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Supplement
Animals
The studies were performed on male SHR and WKY of the Heidelberg sub-strains (SHR/A3 sub-line) from colonies maintained in our animal facility. Animals were studied at 4 and 16 weeks of age. The rats were fed a standard rat chow and received water ad libitum. For tissue collection the rats were anesthetized with 4% isoflurane in oxygen.
Preparation of Proximal Tubule Cells
Proximal tubule cells (PT) were isolated using the method described by Seri et al 1. After anesthesia, the kidneys were removed, rapidly cooled in ice-cold saline and the cortex was dissected and minced on ice to a paste-like consistency. The minced cortical tissue was incubated with Hanks’ balanced salt solution supplemented with 0.075g/100ml Type I collagenase, 500mol/l pyruvic acid, 1mMol/l lactic acid, and 1mMol/l sodium butyrate pH7.4. The incubation occurred for 45 minutes at 37°C with continuous bubbling with 95% O2-5% CO2. The incubation was ended by placing the cells on ice and then pouring them sequentially through sieves of 100, 70, and 40µm pore size to obtain a PT enriched cell suspension. The PT fragments were washed 3 times by centrifugation at 600 rpm at 4°C in a Beckman CS-6KR centrifuge. After the final centrifugation the cells were resuspended in 250L ice-cold homogenization buffer containing 250mMol/l sucrose, 3mMol/l imidazole, 1mMol/l phenylmethylsulfonylfluoride, phosphatase inhibitors (1mMol/L sodium vanadate, 10 mMol/L sodium pyrophosphate, 10mMol/L beta glycerophosphate, and 10mMol/L sodium fluoride, only samples probed with McK1), and a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, Mini-protease inhibitor).
Preparation of sub-cellular membrane fractions
Endosomes were fractionated on a sucrose flotation gradient using the method described by Gorvel et al 2. The cells were homogenized for one minute on ice using a motorized pestle and repeatedly passed through an insulin syringe before being centrifuged for 5 minutes at 4°C and 3,000g. The resulting post nuclear supernatant (PNS) was removed and saved, the pellet re-suspended, and homogenization repeated with the resulting PNS being pooled with the first. The PNS (500µl) was mixed with 900µl of a 62% sucrose, 3mMol/L imidazole solution to form a 40% sucrose solution that was loaded at the bottom of a 5ml ultracentrifuge tube. Next, 16% (1.4ml) and 10% (1ml) sucrose in 3mMol/l imidazole and 0.5mMol/l EDTA in 2H2O and homogenization buffer (0.8ml) were sequentially layered over the 40% sucrose to complete the gradient. The gradients were centrifuged for 1hr at 35,000 rpm in a Beckmann SW50.1 rotor. Early endosomes (EE) were collected at the homogenization buffer-10% sucrose interface and concentrated by diluting 1:5 in phosphate buffered saline (PBS) followed by centrifugation in a Sorvall AT100-4 rotor (4°C, 100,000g). Late endosomes (LE) were collected at the 10% sucrose-16% sucrose interface. A third fraction containing cell ghosts, mitochondria, and BLM was collected at the 16% sucrose-40% sucrose interface. The BLM was further enriched, using the technique described by Hammond et al, by centrifugation (4°C, 20,000g) after which the supernatant and the light ring of membrane layered on top of a denser, brown pellet containing mitochondria and cell ghosts was removed and centrifuged in a Sorvall AT100-4 rotor (4°C, 40,000g) 3. The resulting pellet was resuspended in 12mMol/l HEPES, 300mMol/l mannitol, and Roche Mini protease inhibitors to which 200µl of Percoll was added and the mixture was centrifuged in a Beckman 50.1 rotor (4°C, 27,000 rpm). BLM was collected at the buffer-Percoll interface. Protein amount per fraction was determined using the bicinchoninic acid assay (Pierce Chemical Co.). Identification of early endosomal, late endosomal, and basolateral membrane preparations were supported by Western blots probing with antibodies to marker proteins known to be differentially distributed across these fractions (rab5a (Santa Cruz Biotechnology) for early endosomes, insulin-like growth factor receptor II (Transduction Laboratories) for late endosomes, and rat organic anion transporter I (Chemicon) for basolateral membranes 4-6(Figure I).
Cell Surface Biotinylation
Cell surface biotinylation was performed using a modification of the technique described by Efendiev et al 7. Briefly, proximal tubule cells were prepared as stated except that the cells were suspended in PBS pH 8.0 after the final centrifugation. An aliquot of the cells was disrupted by sonication and the protein concentration determined by the Bradford assay (Bio-Rad). Cells were diluted (1mg cell protein) to 970µL of PBS pH 8.0. EZ-Link Sulfo-NHS-Biotin (Pierce, Rockford, Il) was added to the cells to a final concentration of 1.5mg/mL and mixed by continuous rotation for 1 hour at 4ºC. Incubation was terminated by removal of free Sulfo-NHS-biotin by repeated rinsing and centrifugation. Following the final centrifugation the cells were resuspended in buffer (20mM Tris, 2mM EDTA, 2mM EGTA, 30mM sodium pyrophosphate, pH 7.4) plus protease inhibitors. Cells were disrupted by repeated freeze-thawing and sonication. The cell lysates were cleared by centrifugation for 5 minutes at 14,000g, supernatants were transferred to clean tubes, and protein concentration was determined by Bradford assay. Supernatant protein (70µg) was diluted to 600µL in PBS pH 7.4 and incubated with streptavidin-coated paramagnetic beads (Promega) for 2 hours, rotating at 4ºC. The paramagnetic beads were washed three times with the same buffer to which was added 0.1% SDS and 1% Triton X-100. This was followed by a wash with 50mM Tris-HCl pH 7.4, and resuspension in Laemmli sample buffer (Bio-Rad). Isolated proteins were separated from the biotin-streptavidin bead complex by incubation at 60ºC for 15 minutes.
Western Blots to Quantitate Na+,K+-ATPase Subunit Protein Abundance.
For the sub-cellular fractions, PNS’s, and cell lysates uniform amounts of protein per sub-cellular fraction were mixed with Laemmli buffer and loaded into each lane of a 10% (a1) or 15% (g) SDS PAGE gel, electrophoresed, and transferred onto nitrocellulose. For the biotinylated proteins, equal volumes (7.5µL) were loaded onto SDS PAGE gels. We confirmed that comparable amounts of protein had been loaded onto each gel by silver staining. The blots were blocked with 5% milk in PBS-0.1% Tween-20, probed with 1°antibody (1:2,000 dilution), then probed with 2° antibody (1:2,000) coupled to horseradish peroxidase. Na+,K+-ATPase a1 subunit protein was identified by Western blot using a polyclonal antiserum raised against an Na+,K+-ATPase a1 oligopeptide 8 (anti-NASE, gift of Dr. T. Pressley, Texas Tech University Medical School) or a monoclonal antibody (McK1, gift of Dr. K. Sweadner, Massachusetts General Hospital). McK1 binds to the amino acid sequence DKKSKK of the rat a1 9. This epitope includes the Ser-18 residue that is the target of PKC-mediated regulatory phosphorylation. Phosphorylation of this residue abolishes McK1 binding 10. Na+,K+-ATPase g subunit protein was identified by Western blot using a polyclonal antiserum raised against the 10-residue C terminus of the Na+,K+-ATPase g subunit (gamma C32-4, gift of Dr. R. Blostein, McGill University, Canada).
Western blots were detected using X-ray film with the ECL-Plus detection reagent kit (Amersham Biosciences). The resulting images were scanned using a Hewlett Packard ScanJet 4400c scanner and band intensity was converted into densitometry units using NIH Image 1.58 software, Relative abundance of a1 in each sub-cellular fraction was determined as density units per sub-cellular fraction/sum of density units of all three fractions for that sample x 100%. This approach controlled for blot to blot variation and, therefore, allowed for the comparison of the percent abundance of a1 in each set of fractions (EE, LE, and BLM isolated from one animal) across multiple Western blots. Comparisons of percent abundance in EE, LE, or BLM between SHR and WKY were made by the unpaired Student’s t-test and the null hypothesis of no difference between fractions was rejected at the 95% confidence threshold.
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References
1. Seri I, Kone BC, Gullans SR, Aperia A, Brenner BM, Ballermann BJ. Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells. Am J Physiol. 1988;255:F666-F6773.
2. Gorvel JP, Chavrier P, Zerial M, Gruenberg J. rab5 controls early endosome fusion in vitro. Cell. 1991;64:915-925.
3. Hammond TG, Verroust PJ, Majewski RR, Muse KE, Oberley TD. Heavy endosomes isolated from the rat renal cortex show attributes of intermicrovillar clefts. Am J Physiol. 1994;267:F516-F527.
4. Chavrier P, Parton RG, Hauri HP, Simons K, Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990;62:317-329.
5. Griffiths G, Matteoni R, Back R, Hoflack B. Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells. J Cell Sci. 1990;95( Pt 3):441-461.
6. Kojima R, Sekine T, Kawachi M, Cha SH, Suzuki Y, Endou H. Immunolocalization of multispecific organic anion transporters, OAT1, OAT2, and OAT3, in rat kidney. J Am Soc Nephrol. 2002;13:848-857.
7. Efendiev R, Bertorello AM, Pressley TA, Rousselot M, Feraille E, Pedemonte CH. Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Biochemistry. 2000;39:9884-9892.
8. Pressley TA. Phylogenetic conservation of isoform-specific regions within alpha-subunit of Na(+)-K(+)-ATPase. Am J Physiol. 1992;262:C743-C751.
9. Arystarkhova E, Sweadner KJ. Isoform-specific monoclonal antibodies to Na,K-ATPase alpha subunits - Evidence for a tissue-specific post-translational modification of the alpha subunit. J Biol Chem. 1996;271: 23407-23417.
10. Feschenko MS, Sweadner KJ. Phosphorylation of Na,K-ATPase by protein kinase C at Ser18 occurs in intact cells but does not result in direct inhibition of ATP hydrolysis. J. Biol. Chem. 1997;272:17726-17733.
Figure Legend.
Figure I. Validation of EE, LE, and BLM preparations by Western blot using antibodies against proteins enriched in each organelle (Rab5a, insulin-like growth factor receptor 2, and rat organic anion transporter 1 respectively).
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EE LE BLM
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