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Am J Physiol Renal Physiol 290: F550-F560, 2006. First published September 27, 2005; doi:10.1152/ajprenal.00151.2005
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Immunolocalization of ectonucleotidases along the rat nephron

Renu M. Vekaria,1 David G. Shirley,1 Jean Sévigny,2 and Robert J. Unwin1

1Department of Physiology and Centre for Nephrology, Royal Free and University College Medical School, London, United Kingdom; and 2Centre de Recherche en Rhumatologie et Immunologie, Université Laval, Sainte-Foy, Québec, Canada

Submitted 12 April 2005 ; accepted in final form 2 September 2005


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Evidence is accumulating that extracellular nucleotides act as autocrine/paracrine agents in most tissues, including the kidneys. Several families of surface-located enzymes, collectively known as ectonucleotidases, can degrade nucleotides. Using immunohistochemistry, we have examined the segmental distribution of five ectonucleotidases along the rat nephron. Perfusion-fixed kidneys were obtained from anesthetized male Sprague-Dawley rats. Cryostat sections of cortical and medullary regions were incubated with antibodies specific to the following enzymes: ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1, NTPDase2, NTPDase3, ectonucleotide pyrophosphatase phosphodiesterase 3 (NPP3), and ecto-5'-nucleotidase. Sections were then costained with Phaseolus vulgaris erythroagglutinin (for identification of proximal tubules) and antibodies against Tamm-Horsfall protein (for identification of thick ascending limb), calbindin-D28k (for identification of distal tubule), and aquaporin-2 (for identification of collecting duct). The tyramide signal amplification method was used when the ectonucleotidase and marker antibody were raised in the same species. The glomerulus expressed NTPDase1 and NPP3. The proximal tubule showed prominent expression of NPP3 and ecto-5'-nucleotidase in most, but not all, segments. NTPDase2 and NTPDase3, but not NPP3 or ecto-5'-nucleotidase, were expressed in the thick ascending limb and distal tubule. NTPDase3, with some low-level expression of ecto-5'-nucleotidase, was also found in cortical and outer medullary collecting ducts. Inner medullary collecting ducts displayed low-level staining for NTPDase1, NTPDase2, NTPDase3, and ecto-5'-nucleotidase. We conclude that these ectonucleotidases are differentially expressed along the nephron and may play a key role in activation of purinoceptors by nucleotides and nucleosides.

ectonucleotide pyrophosphatase phosphodiesterase 3; ectonucleoside triphosphate diphosphohydrolases 1–3; ecto-5'-nucleotidase; immunohistochemistry

EXTRACELLULAR NUCLEOTIDES (ATP, ADP, UTP, and UDP), as well as the nucleoside adenosine, are widely accepted as autocrine or paracrine signaling agents in most tissues. They act on a group of receptors, collectively known as purinoceptors, found on most epithelial cells. The purinoceptor family consists of ligand-gated P2X receptors, G protein-coupled P2Y receptors, and P1 (adenosine) receptors. Each family comprises a number of subtypes. Activation of these receptors has been found to modulate ion transport in many epithelial systems, including the kidney (27). Stimulation of apical P2 receptors in the proximal tubule inhibits bicarbonate reabsorption (2) and in vitro studies have demonstrated that P2 receptors can inhibit basolateral Na+-K+-ATPase activity in a proximal tubule cell line (17). In more distal regions of the nephron, in vivo (41) and in vitro (26) perfusion studies have shown that activation of apical P2 receptors inhibits sodium reabsorption in the collecting duct. Activation of P1 receptors expressed in the proximal tubule augments sodium and water reabsorption (46), whereas in vitro studies have shown that stimulation of P1 (A1) receptors in the thick ascending limb (TAL) of the loop of Henle (3) and the collecting duct (47) inhibits sodium reabsorption.

The extent of activation of purinoceptors is influenced by ectonucleotidases located on the surface membranes of epithelial and endothelial cells. Four families are known to exist: ectonucleotide pyrophosphatase phosphodiesterases (NPP1, NPP2, and NPP3), ectonucleoside triphosphate diphosphohydrolases (NTPDase1, NTPDase2, NTPDase3, NTPDase4, NTPDase5, NTPDase6, NTPDase7, and NTPDase8), ecto-5'-nucleotidase, and alkaline phosphatase. These families of enzymes differ in their hydrolysis pathways and/or their affinities for nucleotides. The presence of ectonucleotidases in specific segments of the nephron may have a significant and regulatory influence on the stimulation of tubular purinoceptor subtypes, inasmuch as the generation of hydrolysis products of nucleotides (in particular, ADP and adenosine) will preferentially activate ADP-sensitive P2 and P1 receptors.

Knowledge of the distribution of ectonucleotidases along the nephron is fragmentary and incomplete. Some laboratories have detected mRNA for a number of ectonucleotidases in kidney tissue homogenates (8, 10, 19), and others have demonstrated their expression in the renal vasculature (28). Very recently, Kishore and colleagues (20) identified NTPDase1 and NTPDase2 in rat and mouse kidney; alkaline phosphatase is known to be present in the proximal tubular brush-border membrane of most species including the rat (4); NPP1 has been identified in proximal and distal tubules of the mouse (14); and ecto-5'-nucleotidase expression has been documented in a number of nephron segments in the rat (11). However, a comparative study of the distribution of members of the ectonucleotidase families along well-defined nephron segments has not been reported.

The present study has used immunohistochemistry to examine the expression of five major ectonucleotidases (NTPDase1, NTPDase2, NTPDase3, NPP3, and ecto-5'-nucleotidase) along the rat nephron. Antibodies specific to well-defined segments of the nephron have been used to examine the distribution of these five enzymes in the proximal tubule, TAL, distal tubule, and collecting duct. We were able to confirm the presence of these enzymes in the kidney and to localize the specific expression of each of them along the renal tubule.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Ectonucleotidase Antibodies

Polyclonal antibodies for NTPDase1, NTPDase2, NTPDase3, and ecto-5'-nucleotidase were raised in rabbits by direct intramuscular or subcutaneous injection of the encoding cDNA ligated into the plasmid pcDNA3 by a protocol described elsewhere (21, 40). NPP3 antibody was also raised in rabbits against affinity-purified native protein as described previously (29).

The use and specificity of antibodies to NTPDase1 and NTPDase2 (20, 40), ecto-5'-nucleotidase (21), and NPP3 (31, 32) have been extensively described in previous studies.

Specificity of NTPDase3 Antibodies

Transfection and Western blotting procedures. Human embryonic kidney (HEK 293) cells were prepared and transient transfection with NTPDase3 cDNA constructs was carried out as described previously for NTPDase1 in COS-7 cells (18). Protein samples of NTPDase3-transfected cells and untreated HEK 293 cells were resuspended in NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen, Burlington, ON, Canada) under nonreducing conditions and separated on a NuPAGE 4–12% Bis-Tris gel. The separated proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) by electroblotting according to the manufacturer's recommendation (Invitrogen) and then blocked with 2.5% nonfat milk in PBS (in mM: 10.1 Na2HPO4, 1.8 KH2PO4, 136.9 NaCl, and 2.7 KCl) containing 0.15% Tween 20 (pH 7.4) overnight at 4°C. The membrane was subsequently probed for NTPDase3 by incubation with rabbit anti-NTPDase polyclonal antibody (RN3-1) for 90 min at a dilution of 1:2,000, and the bands were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham Biosciences, Baie d'Urfé, PQ, Canada) for 1 h at a dilution of 1:10,000 followed by Lightning Western Blot Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences, Boston, MA). Positively stained bands were detected only in NTPDase3-transfected cell lines (Fig. 1).



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  		Fig. 1. Western blot showing cell lysates from HEK 293 cells transiently transfected (NTPDase 3) or nontransfected (vector; negative control) with rat NTPDase3 cDNA constructs. Proteins were fractionated on NuPAGE 4–12% Bis-Tris gel under nonreducing conditions, transferred to an Immobilon-P membrane, and incubated with NTPDase3 primary antibody (RN3-1) or its preimmune control (not shown). Stained bands were exclusively detected in NTPDase3-transfected cell extracts.

 
Immunocytochemistry. COS cells were fixed in 10% phosphate-buffered formalin mixed with cold acetone. Briefly, cells were incubated in a blocking solution of 7% normal goat serum in PBS for 30 min and then incubated overnight at 4°C with primary antibodies. The cells were then incubated with 0.15% H2O2 in PBS for 10 min and incubated with avidin-biotin blocking kit (Vector Laboratories, Burlington, ON, Canada) and then with a biotin-labeled goat anti-rabbit secondary antibody. The complex avidin-biotinylated horseradish peroxidase (Vector Laboratories) was added to optimize the reaction. Peroxidase activity was revealed using 3,3'-diaminobenzidine (Sigma, St. Louis, MO) as a substrate. Cells were counterstained with aqueous hematoxylin (Biomeda, Foster City, CA) in accordance with the manufacturer's protocol. Staining was seen only in transfected cells (Fig. 2).



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  		Fig. 2. Immunocytochemistry of intact COS-7 cells transfected with NTPDase3 cDNA constructs (A and C) and nontransfected COS-7 cells (B). Cells were incubated with NTPDase3 antibody (RN3-1, A and B) or preimmune serum (C). A positive signal could be seen only with NTPDase3-transfected cells incubated with NTPDase3 antibody (A). No staining was seen in negative controls (B and C).

 
Other Markers

The proximal tubule marker Phaseolus vulgaris erythroagglutinin (PVE) was purchased from Vector Laboratories (Burlingame, CA). The proximal tubule marker for the S3 segment, neutral endopeptidase (NEP) antibody, was a gift from Prof. P. Ronco and has been previously characterized (38); the TAL marker Tamm-Horsfall protein antibody was purchased from Biogenesis (Poole, UK); the distal tubule marker calbindin-D 28K antibody was purchased from Swant (Bellinzona, Switzerland); and the collecting duct marker aquaporin 2 (AQP2) was a gift from Dr. D. Marples. The specificity of this AQP2 antibody has been previously demonstrated by immunocytochemistry (D. Marples, personal communication). Secondary antibodies for donkey anti-sheep CY3, donkey anti-rabbit CY3, and biotinylated donkey anti-rabbit and goat anti-mouse FITC were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Streptavidin-fluorescein FITC was purchased from Amersham Biosciences (Chalfont St. Giles, Bucks, UK).

Tissue Preparation

All animal procedures were approved by the Institutional Ethical Committee and were carried out under a UK Home Office License. Male Sprague-Dawley rats were anesthetized with thiopentone sodium (Link Pharmaceuticals, Horsham, UK; 100 mg/kg ip). The left kidney was perfused via the abdominal aorta, first with Hanks' balanced salt solution and then with a periodate-lysine paraformaldehyde fixative containing 2% paraformaldehyde, 0.075 M lysine, and 0.01 M sodium periodate solution, pH 7.4 (30). The perfusion-fixed kidney was isolated and left overnight in 7% sucrose at 4°C. Then it was embedded in Tissue-Tek compound and snap frozen in prechilled isopentane followed by liquid nitrogen. Sections (10 µm thick) were cut serially using a cryostat (Leica, Oberkochen, Germany) and mounted onto gelatin-coated glass slides.

Immunofluorescence

Frozen cryostat sections were allowed to air dry for several minutes at room temperature and washed three times for 5 min in 0.1 M PBS. Tissue sections were then incubated in 10% normal horse serum (NHS) prepared in PBS containing 0.05% merthiolate (NHS-PBS merthiolate) for 30 min at room temperature. These sections were incubated overnight at room temperature with antibody raised against NTPDase1 (1:600 dilution), NTPDase2 (1:1,000 dilution), NTPDase3 (1:50 dilution), NPP3 (1:400 dilution), or ecto-5'-nucleotidase (1:400 dilution), each diluted in 10% NHS-PBS merthiolate. They were then incubated with CY3-conjugated donkey anti-rabbit (1:400 dilution) immunoglobulin antibody (labeled red) or biotinylated donkey anti-rabbit antibody (1:500 dilution) for 60 min at room temperature. Sections that had been incubated with biotinylated donkey anti-rabbit antibody were incubated with streptavidin-fluorescein (FITC-green fluorophore, 1:200 dilution) diluted in 1% NHS-PBS merthiolate for 30 min at room temperature.

Proximal Tubule Staining

Sections that had been incubated with CY3-conjugated donkey anti-rabbit antibody (see above) were incubated overnight with the biotinylated lectin PVE (specific to the proximal tubule) diluted 1:1,500 in 10% NHS-PBS merthiolate and then for 30 min with streptavidin-fluorescein (FITC-green fluorophore) diluted in 1% NHS-PBS merthiolate.

Proximal Tubule S3 Segment Staining

Sections that had been incubated with CY3-conjugated donkey anti-rabbit antibody (see above) were incubated overnight with antibody specific to NEP (specific for the S3 segment of the rat proximal tubule) diluted 1:1,000 in 10% NHS-PBS merthiolate and then for 1 h with FITC-green-conjugated goat anti-mouse antibody diluted 1:200 in 1% NHS-PBS merthiolate.

TAL Staining

Sections that had been previously incubated with the biotinylated donkey anti-rabbit antibody and then with streptavidin-fluorescein (FITC-green fluorophore) were incubated overnight with antibody specific to Tamm-Horsfall protein (specific for the TAL) diluted 1:1,500 in 10% NHS-PBS merthiolate and then for 1 h with CY3-conjugated donkey anti-sheep (1:400) diluted in 1% NHS-PBS merthiolate.

All incubations were separated by three 5-min washes in 0.1 M PBS. The slides were mounted in Citifluor (Citifluor, London, UK) solution and then examined and photographed using a microscope (Zeiss Axioplan, Oberkochen, Germany) fitted with a digital camera (model DC 200, Leica, Heerbrugg, Switzerland).

Double-Immunofluorescence Labeling of Ectonucleotidases and Tubular Markers Using Unconjugated Primary Antibodies Raised in the Same Species

Tyramide signal amplification immunohistochemistry was adopted under circumstances where the marker antibody used to identify tubular segments (calbindin-D 28K, a marker for the distal tubule, and AQP2, a marker for the principal cells of the collecting duct) was raised in the same host species (rabbit) as the ectonucleotidase antibody. If we had used the conventional method (as described above for labeling the proximal tubule and the TAL), the secondary antibody (i.e., CY3-conjugated donkey anti-rabbit IgG) would have been unable to differentiate between the ectonucleotidase and marker antibody because of cross-reactivity.

Frozen sections were rinsed in 0.1 M PBS (3 times for 5 min each), incubated with 10% NHS-PBS merthiolate for 30 min, and then incubated overnight at room temperature with antibody specific to NTPDase1 (1:9,000), NTPDase2 (1:12,000), NTPDase3 (1:1,200), NPP3 (1:4,000), or ecto-5'-nucleotidase (1:4,000), prepared at a dilution (in 10% NHS-PBS merthiolate) that could not be detected by the conventional immunofluorescence protocol described above but could be detected using tyramide signal amplification.

The sections were subsequently incubated with biotinylated donkey anti-rabbit antibody for 60 min at room temperature. In accordance with the manufacturer's protocol, the sections were incubated with the supplied horseradish peroxidase-conjugated streptavidin (diluted 1:100 with 1% NHS-PBS merthiolate) for 30 min at room temperature and then incubated with the supplied fluorophore tyramide (Perkin Elmer) prepared diluted 1:50 with the supplied amplification reagent at room temperature for 3–7 min.

The same sections were then incubated overnight at room temperature with a marker antibody: calbindin-D 28K (1:1,500) or AQP2 (1:1,000) antibody diluted with 10% NHS-PBS merthiolate. This marker antibody was labeled using CY3-conjugated donkey anti-rabbit immunoglobulin antibody (labeled red) and incubated for 60 min at room temperature.

Again, all incubations were separated by three 5-min washes in 0.1 M PBS. Slides were then mounted in Citifluor solution and examined and photographed using a Zeiss Axioplan microscope fitted with a digital camera (model DC 200, Leica).

Secondary Antibody Control Experiments

Control experiments in which each secondary antibody was directly incubated with kidney tissue sections (i.e., without prior incubation with primary antibodies) were performed. No positive staining was observed (data not shown).


   RESULTS
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DISCUSSION
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Glomerulus

Prominent immunostaining was found in the glomerulus for NTPDase1 (Fig. 3A) and NPP3 (see Fig. 6A). No fluorescence was found for any other ectonucleotidase.



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  		Fig. 3. A: staining for NTPDase1 in glomerulus. Magnification x20, scale bar 100 µm. B: staining for NTPDase1 in inner medullary collecting ducts. C: section shown in B stained for aquaporin (AQP) 2. D: merged image of B and C stained for NTPDase1 (green) and AQP2 (red). Colocalized staining of NTPDase1 and AQP2 is yellow. Magnification x20, scale bar 25 µm.

 


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  		Fig. 6. A: staining for nucleotide pyrophosphatase phosphodiesterase (NPP) 3 in glomerulus. Magnification x20, scale bar 50 µm. B: staining for NPP3 in proximal tubules. Arrows, positively stained proximal tubules lacking expression of NPP3. C: section shown in B stained for Phaseolus vulgaris erythroagglutinin. Magnification x20, scale bar 100 µm. D: staining for NPP3 in pars recta. E: section shown in D stained for neutral endopeptidase. Magnification x20, scale bar 100 µm.

 
Proximal Tubule

Indirect immunofluorescence labeling with antibodies specific to ectonucleotidases and the proximal tubule marker (PVE) showed prominent staining for NPP3 and ecto-5'-nucleotidase, but not for NTPDase1, NTPDase2, or NTPDase3. Staining for NPP3 (see Fig. 6, B and C) and ecto-5'-nucleotidase (see Fig. 7, A and B) was found predominantly on the apical side of the tubules, although staining in the peritubular space was also seen for ecto-5'-nucleotidase. Not all positively stained proximal tubular segments stained for NPP3 or ecto-5'-nucleotidase, suggesting that these enzymes are localized to specific proximal tubular subsegments.



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  		Fig. 7. A: staining for ecto-5'-nucleotidase in proximal tubules. Arrows, positively stained proximal tubules lacking expression of ecto-5'-nucleotidase. B: section shown in A stained for Phaseolus vulgaris erythroagglutinin. G, glomerulus. Magnification x20, scale bar 100 µm. C: staining for ecto-5'-nucleotidase in cortical labyrinth and outer stripe of outer medulla. D: section shown in C stained for neutral endopeptidase. Note staining in medullary rays and outer stripe. Magnification x10, scale bar 200 µm. E: staining for ecto-5'-nucleotidase in distal tubules. Arrows, low-level expression of ecto-5'-nucleotidase in positively stained distal tubules. F: section shown in E stained for calbindin-D 28K. Magnification x10, scale bar 50 µm. G: staining for ecto-5'-nucleotidase in outer medullary collecting ducts. H: section shown in G stained for AQP2. I: merged image of G and H stained for ecto-5'-nucleotidase (green) and AQP2 (red). Arrows, presumed intercalated cells. Magnification x20; scale bar 25 µm. J: staining for ecto-5'-nucleotidase in inner medullary collecting ducts. K: section shown in J stained for AQP2. L: merged image of J and K stained for ecto-5'-nucleotidase (green) and AQP2 (red). Arrows, presumed intercalated cells. Magnification x20, scale bar 25 µm.

 
To investigate this further, the monoclonal antibody to neutral endopeptidase (NEP), which in the rat is localized to the S3 segment of the proximal tubule (38), was used in conjunction with the NPP3 and ecto-5'-nucleotidase antibodies. NPP3 staining was found to colocalize with NEP (see Fig. 6, D and E), suggesting that NPP3 is exclusively or predominantly expressed in the S3 segment of the proximal tubule. In contrast, costaining for ecto-5'-nucleotidase and NEP was found in some, but not all, positively stained S3 segments (see Fig. 7, C and D).

Thick Ascending Limb of Henle

Staining of the TAL with Tamm-Horsfall protein as a marker revealed expression of NTPDase2 (Fig. 4, A and B) and NTPDase3 (Fig. 5, A and B). This staining was not exclusive to the apical surface; some staining, particularly for NTPDase2, was also found on the basolateral surface. No immunoreactivity for NTPDase1, NPP3, or ecto-5'-nucleotidase was observed in this nephron segment.



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  		Fig. 4. A: staining for NTPDase2 in thick ascending limbs. B: section shown in A stained for Tamm-Horsfall protein. Magnification x20, scale bar 100 µm. C: staining for NTPDase2 in distal tubules. Arrows, positively stained distal tubules lacking expression of NTPDase2. D: section shown in C stained for calbindin-D 28K. Magnification x20, scale bar 100 µm. E: staining for NTPDase2 in inner medullary collecting ducts. F: section shown in E stained for AQP2. G: merged image of E and F stained for NTPDase2 (green) and AQP2 (red). Colocalized staining of NTPDase1 and AQP2 is yellow. Magnification x20, scale bar 25 µm.

 


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  		Fig. 5. A: staining for NTPDase3 in thick ascending limbs. B: section shown in A stained for Tamm-Horsfall protein. Magnification x20; scale bar 100 µm. C: staining for NTPDase3 in distal tubules. D: section shown in C stained for calbindin-D 28K. Magnification x20, scale bar 100 µm. E: staining for NTPDase3 in outer medullary collecting ducts. F: section shown in E stained for AQP2. G: merged image of E and F stained for NTPDase3 (green) and AQP2 (red). Arrows, presumed intercalated cells. Magnification x10, scale bar 50 µm. H: staining for NTPDase3 in inner medullary collecting ducts. I: section shown in H stained for AQP2. J: merged image of H and I stained for NTPDase3 (green) and AQP2 (red). Colocalized staining of NTPDase1 and AQP2 is yellow. Magnification x10, scale bar 800 µm.

 
Distal Tubule

The distal tubule stained for NTPDase2 (Fig. 4, C and D) and NTPDase3 (Fig. 5, C and D), predominantly on the apical side, and showed no staining for NPP3 or NTPDase1. Staining for NTPDase2 was variable: not every positively stained distal tubular segment expressed this enzyme. Very low-level apical expression was also observed for ecto-5'-nucleotidase in some, but not all, segments of positively stained distal tubules (Fig. 7, E and F).

Collecting Duct

Cortical and outer medullary collecting ducts displayed sparse staining for NTPDase3, which did not colocalize with AQP2-stained cells, suggesting that the enzyme is confined to intercalated cells (Fig. 5, E–G). A similar pattern of low-level staining was also observed for ecto-5' nucleotidase (Fig. 7, G–I). No other ectonucleotidases were identified in these regions.

Inner Medullary Collecting Duct

In this segment, ecto-5'-nucleotidase was found again in intercalated cells (Fig. 7, J–L), whereas low-level staining for NTPDase3 was found in most principal cells (Fig. 5, H–J). In addition to these two enzymes, some principal and intercalated cells of the inner medullary collecting duct showed sparse expression of NTPDase1 (Fig. 3, B–D) and NTPDase2 (Fig. 4, E–G).


   DISCUSSION
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 ABSTRACT
 METHODS
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GRANTS
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The existence of purinoceptors (P1 and P2) along the apical and basolateral membranes of the renal tubule is well established (42, 44), and, as indicated above, there is good evidence that activation of these receptors elicits a range of physiological responses. It seems probable that the hydrolysis of native nucleotides by ectonucleotidases along the nephron will result in a constantly shifting activation of different purinoceptor subtypes, thereby modifying the final physiological response. Thus a detailed knowledge of the types of ectonucleotidase present in each nephron segment is essential to our understanding of purinoceptor function. This concept is illustrated by in vitro studies examining the effect of ectonucleotidases on ADP-sensitive platelet aggregation (40) and activation of ADP-sensitive cellular P2Y1 receptors (1). These studies demonstrated that, in the presence of extracellular ATP, expression of NTPDase1 (which hydrolyzes ATP and ADP with almost equal preference) reduced platelet aggregation and activation of P2Y1 receptors, whereas expression of NTPDase2 (which preferentially hydrolyzes ATP) had the opposite effect.

The only ectonucleotidases with distribution along the nephron that has been described in detail are alkaline phosphatase and ecto-5'-nucleotidase. Alkaline phosphatase has broad substrate specificity, capable of metabolizing ATP, ADP, and AMP to adenosine. Because it is glycosylphosphatidylinositol anchored, it may also exist in a soluble form. In the kidney, alkaline phosphatase has been identified in the brush-border membrane of the proximal tubule in most species, including the rat (4). Ecto-5'-nucleotidase dephosphorylates AMP to adenosine and is also glycosylphosphatidylinositol anchored. It has been shown previously to be present in the brush-border membrane of the proximal tubule and in the apical membrane and apical cytoplasm of intercalated cells in the connecting tubule and collecting duct, as well as in the peritubular space (11, 24).

Only limited information is available concerning the distribution of the other two families of ectonucleotidases (NTPDases and NPPs) along the nephron. Of the eight-member NTPDase family, only NTPDase1, NTPDase2, NTPDase3, and NTPDase8 are concerned with the hydrolysis of extracellular nucleotides: NTPDase4, NTPDase5, NTPDase6, and NTPDase7 are believed to be localized to intracellular membranes such as the Golgi apparatus and the endoplasmic reticulum (5, 22, 33, 43, 45). A recent study has provided limited information on the tubular distribution of NTPDase1 and NTPDase2 (20), but little is known about NTPDase3. Of the NPP family, the intrarenal distribution of NPP1 has previously been examined in the mouse (14), but no information is available on NPP2 or NPP3 in the kidney. Unfortunately, there are no antibodies available that would allow the study of the distribution of NPP1 or NPP2 in the rat. Therefore, in the present study, we examined the intrarenal distribution of NTPDase1, NTPDase2, NTPDase3, NPP3, and, for comparison, ecto-5'-nucleotidase. A key feature of our approach has been the use of specific markers to identify each nephron segment. The main findings are summarized in Fig. 8.



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  		Fig. 8. Summary of distribution of ectonucleotidases along rat nephron.

 
NTPDase1, NTPDase2, and NTPDase3

All members of the NTPDase family hydrolyze ATP and ADP to AMP, but they differ in their preferences for these substrates (22). As indicated above, NTPDase1 hydrolyzes ATP and ADP with almost equal preference (49). However, hydrolysis of ATP by rat NTPDase1 largely proceeds directly to AMP (48). In the present study, NTPDase1 was prominently expressed in the glomerulus and peritubular space, with some low-level staining in the inner medullary collecting ducts; this enzyme was not present in any of the other nephron segments examined. Our findings are consistent with those of a recent study in rat and mouse in which glomerular mesangial cells and/or glomerular capillary membranes, as well as peritubular capillaries, stained for NTPDase1 (20). The same study also identified NTPDase1 in thin ascending loop of Henle, but (owing to the lack of a suitable marker for this nephron segment) we were unable to confirm this finding. Lemmens et al. (28) identified this enzyme in the blood vessel walls of glomerular and peritubular capillaries of the porcine kidney, suggesting conserved renal distribution of NTPDase1 among species.

In contrast to NTPDase1, NTPDase2 has a significantly higher (~30-fold) preference for the hydrolysis of ATP over ADP (48). Kishore and colleagues (20) observed NTPDase2 labeling in tubular structures in kidneys from rats and mice. We have identified these tubular segments as the TAL and the distal tubule, with some low-level enzyme expression in the inner medullary collecting ducts. Staining in these regions of the nephron was not confined to the apical membrane but was also distributed throughout the cell cytoplasm. Colocalization of NTPDase2 with the distal tubular marker calbindin-D28k was variable. This variation in distribution might be explained by the presence of calbindin-D28k in both the distal convoluted tubule and the connecting tubule (6); given its prominent expression in the TAL, it seems possible that NTPDase2 is expressed only in the distal convoluted tubule and not the connecting tubule.

NTPDase3 was found in all postproximal segments of the nephron examined, including TAL, distal tubule, and collecting duct. In the cortical and outer medullary collecting duct, this enzyme appears to be confined to intercalated cells, whereas in the inner medullary collecting ducts it was found in principal cells. Catalytically, this ectonucleotidase has a higher preference (~3-fold) for the hydrolysis of ATP over ADP (48).

The functional significance of the distribution of NTPDase2 and NTPDase3 is unclear. However, the presence of NTPDase1 in the glomerulus and peritubular space raises a number of possibilities. In the glomerulus, NTPDase1 (and other ectonucleotidases such as NPP3) may modulate P2 receptor-dependent contraction and relaxation of mesangial cells (16), thereby influencing the capillary surface area available for filtration. These enzymes may also influence other glomerular purinoceptor responses, such as P2Y-dependent cell proliferation of mesangial cells, or may serve in the protection of these cells by preventing ATP from reaching concentrations that activate the apoptotic P2X7 receptor (13). In glomerulonephritis, these glomerular ectonucleotidases may have an anti-inflammatory role, as indicated by in vivo studies in which inactivation of ectonucleotidases (or application of ATP or ADP analogs) in glomeruli of nephritic kidneys caused an increase in platelet aggregation (36, 37).

It is also possible that NTPDase1, along with ecto-5'-nucleotidase, is involved in tubuloglomerular feedback (TGF), whereby changes in renal perfusion pressure ultimately cause compensatory changes in afferent arteriolar resistance and glomerular filtration rate is autoregulated. ATP concentrations in renal interstitial fluid increase in response to elevations in renal arterial perfusion pressure (34), and ATP can act directly on P2X1 receptors on the afferent arteriole to induce constriction (15). However, strong evidence also exists for adenosine (acting via A1 receptors) being the chemical mediator in this response (35, 39). In this context, it is possible that NTPDase1, expressed in the peritubular space, might convert any unbound or excess ATP to AMP. The latter, being a suitable substrate for the enzyme ecto-5'-nucleotidase, also expressed in the peritubular space (see below), could then be converted to adenosine to cause or augment the vasoconstrictive response in TGF.

NPP1, NPP2, and NPP3

NPP1, NPP2, and NPP3 are also able to catalyze ATP and ADP to AMP but vary in their affinities for the nucleotide substrates. Of these three well-characterized members, only NPP1 has been identified previously in the kidney. Immunohistological studies showed strong expression of this enzyme in the basal epithelium of the distal tubule of the mouse, with low-level staining in the proximal convoluted tubule (14). Although Northern blot data indicated the presence of NPP2 mRNA in rat kidney (10), lack of a suitable antibody precluded our mapping the expression of NPP1 or NPP2 along the rat renal tubule.

We observed prominent staining for NPP3 in the brush-border membrane of the proximal tubule. However, its expression in this part of the nephron varied, inasmuch as some positively identified proximal segments displayed little or no staining for the enzyme. Subsequent colocalization studies using NEP antibody as a specific marker of the S3 segment in the rat (9, 38) showed prominent expression of NPP3 in every case, indicating that those segments of the proximal tubule not staining for NPP3 are likely to be within S1 or S2 regions and that NPP3 is expressed predominantly in the S3 segment of the proximal tubule. In addition to the proximal tubule, NPP3 was also expressed in glomeruli. A unique feature of the NPP ectonucleotidases is their ability to hydrolyze the phosphodiester bonds of nucleic acids, suggesting a protective role against blood-borne DNA- or RNA-based viruses; alternatively, they may serve to salvage purines liberated during injury or cell death (49).

Two additional members of the NPP family, NPP4 and NPP5, have been identified (12). However, no information is available concerning their expression in the kidney.

Ecto-5'-Nucleotidase

We found prominent immunostaining of ecto-5'-nucleotidase in the apical membrane of the proximal tubule. However, as with NPP3, staining was variable: not all positively stained proximal tubular segments expressed ecto-5'-nucleotidase. Further investigation, again with the NEP antibody specific to the S3 segment of the rat proximal tubule, showed that, in contrast to NPP3, there was only modest and variable costaining with NEP. This suggests that ecto-5'-nucleotidase expression in the proximal tubule is restricted mainly to the S2 and, possibly, S1 segments.

Le Hir and Kaissling (25) reported high ecto-5'-nucleotidase activity in the S1 segment of the proximal tubule of the rat, decreasing progressively in the S2 segment. In the outer stripe of the outer medulla, the S3 segment showed expression of ecto-5'-nucleotidase in most, but not all, rats, whereas staining in the S3 segments of the medullary rays was weak or absent (25), which is consistent with our findings. If adenosine were to be produced by ecto-5'-nucleotidase or alkaline phosphatase in the proximal tubule, it would be expected to activate the adenosine (A1) receptors also found in the proximal tubule (42), resulting in increased sodium and water reabsorption (46). Adenosine produced in this region might travel downstream or, alternatively, be taken up by nucleoside transporters in the apical membrane of proximal tubule cells (23).

In the present study, low-level staining for ecto-5'-nucleotidase was also seen in the cortical and medullary collecting ducts. Some low-level staining of this enzyme was also found in some, but not all, positively stained distal tubular segments. As previously indicated, calbindin-D28k is expressed in both the distal convoluted tubule and the connecting tubule. Given that ecto-5'-nucleotidase is expressed in the collecting duct, it seems possible that it is present in the connecting tubule, rather than the distal convoluted tubule. Gandhi et al. (11) attributed the sparse staining of this enzyme in the connecting tubule and the collecting duct to intercalated cells, which is consistent with our observations. Adenosine A1 receptors have been identified in the medullary collecting ducts (42). Contrary to their effects in the proximal tubule, stimulation of collecting duct A1 receptors reduces sodium reabsorption (47). Overall, renal A1 receptor inhibition is natriuretic and diuretic, because the effect on proximal tubular reabsorption predominates (46). Interestingly, staining of ecto-5'-nucleotidase was markedly lower in the collecting duct than in the proximal tubule; on this basis, it seems possible that higher concentrations of endogenous adenosine may explain the overriding effects of A1 receptor inhibition in the proximal tubule over the collecting ducts.

Prominent ecto-5'-nucleotidase staining was also observed outside the tubule, in the peritubular space. In an extensive previous study (24), this peritubular staining was attributed to interstitial fibroblasts. Located between tubules, peritubular capillaries, and arteriolar smooth muscle cells, these fibroblasts may serve to convert nucleotides (released from nerve endings and tubules) to adenosine. This would affect nearby adenosine-sensitive tissues such as the renin-producing granular cells and erythropoietin-producing cells (25). The possible role of ecto-5'-nucleotidase in TGF has already been discussed. In this connection, ecto-5'-nucleotidase knockout mice have been shown to have a blunted TGF response (7).

Overall, it seems as though expression of adenosine receptors in the nephron coincides with the presence of adenosine-producing ectonucleotidases, allowing the possibility that these enzymes serve to produce adenosine to activate adjacent receptors. On the other hand, coexpression of nucleoside transporters with these adenosine-producing enzymes in the proximal tubule suggests an alternative function for the ectonucleotidases in recycling/scavenging nucleotides released from cells or filtered at the glomerulus.

In conclusion, the distribution of the ectonucleotidases we studied varies significantly along the different regions of the nephron. Reasons for this pattern of distribution remain unclear, but their varying hydrolysis pathways suggest that these enzymes may be strategically placed so as to influence the activity of P2 and P1 purinoceptors through the generation, or hydrolysis, of agonists such as ATP, ADP, or adenosine.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The National Kidney Research Fund and St. Peter's Trust for Kidney, Bladder, and Prostate Research funded this work.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Maurice for providing antibodies to NPP3; J. Pelletier for contributing to the production of antibodies to NTPDase1, NTPDase2, NTPDase3, and ecto-5'-nucleotidase; Dr. D. Marples for providing antibodies to aquaporin-2; Dr. P. Ronco for providing antibodies to NEP; Drs. J. Marks and H. Zimmermann for helpful advice; and Prof. G. Burnstock for support and encouragement.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Vekaria, Dept. of Physiology and Centre for Nephrology, Royal Free and Univ. College Medical School, Rowland Hill St., London NW3 2PF, UK (e-mail: r.vekaria{at}medsch.ucl.ac.uk)

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.


   REFERENCES
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