Cell cultures may be helpful in studying the cellular mechanisms of ion transport regulation in kidney proximal epithelium. However, several conditions are mandatory: Proximal cells in culture must be highly differentiated, displaying the cardinal features of the corresponding parent cells, that is, the morphological, enzymatic, and electrical properties, the specific apical transport pathways such as the Na/H antiporter NHE-3, the type II Na-Pi (Na-Pi-2), and Na-glucose cotransporters, the specific hormone receptors, and their corresponding cellular transduction pathways. Primary cultures generally rapidly dedifferentiate with time and do not express NHE-3 or type II Na-Pi transporters. Proximal tubular-like cell lines of undefined origin have been used extensively, particularly opossum kidney (OK) cell lines, which exhibit apical Na/H antiporter and type II Na-Pi cotransport (Na-Pi-4) with high homology with NHE-3 and Na-Pi-2, respectively1,2. Cell lines of well-defined proximal origin have also been established from primary cultures using simian virus-40 (SV40) transformation. Unfortunately, these cell lines (MCT and RKPC2) do not express NHE-33,4. It has been shown recently that cortical collecting duct cell lines from transgenic mice are more successful with regard to transport functions than cells immortalized in vitro5. It has been suggested that differentiated functions that may not be needed for survival in cultured cells are likely to be lost with in vitro transformation, whereas such functions could be essential for the survival of the entire organism and therefore must be retained in the transgenic animal5. This led to our trying to develop a nontransformed cell culture from a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4), which contains the SV40 T-antigen coding sequence downstream from the E1A adenovirus enhancer6. The E1A promoter directs the low-level expression of the T antigen in all three germ-layer derivatives7. The E1A-T construct has been shown to promote immortalization but not transformation. It has been successfully used to immortalize cells at the stem cell stage while still allowing differentiation along multiple pathways to occur after induction8,9,10. Mice transgenic for PK4 plasmid have already permitted an immortalized submandibular cell line retaining polarity and functional properties11. To extend the immortalizing strategy, we reasoned that differentiation should come from interactions between cells of different embryonic origin to start from whole kidney cortex. This article is a structural and functional characterization of mouse kidney cortical cells (MKCCs). After more than 12 months in culture, MKCCs retain the differentiated properties of polarized proximal cells. They express functional NHE-3 exchanger and type II Na-Pi and Na-glucose cotransporters, and exhibit functional receptors for parathyroid hormone (PTH) and angiotensin II (Ang II).
METHODS
Cell culture
Kidneys were removed under sterile conditions from a three-month-old female mouse transgenic for the plasmid PK4. Cortical slices were dissected and dissociated for six incubation periods of seven minutes at 37°C in buffer containing collagenase (0.5 mg/ml, type A; Boehringer Mannheim, Mannheim, Germany), 0.1% bovine serum albumin (BSA) and (in mM) NaCl 112, KCl 5.4, NaHCO3 25, MgSO4 0.4, MgCl2 0.5, CaCl2 1.2, N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES) 10, KH2PO4 0.2, K2HPO4 0.8, Na2HPO4 0.3, and glucose 5. Tubule fragments were washed, resuspended in defined medium [Dulbecco's modified Eagle's medium (DMEM): Ham's F12; 1:1, vol/vol], sodium selenite (2.10-8M), nonessential amino acids, glutamine (4 mM), HEPES (33 mM), NaHCO3 (25 mM), penicillin/streptomycin (50 UI/ml), fungizone (0.25 
g/ml), supplemented with insulin (0.5 g/ml), triiodothyronin (1 M), transferrin (5 g/ml), cholera toxin (10 ng/ml), epidermal growth factor (10 ng/ml), dexamethasone (50 nM), and 5% fetal calf serum. They were seeded on culture dishes coated with extracellular matrix basement membrane (matrix derived from Engelbreth-Holm-Swarm mouse tumor; Harbor Bio-products, Norwood, MA, USA) and incubated in a humidified 5% CO2/95% air atmosphere at 37°C. The culture medium was changed every two days. For regulation studies, the cells were rendered quiescent by incubation in DMEM/Ham's F12 (1:1) with penicillin/streptomycin for 24 hours. Data were obtained on passages 7 to 17.
Electron microscopy
Cells grown on semipermeable filters (Nunc polycarbonate, 0.4 m pore size, 4.2 cm2 diameter) were washed with phosphate-buffered saline (PBS; in mM: 137 NaCl, 2 KCl, 8 Na2HPO4, and 1.5 KH2PO4) and fixed with 2.5% glutaraldehyde in PBS 0.1 M, pH 7.4, for 30 minutes. They were postfixed with 1% osmium tetroxide and embedded in Epon 812. Samples were thin sectioned, counterstained with uranyl acetate and lead citrate, and observed with a Zeiss EM10 transmission electron microscope.
Transepithelial electrical resistance measurements
Experiments were performed on confluent cells grown on semipermeable filters. Transepithelial resistance was measured 15 days after seeding using a Millicell Electrical Resistance System (ERS Millipore, Bedford, MA, USA) and dual silver/silver chloride (Ag/AgCl) electrodes.
Immunological techniques
Immunoblot analysis of protein kinase C isozymes, Na/H exchanger isoforms, and Na-Pi cotransport
The cells were scraped into the homogenization buffer consisting of ethylenediaminetetraacetic acid (EDTA) 5 mM/tris (hydroxymethyl)aminomethane (Tris) 10 mM, pH 7.4, with leupeptin (12.5 g/ml), 4-(2aminoethyl)benzenesulfonyl fluoride (0.2 mM), benzamidine (10 mM), aprotinin (0.1 g/ml), and dithiothreitol (DTT, 2 mM). To detect Na/H exchanger isoforms and Na/Pi cotransport, a membrane fraction was prepared. The homogenate was centrifuged (500 g for 10 minutes), and the resulting supernatant was centrifuged at 200,000 g for 20 minutes. The pellet representing the membrane fraction was solubilized in Laemmli buffer. To detect protein kinase C (PKC) isoforms, the homogenate was solubilized directly in Laemmli buffer, heated at 90°C for 10 minutes, and stored at -80°C until use. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% polyacrylamide gels according to Laemmli12. Proteins on the gel were electrophoretically transferred onto nitrocellulose membranes (0.45 mm; Schleicher & Schuell, Dassel, Germany) using a Bio-Rad apparatus. Anti-PKC isozyme polyclonal antibodies were purchased from Life Technologies (Grand Island, NY, USA) for 
and 
and from Santa Cruz (Santa Cruz, CA, USA) for 
and 
. Rabbit polyclonal antibody against NHE-1 was a generous gift of Dr. S. Grinstein, Hospital for Sick Children, Toronto, Canada. Rabbit polyclonal antibodies against NHE-2, NHE-3, and NHE-4 and rabbit antisera against Na-Pi-2 have been characterized previously13,14,15. After treatment with the specific antibody, the blots were incubated with a horseradish peroxidase-conjugated goat antirabbit antibody. Proteins were detected with the enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA) according to the manufacturer's protocols.
Immunocytochemistry
Cells were fixed with either 3% paraformaldehyde in PBS for 30 minutes at room temperature or in cold methanol for seven minutes, washed for five minutes with 50 mM NH4Cl, permeabilized with 0.1% Triton X-100 for one minute, and incubated with 10% pig serum, 1% BSA for 10 minutes to block nonspecific antibody binding. Indirect immunofluorescence was carried out using several antibodies: rabbit anti–Na-Pi-2 antiserum (discussed earlier in this article), rabbit anti-NHE-3 antiserum (generous gift of Dr. R. Alpern and Dr. O. Moe, Southwestern Medical Center, Dallas, TX, USA), mouse monoclonal antibody against SV40 T-antigen, rabbit polyclonal antibody against ZO1 (Zymed Laboratories, Inc., San Francisco, CA, USA), monoclonal antibody against cytokeratins (clone ZSK3, pan anticytokeratin; Zymed), and rabbit polyclonal antibody against PKC (Santa Cruz). Cells were incubated overnight at 4°C with primary antibody diluted in PBS containing 1% serum and 0.1% BSA. Treatment with the specific antibody was followed by the addition of the appropriate fluorescein-conjugated antiserum, except for cytokeratin, which was revealed after amplification using biotinylated antimouse IgG (Amersham) and then FluorolinkTMCyTM2-labeled streptavidin (Amersham). Actin filaments were visualized with phalloidin TRITC-labeled (Sigma, St. Louis, MO, USA) at 0.1 g/ml. In some experiments, the cell surface was labeled by incubating the monolayers with TRITC-conjugated lectin PHA-E (50 g/ml; Sigma) before permeabilization. The cells were washed with PBS and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA).
Laser scanning confocal microscopy
The relative distributions of actin and PKC, lectin and Na-Pi-2, actin and NHE-3 in cortex cells were observed with a Zeiss LSM 510 confocal microscope using a 
63 objective. ZO1 and cytokeratin were detected using a Leica TCS-NT SP confocal microscope. The cells were analyzed viewing horizontal and vertical (xz-series) sections through the cells.
Reverse transcriptase/polymerase chain reaction
Cellular RNA was extracted by guanidine thiocyanate solubilization followed by phenol/chloroform precipitation using the method of Chomczynski and Sacchi16. Primers of the four NHE isoforms, used in a previous study17, were chosen from the published rat sequences. They recognized mouse sequences as verified by dideoxy-sequencing of polymerase chain reaction (PCR) products. Reverse transcription (RT) was performed in a total volume of 22 l by incubating RNA at 37°C for one hour with 200 U Moloney murine leukemia virus (MMLV) reverse transcriptase, 20 pmol of downstream primer, 4 g of yeast tRNA, 2.5 mM each deoxyribonucleotide (dNTP), 10 mM DTT, and 2 U of ribonuclease inhibitor in buffer containing 50 mM Tris-HCl, 75 mM KCl, and 3 mM MgCl2. The reaction was stopped by incubation for five minutes at 95°C. For PCR, 10 l of the cDNA solution were supplemented with 5 l of 10 PCR buffer, 5 l of a 25 mM MgCl2 solution, 10 pmol of each primer, 1 l of a 25 mM dNTP solution, and 1.25 U Taq polymerase in a final volume of 50 l. Samples were overlayered with mineral oil and were denatured at 94°C for four minutes. After that, 35 cycles consisting of denaturing at 94°C (1 min), annealing at 60°C (1 min), and extension at 72°C (1.5 min) were performed. PCR was completed by a final extension step of 10 minutes at 72°C. Samples of PCR products were size fractionated on 1.5% agarose gels stained with ethidium bromide.
Enzymatic studies
Alkaline phosphatase
Cells were washed with PBS, fixed with 3% paraformaldehyde in PBS for four minutes at 4°C, and stained for alkaline phosphatase with a solution of nitroblue tetrazolium (150 g/ml; Sigma) and 5 bromo-4 chloro-3 indolyl phosphate (300 g/ml; Sigma) for 30 minutes in the dark. For controls, cells were treated with levamisole (1 mM; Sigma), an alkaline phosphatase inhibitor.
Adenylate cyclase activity
Cells were preincubated for 15 minutes in a medium containing (in mM) NaCl 110, KCl 3, MgSO4 1, CaCl2 1, HEPES 10, KH2PO4 0.2, K2HPO4 0.8, NaHCO3 25, glucose 5, alanine 5, and pyruvic acid 5 with 1 mM 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor; Sigma) at 37°C in 5% CO2/95% air atmosphere, prior to the addition of PTH (PTH 1–34, 10-7M; Sigma) or arginine vasopressin (AVP; 10-7M; Sigma). The reaction was stopped after 15 minutes by removing the incubation medium and adding ice-cold 5% HClO4. The cells were scraped and centrifuged. The supernatants containing cAMP were neutralized with saturated K2CO3, and cAMP was measured by radioimmunoassay (RPA 509 Amersham). The pellets were solubilized in 200 l NaOH N for protein assay.
Transport studies
Glucose and phosphate uptake
Uptake of -methylglucopyranoside (AMG; a nonmetabolizable analogue of D-glucose) or uptake of phosphate was assayed in the absence of NaCl or with NaCl alone or in combination with specific inhibitors namely phlorizin (for AMG) or sodium arsenate (for phosphate) at room temperature. Cells were washed in a sodium-free buffer containing (in mM) choline chloride 140, KCl 3, CaCl2 1, MgCl2 1, HEPES 10, pH 7.4. The cells were then incubated in 1 ml of uptake media containing (in mM) NaCl 140, KCl 3, CaCl2 1, MgCl2 1, HEPES 10, pH 7.4, supplemented with [14C]AMG (0.5 mM, 0.2 Ci/ml; Amersham) or K2H[32PO4] (0.1 mM, 0.1 Ci/ml; Amersham), plus or minus 0.5 mM phlorizin (Sigma) or 10 mM sodium arsenate, or in a sodium-free buffer. After 5, 10, 15, or 20 minutes of incubation, transport was stopped by removing the incubation medium and washing the cells three times in an ice-cold solution containing (in mM) NaCl 100, Tris/HEPES 1, pH 7.4. The cells were solubilized for one hour in 0.5 ml NaOH N/2 and neutralized with 0.5 ml HCl N/2 before splitting the samples for counting and for protein assay.
Uptake of 22Na
Na/H activity was assayed by a proton gradient-stimulated initial rate of 22Na uptake after cell acidification by a NH4 prepulse (adapted from18). Cells were washed and incubated for 30 minutes at 37°C in Na/NH4Cl medium containing (in mM) NaCl 110, NH4Cl 30, HEPES/Tris 10, pH 7.4, KCl 3, CaCl2 1, MgCl2 1, KH2PO4 0.2, K2HPO4 0.8, glucose 5, alanine 5, pyruvic acid 5, and BSA 0.01%. Then the cells were incubated for an additional period of 10 minutes in the same medium without Na (equimolar substitution by TMACl). The medium was removed, and 1 ml of the uptake medium was added (in mM):22NaCl 1 (Amersham); 0.05 Ci/l, HEPES/Tris 10, pH 7.4, KCl 3, CaCl2 1, MgCl2 1, TMA-Cl 140, and ouabain 2.10-3M, amiloride 10-6M, bafilomycin 4.10-8M with or without Na/H inhibitors [ethylisopropylamiloride (EIPA); Sigma] or HOECHST 642, (HOE, generous gift of Dr. H.J. Lang, Hoechst, Frankfurt, Germany). Under these conditions of acid loading,22Na uptake was linear with time for 20 minutes. Therefore, a time course of six minutes was selected. The reaction was stopped by three washes with an ice-cold stop solution (in mM): mannitol 280, Tris/HEPES 20, pH 7.4, and amiloride 0.5. The cells were solubilized for one hour in 0.5 ml NaOH N/2 and neutralized with 0.5 ml HCl N/2 before splitting the samples for counting and for protein assay.
RESULTS
Immortalized mouse kidney cortical cells have features of polarized epithelial cells
Cells obtained from cortical slices could be replated and still divide over two weeks in exponentially growing conditions. The number of cells doubled every 18 hours. SV40 large T-antigen was detected in the nucleus of all the cells Figure 1 a, b. A small number of morphologically distinct cell types were visible, and many islands of cells presented a cobblestone appearance with multiple dome formation. More than 80% were positive for alkaline phosphatase, a typical marker of luminal membranes, and 100% of the cells were positive for cytokeratin (keratin 10, 17, 18, and 19), indicating that the cells retained an epithelial-like phenotype Figure 1 c, d. A small population (approximately 10 cells) was selected from an island of the epithelial-like cells. These cells, now referred to as MKCCs, exhibit an homogeneous and stable phenotype after replating for more than 12 months under standard culture conditions. They had no tumorigenic potential after subcutaneous or intraperitoneal injections into athymic mice (107 cells per mouse, N = 3) and irradiated syngeneic mice (N = 3). When plated on culture dishes, they grew as a single monolayer and formed domes soon after they reached confluency Figure 1e. When transferred on semipermeable filters coated with extracellular matrix, they formed a homogeneous polarized epithelium, as demonstrated by ultrastructural study. Cells exhibited polarity with an apical domain bearing many microvilli Figure 2a. Moreover, junctional complexes could be visualized Figure 2 b, c. The presence of tight junctions was revealed by anti-ZO1 antibodies Figure 1f. Abundant mitochondria and endocytotic vesicles were observed, consistent with active metabolism. Furthermore, electrophysiological measurements indicated that the monolayers developed a low transepithelial electrical resistance ranging between 35 and 54 
/cm2, in agreement with data obtained in proximal tubule19. Taken together, these results indicated that immortalized MKCCs retained in vitro the structural features of polarized proximal cells.
Figure 1.
Light phase and confocal microscopy of mouse kidney cortical cells (MKCCs) showing features of epithelial cells. (A) Immunochemical staining using an antibody against the large T antigen of simian virus 40 (SV40), the nuclei of all cells are stained (100). (B) Light phase contrast microscopy illustration of the corresponding field (100). (C) Staining for alkaline phosphatase (insert: negative control with levamisole, 10). (D) Confocal microscopy images of indirect immunofluorescence using an antibody to cytokeratins (700). (E) Light phase contrast microscopy showing a confluent epithelial monolayer with cobblestone appearance and multiple dome formation (arrows; 30). (F) Confocal microscopy image of indirect immunofluorescence showing the presence of ZO1 protein at the apical–basolateral boundary (500).
Figure 2.
Electron microscopy of mouse kidney cortical cells (MKCCs) grown on filter supports showing features of polarized proximal cells (counterstained with lead and uranyl). (A) Continuous monolayer of epithelial cells adherent to the filter (F). The cells present large nuclei (N) with conspicuous nucleoles, vacuoles (V) within the cytoplasms, and a well-developed brush border at the apex (10,000). (B) The tubular cells show microvilli, many mitochondria (M), vacuoles (V), and numerous endocytotic vesicles (thin arrows). At the apex, the intercellular cleft is closed by the junctional complex (large arrow; 30,000). (C) Higher power view showing the junctional complex occluding the intercellular cleft (large arrow) at the apex (100,000).
Polarized mouse kidney cortical cells express functional Na-glucose, Na-Pi cotransport, and Na/H exchangers
Na-Pi and Na-glucose cotransporters
The presence of Na-glucose and Na-phosphate cotransports characteristic of the proximal tubule was examined by Na-dependent [14C]AMG or [32phosphate] uptake measurements, respectively. A Na-dependent [14C]AMG uptake was observed Figure 3. It was 96% inhibited by Na removal and 97% by 0.5 mM phlorizin, a specific inhibitor of Na-glucose cotransport, indicating the presence of a functional Na-glucose cotransport.
Figure 3.
Sodium-dependent glucose uptake in mouse kidney cortical cells (MKCCs). Glucose transport was measured by [14C]-methylglucopyranoside (AMG) uptake on confluent cells, as described in the Methods section. Values are means 
SE of three independent experiments. Symbols are: (
) 140 mM NaCl; (
) NaCl + phlorizin 0.5 mM; (
) 0 Na.
Moreover, a Na-dependent [32phosphate] uptake was observed Figure 4a. It was 80% inhibited by Na removal and 87% by 10 mM arsenate, a specific inhibitor of Na-Pi cotransport, indicating the presence of a functional Na-Pi cotransport. The type II Na-Pi protein, which is normally present in brush borders of proximal tubules15, was detected by Western blot analysis in a membrane fraction Figure 4b. Indeed, rabbit antiserum against Na-Pi-2 reacted with a 87 kD protein. Furthermore, indirect immunofluorescence with confocal microscopy analysis showed the labeled Na-Pi-2 protein within the cells and on the apical membrane Figure 4c.
Figure 4.
Sodium-phosphate cotransport in mouse kidney cortical cells (MKCCs). (A) Phosphate transport was measured by K2[32PO4] uptake on confluent cells as described in the Methods section. Values are means SE of three experiments. Symbols are: () 140 mM NaCl; () arsenate 10 mM; () 0 Na. (B) Detection of Na-phosphate protein by Western blot using anti–Na-Pi-2 antisera. Cell lysates were homogenized, and a membrane fraction was prepared. Proteins were subjected to 7.5% SDS-PAGE and immunoblotting with antiserum (1/100 dilution). Molecular mass standards are indicated on the left. Results are representative of three experiments. (C) Confocal microscopy image of indirect immunofluorescence showing the subcellular distribution of Na-phosphate protein. The cells were fixed and immunostained with anti–Na-Pi-2 antiserum (1/100 dilution) and PHA-lectin-TRITC (50 g/ml), as described in the Methods section. Shown is an xz section calculated from a set of xy images (bottom, plane of Petri dish).
Na+/H+ exchangers
The presence of mRNA of the four Na/H exchangers normally expressed in the kidney cortex was examined first by reverse transcription-PCR (RT-PCR) analysis. The amplification products of predicted size were detected for NHE-1, NHE-2, and NHE-3 Figure 5a. We then carried out Western blot analysis to verify that NHE mRNAs were effectively translated. Antibodies against NHE-1, NHE-2, and NHE-3 reacted with at 111, 81, and 75 kD proteins, respectively Figure 5b. NHE-4 was not detected by RT-PCR or by Western blot.
Figure 5.
NHE antiporters in mouse kidney cortical cells (MKCCs). (A) Detection of NHE mRNAs by reverse transcription-polymerase chain reaction. RNA was isolated from cell cultures. cDNAs were prepared, and PCR reactions were carried out using standard conditions (discussed in the Methods section; C, control using mouse whole kidney homogenate; T, kidney cortex cells from transgenic mouse). Each reaction was performed in the presence (+) or absence (-) of reverse transcriptase. Molecular weight markers are indicated on the left. Results are from one representative experiment out of three. (B) Detection of NHE proteins by Western blots using affinity purified anti NHE-1, NHE-2, NHE-3, or NHE-4 antibodies. Cell lysates were homogenized, and a membrane fraction was prepared. Proteins were subjected to 7.5% SDS-PAGE and immunoblotting with specific antibodies (1/200, 1/200, 1/100, and 1/500 dilution for NHE-1, NHE-2, NHE-3, and NHE-4, respectively). Molecular mass standards are indicated on the left. Results are from one representative experiment out of three. (C) Na/H activity was assayed by outward proton gradient-stimulated initial rate of 22Na uptake after cell acidification by NH4 prepulse, as described in the Methods section. Results expressed in percentage of the activity sensitive to 300 M EIPA are means SE of six experiments. (D) Confocal microscopy image of indirect immunofluorescence showing the subcellular distribution of NHE-3 protein. The cells were fixed and immunostained with anti-NHE-3 antiserum (1/1000 dilution) and phalloidin-TRITC (0.1 g/ml) as described in the Methods section. Shown is an xz section calculated from a set of xy images (bottom, plane of Petri dish).
The involvement in the Na+ uptake of each of the three antiporters expressed in MKCCs was then estimated by using the antagonist HOE 642, which presents different inhibition constants for NHE-1 (Ki = 0.01 M), NHE-2 (Ki = 3 M), and NHE-3 (Ki = 1 mM)20,21. The outward proton gradient-stimulated 22Na uptake sensitive to 300 M EIPA was considered as the total Na/H activity. It was calculated by the difference between the total 22Na uptake (16.08 2.20 nmol/mg protein/6 min, N = 6) and the residual 22Na uptake, resistant to 300 M EIPA (2.53 0.01 nmol/mg protein/6 min, N = 6). One M HOE-sensitive Na uptake was attributed to NHE-1. One hundred M HOE-sensitive Na uptake minus NHE-1 was attributed to NHE-2, and 100 M HOE-resistant Na uptake minus residual component was attributed to NHE-3. As shown in Figure 5c, Na uptakes corresponding to each isoform were present in MKCCs. Furthermore, indirect immunofluorescence with confocal microscopy analysis showed intracellular and apical membrane staining of the labeled NHE-3 protein Figure 5d.
Together, these results showed that MKCC culture displays three major apical transporters specific of proximal tubular cells, Na-glucose, type II Na-Pi cotransporters, and NHE-3 antiporter.
Mouse kidney cortical cells are sensitive to parathyroid hormone and angiotensin II
Proximal NaHCO3 absorption is regulated by hormones including PTH and Ang II22. In proximal tubule cells, PTH receptors are positively coupled to adenylate cyclase through Gs23. Thus, to know whether the PTH receptors were present and functional in MKCCs, cAMP accumulation was determined in response to the hormone. As shown in Figure 6a, the treatment of the cells with 10-7M PTH elicited a tenfold increase in cAMP, as observed in freshly isolated cortical tubules24. It should be noted that 10-7M AVP weakly stimulated cAMP in these cells (a less than twofold increase). This stimulation remained trivial compared with that elicited by AVP in freshly isolated segments of the distal nephron (10- to 20-fold increase)25. Because type II Na-Pi-2 cotransporter is specifically inhibited by PTH in rat proximal tubules26, we tested whether PTH was able to decrease the Na-dependent phosphate uptake in MKCCs. As shown in Figure 6b, the treatment of MKCC for 15 minutes with 10-7M PTH inhibited the Na-dependent phosphate uptake by approximately 20%, indicating that the type II Na-Pi was appropriately regulated in MKCCs. Because apical Na/H activity is specifically inhibited by PTH22 in proximal tubules, we tested whether PTH was able to decrease the outward proton gradient-stimulated 22Na uptake representing NHE-3 activity. As shown in Figure 6c, the treatment of MKCCs for 45 minutes with 10-7M PTH inhibited 22Na uptake by approximately 24%.
Figure 6.
cAMP response to parathyroid hormone (PTH) and arginine vasopressin (AVP) in mouse kidney cortical cells (MKCCs). (A) Hormone-induced cAMP accumulation. cAMP was measured in confluent cells in the absence or presence of PTH and AVP. Cells were preincubated for 15 minutes with 1 mM IBMX, and then PTH or AVP (10-7M) were added for an additional 15 minutes in the continual presence of IBMX. Values are means SE of seven experiments. *P < 0.001. (B) Sensitivity of the Na-phosphate uptake to 10-7M PTH. Values are means SE of three experiments. *P < 0.05. (C) Sensitivity of NHE-3 activity to PTH 10-7M. PTH was present during each step of the acidifying protocol.22Na uptake was performed in the presence of 10 mM Na. It was linear over five minutes. Na/H activity was assayed by outward proton gradient-stimulated initial rate of 22Na uptake after cell acidification by NH4 prepulse, as described in the Methods section. Results are means SE of five experiments. *P < 0.02 by paired t-test.
In proximal tubular cells, Ang II receptors are coupled to phospholipase C, specific of phosphatidylinositol bisphosphate, leading to the development of a Ca++ signal and PKC activation. First, we investigated whether the repertoire of PKC normally present in the kidney cortex also occurs in MKCC cells. As shown in Figure 7, PKC , , , and were detected by Western blot analysis, with molecular masses of 80, 80, 90, and 72 to 80 kD, respectively. These PKC isoforms are normally present in intact cortical tubular cells27. Regarding PKC, two proteins were detected above 80 kD that could be phosphorylated forms. It has been shown previously that PKC was translocated toward the membrane in response to Ang II in freshly isolated cortical tubules27. A confocal microscopy approach was used to determine if such PKC translocation occurs in MKCCs in response to Ang II. Phalloidin was used to characterize the polarized epithelia with organization of the plasma membrane into apical and basolateral domains (data not shown). PKC was localized by immunofluorescence predominantly in the cytosol of cortex cells under nonstimulated conditions Figure 8a. When cells were treated with Ang II (10 min, 10-7M), the fluorescence intensity was concentrated toward the basolateral and the apical membranes Figure 8b. These data provided evidence for Ang II receptors functionally coupled to PKC.
Figure 7.
Western blot analysis of PKC isoforms in mouse kidney cortical cells (MKCCs). Cell lysates were homogenized and proteins were subjected to 7.5% SDS-PAGE and immunoblotting with specific antibodies (1/1000 for each anti-PKC antibody tested). *Antibody was specifically blocked by the antigenic peptide. Results are representative of three experiments. Molecular mass standards are indicated on the left.
Full figure and legend (11K)Figure 8.
Angiotensin II-induced protein kinase C (PKC)-
translocation in mouse kidney cortical cells (MKCCs). Indirect immunofluorescence using anti-PKC FITC-conjugated antibody and visualized by confocal fluorescence microscopy. (A) Controls. (B) Angiotensin II 10-7M for 10 minutes. FITC staining becomes restricted to membranes.
Taken together, these data showed that MKCCs possess functional receptors for two major peptide hormones regulating the type II Na-Pi (PTH) and NHE-3 (PTH and Ang II) in the proximal tubules.
DISCUSSION
We have developed a nontransformed cell culture derived from kidney cortex of mice transgenic for the SV40 T antigen, which is referred to as MKCCs. Cells display characteristics of differentiated proximal tubular cells: they formed polarized monolayers with distinct apical and basolateral domains; they showed microvilli at their apical surface; and they formed tight junctions. The electrical resistance of the monolayers is consistent with the formation of a leaky epithelium and is close to the values reported in intact proximal tubules28. Cells expressed a set of ion transport pathways specific of the proximal tubule. They exhibit sodium-dependent pathways coupled to glucose, phosphate, and H+. The Na-dependent phosphate uptake shows the features of the apical type II Na-Pi cotransport, the isoform relevant for Pi reabsorption in the proximal tubule and regulated by phosphate diet and PTH29,30. Indeed, the phosphate uptake is inhibited by PTH, in agreement with data obtained in brush border membrane vesicles isolated from PTH-treated cortical slices31. The presence of type II Na-Pi in our cell culture has been confirmed by Western blotting and localized both inside the cells and on the apical membrane, as expected15.
Mouse kidney cortical cells also possess functional NHE isoforms. Besides the ubiquitous NHE-1, two additional isoforms were detected by Western blotting that are 98% homologous to rat NHE-2 and NHE-3, both being functional. NHE-3 appeared intracellularly with a fraction in the apical membrane. NHE-2 and NHE-3 are normally located on the apical side of kidney tubules13,32. NHE-2 is also expressed in other proximal tubular cell lines, MCT4 and RKPC233. The presence of NHE-2 in intact proximal tubule is not established. The NHE-2 protein has not been detected in the rat proximal tubule by microscopic immunoperoxydase labeling with anti-NHE-2 polyclonal antibodies13. NHE-3 is considered to be the main isoform responsible for most of Na and HCO3 reabsorption in the proximal tubule. The expression of NHE-3, which is rapidly lost in primary culture as a consequence of the removal of the cells from their proper surrounding, appears also suppressed by transformation. Indeed, MCT and RKPC2 proximal-like tubule cell lines do not express NHE-3. In MKCCs, NHE-3 activity is inhibited by PTH in agreement with data obtained on apical Na/H activity in proximal tubule22 and on NHE-3 activity in OK cells (abstract; Collazo et al, J Am Soc Nephrol 9:A0018, 1998). In view of the important role of NHE-3 for Na and HCO3 reabsorption in proximal tubule, the MKCC system should prove useful for identifying the pretranscriptional or posttranscriptional mechanisms controlling NHE-3 activity.
The main signaling pathways for PTH and for Ang II receptors, normally present in the proximal tubule, are expressed in MKCCs. The magnitude of cAMP response to PTH is similar to that reported in freshly isolated cortical tubules24. PKC , , , and are present as in intact proximal tubular cells26. The presence of Ang II receptors was evidenced by PKC translocation. Under nonstimulated conditions, PKC is mostly cytosolic, with a slight membrane association. Ang II elicited PKC redistribution toward basolateral and apical membranes, supporting a coupling of Ang II receptors with a phospholipase C, as reported in freshly isolated cortical tubules in suspension24.
This cell culture is the first nontransformed cell culture obtained from transgenic mice in which a complete set of ion transport pathways and functional hormonal receptors specific of the proximal tubule have been identified, to our knowledge. Cell lines of proximal tubular origin obtained from transgenic mice that have been recently described include the PKSV-PCT34 and tsMPT35 cell lines. The PKSV-PCT cell line also exhibits a low transepithelial resistance value similar to that of proximal tubule, but the ion transport pathways other than the Na-glucose cotransport were not studied. The tsMPT cell line exhibits a Na-glucose cotransport and a Na/H exchange activity that was not characterized. The Na-Pi cotransport present in the latter cell line is not affected by PTH, suggesting that the Na-Pi cotransport present is not type II Na-Pi.
In summary, we have developed kidney cortical tubular cell culture from mice transgenic for the T antigen of SV40. These cells retain the properties of proximal tubular cells with regard to morphological, enzymatic characteristics and transepithelial electrical resistance. They also possess specific transport pathways of the proximal tubule, Na-glucose and type II Na-Pi cotransports, and NHE-3 isoform of Na/H exchange and display functional receptors for PTH and Ang II. These cells appear to be a powerful in vitro model for studying cellular mechanisms of ion transport in the proximal epithelium and their regulation.
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Acknowledgments
Part of this work was presented in an abstract form at the 1998 meeting of the American Society of Nephrology. We thank Dr. R. Alpern and O. Moe for providing us with anti-NHE-3 antiserum 1568, Dr. S. Grinstein for anti-NHE-1 antibody, Dr. H.L. Lang for HOECHST 642, M.F. Belair for performing electron microscopy, T. Eirinopoulou for Leica confocal laser microscopy, and M. Paing and P. Kitmasher for photographs.
