

Nephron Segmentation

Our study shows that the nephron of the winter flounder consists exclusively of segments belonging to the proximal tubule. A nephron segment with the morphological characteristics of the first segment of the distal nephron (commonly referred to as the diluting segment in most vertebrate kidneys; see Ref. 20) was lacking. This finding is in agreement with observations on two other marine species of flounders (5, 35, 37) but is different from observations in the southern flounder (25), where a distal segment was observed. Furthermore, our quantitative results revealed that <7% of the proximal tubule of winter flounder (segment PI) corresponds to the proximal tubule of other vertebrates, displaying an apical endocytotic apparatus of endosomes and dense tubules, which is functionally linked to the lysosomal apparatus (32). In analogy to experiments with tracer in other fish [plaice (36) and dogfish (23)], we believe that only the short PI portion is involved in reabsorption of filtered substances of high molecular weight. Almost the entire proximal tubule, namely >93%, belongs to the PII, which lacks the morphological correlates of protein uptake present in PI. Instead, the apical cytoplasmic zone contains an extensive system of smooth clear vesicles. In marine elasmobranchs, the corresponding proximal tubule segment (PIIa; see Ref. 19) was shown to secrete salt and fluid (3), similar to the proximal tubule of aglomerular teleost fish.

The prevalence of the secretory proximal tubule segment PII over the reabsorptive first portion of the proximal tubule (PI) clearly is of adaptive value for a marine fish. As a teleost species that predominantly lives in a marine environment, P. americanus relies on a very low glomerular filtration rate to minimize water loss by filtration, and urine can be expected to be mainly formed by secretion of salt and fluid in the secretory PII, comparable to the urine production in aglomerular teleost fish (see, e.g., Ref. 2). The winter flounder has only a limited capability to adapt to water of lower salinity (brackish water), where glomerular filtration is more important than in sea water (12, 40). The low amount of the reabsorptive PI therefore is in accordance with the minor role of glomerular filtration, which has its morphological correlate in the minor development of the glomerulus in size and number. Thus the functional unit of glomerulus and reabsorptive PI is greatly reduced in favor of an elaborate secretory proximal tubule segment. Renal mechanisms of filtration and proximal reabsorption are entirely suppressed in aglomerular fish. Interestingly, the extreme condition of morphologically aglomerular kidney has evolved in a relative of the flounder, the lemon sole (6). Our stereological results indicate that the winter flounder with its very reduced reabsorptive PI and loss of the distal nephron segment is well on the way to an aglomerular fish.

Localization of NaPi-II Along the Proximal Tubule

In the RT-PCR experiments, mRNA of the flounder Na-Pi cotransporter in the proximal tubule was restricted to the PII. NaPi-II-related protein seemed also to be confined to the PII in this species. The apparent lack of NaPi-II in the PI may be surprising in view of the great homology of this transport system to the mammalian NaPi-II-type transporter. It can, however, be explained by the minor development of the PI in this species. Thus our results provide evidence that the secretory PII segment is an important site of phosphate regulation in the kidney of this marine teleost.

Intracellular Distribution of NaPi-II-Related Protein in PII

Immunocytochemistry revealed localization of NaPi-II-related protein predominantly at the basolateral cell region, confirming previous results (28). By the investigation of a larger number of specimens in the present study, a reaction was revealed in an apical cytoplasmic zone of a moderate number of PII cells (10-20%). The flounder renal proximal tubule is, to the best of our knowledge, the only renal epithelium in which a Na-Pi transport system has been described at the basolateral cell pole in this and our previous paper (28). As Pi secretion in teleosts was found to occur in the proximal tubule (7), it may be concluded that the flounder NaPi-II transporter is involved in this process when located at the basolateral membrane.

Stimulation of net secretion of phosphate can be induced in flounder proximal tubule cell culture and chick proximal tubule by substances such as thapsigargin and the calcium ionophore A-23187 and by parathyroid hormone, respectively (1, 17, 31). Based on these studies, we suggested a similar mechanism for phosphate secretion as for phosphate reabsorption in the mammalian proximal tubule. Phosphate exit into the lumen was shown to be voltage gradient dependent (electrically negative) and potassium dependent. Phosphate entry into the cell at the base of the epithelium is Na dependent and was proposed to occur via a Na-Pi cotransporter. Physiological evidence for a Na-dependent symporter for phosphate located in the basolateral membrane was also obtained in a different mammalian epithelium, the sheep parotid gland, which secretes phosphate in excess of the dietary intake (42, 44).

Reabsorption of phosphate by PII cells was observed in cultured flounder epithelial cell layers (11). These cells show morphological similarity with PII cells, including the apical vesicular compartment typical for this cell type. Stanniocalcin and ovine prolactin led to rapid stimulation of net reabsorption of phosphate (39).

Localization of NaPi-II in the CT/CD System

In addition to the proximal tubule, we observed mRNA for NaPi-II in cells of the CT/CD. In several fish, labeling with AK55 revealed specific, peptide-protectable immunostaining in the epithelial cells of these portions of the renal tubule. These observations extend our previous histochemical results (28). The predominantly apical localization in the upper one-third of the cells suggests the presence of NaPi-II transporter in the apical cell membrane as well as in the apical cytoplasmic zone. Thus the presence of NaPi-II in this tubule portion may be indicative of phosphate reabsorption. Reabsorption in the lower renal tubule should alter the phosphate content of the final urine. Therefore, the CT/CD would have a modulating function for net renal Pi excretion. This would be especially important under conditions of reduced glomerular filtration ("aglomerular" urine formation; see below).

Although our results with marine flounder suggest that mechanisms of proximal secretion and subsequent reabsorption are operative in teleosts, similar mechanisms apparently are present, albeit less well developed, in other vertebrates. In this context, it is relevant that NaPi-II mRNA was detected in rat kidney cortical and medullary CDs (8). Moreover, physiological evidence for distal reabsorption of Pi has been presented by experiments with Pi-deprived rats (16, 38) and desert rodents, the gundi, Ctenodactylus vali (41).

Functional Considerations of Phosphate Excretion as Related to Glomerular Filtration

By the paucity or even lack of glomerulus and PI in many marine species, renal control of phosphate involving a mechanism of primary filtration and subsequent reabsorption is not feasible, as clearly shown in the case of aglomerular fish. Therefore, these animals must have the possibility to excrete excess phosphate by an additional tubular mechanism. This is corroborated by the observation of a significant secretion of serum phosphate by renal tubules in carp (26) and in the avian kidney (1). We have obtained evidence of glomerular intermittency of glomerular blood perfusion and/or filtration in conscious carp (15a). This indicates the presence of functionally aglomerular nephrons in kidneys of teleosts with glomerular kidneys, which can explain the highly variable glomerular filtration rate, which is generally observed in fish (25). Glomerular intermittency has been observed in all vertebrate classes from fish (except cyclostomes; see Ref. 33) to birds (for review, see Refs. 10, 21). Proximal tubular secretory mechanisms for salt, water, and other solutes are commonly found in those vertebrate groups, which display intermittent function of glomeruli (10).

In summary, the RT-PCR and immunohistochemical observations with flounder kidney indicate the presence of a NaPi-II cotransport system in two subsequent portions of the renal tubule, the secretory proximal segment PII, and the CT/CD system. By the basolateral localization of NaPi-II-related protein, the PII is identified as the site of tubular phosphate excretion characteristically observed in phosphate-loaded teleosts. In the CT/CD cells, the predominantly apical localization of this transport protein strongly suggests a reabsorptive capacity for Pi in this lower portion of the renal tubule, thus modulating the amount of urinary Pi.

    ACKNOWLEDGEMENTS

We thank Angela Friedrich and Rita Haubrock for help with the histology and the immunohistochemistry and Dr. Hanna Tinel and Alexander Giffey for help with the confocal laser scanning microscope.

    FOOTNOTES

This work was supported by Max-Planck-Gesellschaft, Deutsche Forschungsgemeinschaft (Travel grant EL 92/3-2 to M. Elger), and Mount Desert Island Biological Laboratory (Fellowship to H. Hentschel).

Address for reprint requests: H. Hentschel, Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, D-44139 Dortmund, Germany.

Received 18 February 1997; accepted in final form 31 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Barber, L. E., V. Coric, N. B. Clark, and J. L. Renfro. PTH-sensitive K+- and voltage-dependent Pi transport by chick renal brush-border membranes. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F822-F829, 1993[Abstract/Free Full Text].

2.   Beyenbach, K. W., and M. D. Baustian. Comparative physiology of the proximal tubule. From the perspective of aglomerular urine formation. In: Structure and Function of the Kidney. Comparative Physiology, edited by R. K. H. Kinne. Basel: Karger, 1989, vol. 1, p. 103-142.

3.   Beyenbach, K. W., and E. Frömter. Electrophysiological evidence for Cl- secretion in shark proximal tubule. Am. J. Physiol. 248 (Renal Fluid Electrolyte Physiol. 17): F282-F295, 1985.

4.   Biber, J., M. Custer, S. Magagnin, G. Hayes, A. Werner, M. Lotscher, B. Kaissling, and H. Murer. Renal Na/Pi-cotransporters. Kidney Int. 49: 981-985, 1996[Medline].

5.   Bulger, R. E., and B. F. Trump. Renal morphology of the English sole (Parophrys vetulus). Am. J. Anat. 123: 195-226, 1968[Medline].

6.   Christensen, J. A., R. Taugner, D. S. Meyer, and A. Bohle. The granular epitheloid cells in the kidney of the lemon sole (Pleuronectes microcephalus Donovani). Cell Tissue Res. 249: 137-143, 1987[Medline].

7.   Cliff, W. H., and K. W. Beyenbach. Fluid secretion in glomerular renal proximal tubules of freshwater-adapted fish. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R154-R158, 1988[Abstract/Free Full Text].

8.   Custer, M., M. Lotscher, J. Biber, H. Murer, and B. Kaissling. Expression of Na-Pi cotransport in rat kidney: localization by RT-PCR and immunohistochemistry. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F767-F774, 1994[Abstract/Free Full Text].

9.   Damjanov, I. Biology of disease. Lectin cytochemistry and histochemistry. Lab. Invest. 57: 5-20, 1987[Medline].

10.   Dantzler, W. H. Comparative Physiology of the Vertebrate Kidney. Berlin: Springer Verlag, 1989.

11.   Dickman, K. G., and J. L. Renfro. Primary culture of flounder renal tubule cells: transepithelial transport. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F424-F432, 1986.

12.   Elger, E., B. Elger, H. Hentschel, and H. Stolte. Adaptation of renal function to hypotonic medium in the winter flounder (Pseudopleuronectes americanus). J. Comp. Physiol. [B] 157: 21-30, 1987[Medline].

13.   Elger, M., and H. Hentschel. Light- and electronmicroscopic results on the segmentation of the nephronic tubule of the dogfish, Scyliorhinus caniculus (Abstract). L. Verh. Dtsch. Zool. Ges. 1982: 267, 1982.

14.   Elger, M., and H. Hentschel. Cell junctions in renal tubule of a fresh-water teleost, Salmo gairdneri Rich. Cell Tissue Res. 244: 395-401, 1986.

15.   Elger, M., H. Hentschel, and R. K. H. Kinne. The proximal tubule of the winter flounder, Pseudopleuronectes americanus, as revealed by microdissection and quantitative histology. Bull. Mt. Desert Isl. Biol. Lab. 34: 97-99, 1995.

15a.   Elger, M., R. Kaune, and H. Hentschel. Glomerular intermittency in a freshwater teleost, Carassius auratus gibelio, after transfer to saltwater. J. Comp. Physiol. [B] 154: 225-231, 1984.

16.   Greger, R., F. Lang, G. Marchand, and F. G. Knox. Site of renal phosphate reabsorption. Pflügers Arch. 369: 111-118, 1977[Medline].

17.   Gupta, A., and J. L. Renfro. Control of phosphate transport in flounder renal proximal tubule primary cultures. Am. J. Physiol. 256 (Regulatory Integrative Comp. Physiol. 25): R850-R857, 1989[Abstract/Free Full Text].

18.   Hentschel, H. The kidney of Spinachia spinachia (L.) Flem. (Gasterosteidae). 1. Investigations of juvenile sticklebacks: anatomy, circulation and fine structure. Z. Mikroskop. Anat. Forsch. 91: 4-21, 1977.

19.   Hentschel, H., and M. Elger. Structural organization of the elasmobranch kidney. Verh. Dtsch. Zool. Ges. 75: 263, 1982.

20.   Hentschel, H., and M. Elger. The distal nephron in the kidney of fishes. Adv. Anat. Embryol. Cell Biol. 108: 1-147, 1987[Medline].

21.   Hentschel, H., and M. Elger. Morphology of glomerular and aglomerular kidneys. In: Comparative Physiology. Structure and Function of the Kidney, edited by R. K. H. Kinne. Basel: Karger Verlag, 1989, p. 1-72.

22.   Hentschel, H., E. Kinne-Saffran, R. K. H. Kinne, M. Elger, and J. N. Forrest, Jr. Glycoconjugates in the rectal gland of spiny dogfish, Squalus acanthias, as revealed by lectin binding to cryostat sections. Bull. Mt. Desert Isl. Biol. Lab. 34: 32-35, 1995.

23.  Hentschel, H., and P. Schulte-Holthey. Fluid phase endocytosis of FITC-dextran in the proximal tubule of elasmobranch, Scyliorhinus caniculus (Abstract). Eur. J. Cell Biol. 37, Suppl. 60: 150, 1993.

24.   Herter, P., M. Elger, B. Kohl, L. Renfro, H. Hentschel, R. K. H. Kinne, and A. Werner. Immunohistochemical localisation of NaPi II-cotransport system in the kidney and intestine of winter flounder (Pleuronectes americanus). Bull. Mt. Desert Isl. Biol. Lab. 35: 28-30, 1996.

25.   Hickman, C. P., and B. F. Trump. The kidney. In: Fish Physiology, edited by W. S. Hoar, and D. J. Randall. New York: Academic, 1969, p. 91-239.

26.   Kaune, R., and H. Hentschel. Stimulation of renal phosphate secretion in the stenohaline freshwater teleost: Carassius auratus gibelio bloch. Comp. Biochem. Physiol. A Physiol. 87: 359-362, 1987.

27.   Kempson, S. A., M. Lotscher, B. Kaissling, J. Biber, H. Murer, and M. Levi. Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F784-F791, 1995[Abstract/Free Full Text].

28.   Kohl, B., P. Herter, B. Huelseweh, M. Elger, H. Hentschel, R. K. H. Kinne, and A. Werner. Na-Pi cotransport in flounder: same transport system in kidney and intestine. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F937-F944, 1996[Abstract/Free Full Text].

29.   Levi, M., M. Lotscher, V. Sorribas, M. Custer, M. Arar, B. Kaissling, H. Murer, and J. Biber. Cellular mechanisms of acute and chronic adaptation of rat renal Pi-transporter to alterations in dietary Pi. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F900-F908, 1994[Abstract/Free Full Text].

30.   Loghman-Adham, M., G. T. Motock, P. Wilson, and M. Levi. Characterization of Na+-phosphate cotransporters in renal cortical endosomes. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F93-F102, 1995[Abstract/Free Full Text].

31.   Lu, M., L. E. Barber, and J. L. Renfro. Renal transepithelial phosphate secretion: luminal membrane voltage and Ca2+ dependence. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F624-F631, 1994[Abstract/Free Full Text].

32.   Maunsbach, A. B., and E. I. Christensen. Functional ultrastructure of the proximal tubule. In: Handbook of Physiology. Renal Physiology. Bethesda, MD: Am. Physiol. Soc., 1992, vol. I, sect. 8, chapt. 2, p. 41-108.

33.   McVicar, A. J., and J. C. Rankin. Dynamics of glomerular filtration in the river lamprey, Lampetra fluviatilis. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F132-F138, 1985.

34.   Murer, H., M. Loetscher, B. Kaissling, and J. Biber. Molecular mechanisms in the regulation of renal proximal tubular Na/phosphate transport. Kidney Blood Press. Res. 19: 151-154, 1996.[Medline]

35.   Olsen, S. Ultrastructure of the nephron of a glomerular teleost: "Pleuronectes platessa." Acta Pathol. Microbiol. Scand. Suppl. 212: 81S-96S, 1970.

36.   Ottosen, P. D. Reversible peritubular binding of a cationic protein (lysozyme) to flounder kidney tubules. Cell Tissue Res. 194: 207-218, 1978[Medline].

37.   Ottosen, P. D. Ultrastructure and segmentation of microdissected kidney tubules in the marine flounder, Pleuronectes platessa. Cell Tissue Res. 190: 27-45, 1978[Medline].

38.   Poujeol, P., R. L. Jamison, and C. De Rouffignac. Phosphate reabsorption in juxtamedullary nephron terminal segments. Pflügers Arch. 387: 27-31, 1977.

39.   Renfro, J. L. Solute transport by flounder renal cells in primary culture. In: Cellular and Molecular Approaches to Fish Ionic Regulation, edited by C. M. Wood, and T. J. Shuttleworth. New York: Academic, 1995, p. 147-171.

40.   Renfro, J. L., and A. Gupta. Comparative physiology of phosphate transport across renal plasma membranes. In: Comparative Aspects of Sodium Transport Systems. Comparative Physiology, edited by R. K. H. Kinne. Basel: Karger Verlag, 1990, vol. 7, p. 216-240.

41.   Rouffignac, C. De, L. Bankir, and N. Roinel. Renal function and concentrating ability in a desert rodent, the gundi (Ctenodactylus vali). Pflügers Arch. 390: 138-144, 1981[Medline].

42.   Shirazi-Beechey, S. P., J. I. Penny, J. Dyer, I. S. Wood, P. S. Tarpey, D. Scott, and W. Buchan. Epithelial phosphate transport in ruminants, mechanisms and regulation. Kidney Int. 49: 992-996, 1996[Medline].

43.   Stolte, H., R. G. Galaske, G. M. Eisenbach, C. Lechene, B. Schmidt-Nielsen, and J. W. Boylan. Renal tubule ion transport and collecting duct function in the elasmobranch little skate, Raja erinacea. J. Exp. Zool. 199: 403-410, 1977[Medline].

44.   Vayro, S., R. Kemp, R. B. Beechey, and S. P. Shirazi-Beechey. Preparation and characterisation of basolateral plasma-membrane vesicles from sheep parotid gland. Biochem. J. 29: 843-848, 1991.

45.   Weibel, E. R. Stereological Methods. Practical Methods for Biological Morphometry. London: Academic, 1979.

46.   Werner, A., H. Murer, and R. Kinne. Cloning and expression of a renal Na-Pi cotransport system from flounder. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F311-F317, 1994[Abstract/Free Full Text].

AJP Renal Physiol 274(2):F374-F383
0363-6127/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
M. Carlile, P. Nalbant, K. Preston-Fayers, G. S. McHaffie, and A. Werner
Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs
Physiol Genomics, June 10, 2008; 34(1): 95 - 100.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
P. M. Guerreiro, J. L. Renfro, D. M. Power, and A. V. M. Canario
The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R679 - R696.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
K. W. Beyenbach
Kidneys sans glomeruli
Am J Physiol Renal Physiol, May 1, 2004; 286(5): F811 - F827.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. H. Sugiura, N. K. McDaniel, and R. P. Ferraris
In vivo fractional Pi absorption and NaPi-II mRNA expression in rainbow trout are upregulated by dietary P restriction
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2003; 285(4): R770 - R781.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
C. Graham, P. Nalbant, B. Scholermann, H. Hentschel, R. K. H. Kinne, and A. Werner
Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney
Am J Physiol Renal Physiol, April 1, 2003; 284(4): F727 - F736.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. Werner and R. K. H. Kinne
Evolution of the Na-Pi cotransport systems
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2001; 280(2): R301 - R312.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elger, M.
Right arrow Articles by Hentschel, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elger, M.
Right arrow Articles by Hentschel, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online