T6522

Proximal tubules were isolated as described previously^{41 (link),42 (link)} with several modifications. Briefly, mice were perfused through the thoracic aorta with 20 mL of a cold magnetic bead solution supplemented with 40 μL of Dynabeads M450 (14013, Invitrogen). Kidney cortex was collected and minced into 1-mm³ pieces, which were then digested in HBSS supplemented with 1 mg/mL collagenase I (LS004196, Worthington), 0.75 mg/mL trypsin inhibitor (T6522, Sigma), and 40 U/mL DNase I (D4513, Sigma) at 37 °C for 10 min on a rotator. This digestion step was performed twice and the suspension was passed through cell strainers from 100 to 45 μm to remove large cellular debris. Digested tissues on the cell strainer were collected with cold PBS, and washed three times in cold PBS. The filtrates were placed on a Magnetic Separation Stand (Promega) to remove glomeruli. The long segments of proximal tubules in suspension were used immediately in subsequent experiments.

For experiments in subcutaneous (s.c.) allografts, tumors were weighed and placed in RPMI and finely minced with scissors in a 2 mL tube, which was then washed with up to 2.5 mL of digestion buffer (Tumor Dissociation Kit, Miltenyi, 130-096-730) plus deoxyribonuclease I (300 µg/mL, Sigma, DN25-1G). Dissociation was performed using the protocol suggested by Miltenyi. For KPC tumors, a trypsin inhibitor (250 µg/mL, Sigma, T6522) was added to the digestion buffer. Following the digestion, the samples were passed through a 70 µM strainer filter (Greiner Bio-One, 542–070) washed with MACS buffer (PBS+0.1% FBS and 2 nM EDTA).
Mouse spleens, inguinal and mesenteric lymph nodes were mashed on a 100 µM filter (Greiner Bio-One, 542–000) over a 50 mL tube, using a syringe plug and the filter was washed with MACS buffer and centrifuged (at 4°C as for all the following centrifugation). Red cell lysis buffer (1 mL; 0.15M ammonium chloride; 10 mM potassium hydrogen carbonate; 0.1 mM EDTA; pH 7.4 (adjusted with KOH) was then used to resuspend splenocytes, 3 min at room temperature and then washed with MACS buffer and centrifuged at 300 g for 5 min. Samples were eventually resuspended in 200–400 µL of MACS buffer.

Renin activity assay was performed as previously described.^{9 (link)} Briefly, renin activity in urine and renal inner medulla was determined by the delta value of the Ang I generation using an ELISA kit from the sample incubating at 4 °C and 37 °C for 1 hour, respectively. Total renin content was measured with excessive angiotensinogen plus trypsinization and active renin content with excessive angiotensinogen. Urine and tissue samples were spiked with 1 µmol/L synthetic renin substrate tetradecapeptide (R8129; Sigma-Aldrich, St Louis, MO). After incubation at 37 °C for 18 hours, Ang I generation was assayed by using an Ang I enzyme immunoassay kit (S-1188; Peninsula Laboratories International, SanCarlos, CA), according to the manufacturer’s instructions. The values were expressed as nanograms per milliliter per hour of generated Ang I. For measurement of total renin content, trypsinization was performed to activate prorenin to renin.^{17 (link)} The samples were incubated with trypsin derived from bovine pancreas (100 g/L, T1426, Sigma-Aldrich) in 37 °C for 18 hours. The reaction was then terminated with soybean trypsin inhibitor (100 g/L, T6522; Sigma-Aldrich) at 37 °C for 1 hour. Renin activity was determined in the native condition, active renin content with excessive angiotensinogen, and total renin content with excessive angiotensinogen plus trypsinization.