Protein kinase C-α mediates hypertonicity-stimulated increase in urea transporter phosphorylation in the inner medullary collecting duct

Abstract

The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [(32)P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.

Fig. 1.

Inhibition of protein kinase C (PKC) blocks hypertonic stimulation of UT-A1 phosphorylation. Metabolically labeled (³²P) rat inner medullary collecting ducts (IMCDs) were incubated with 290-mosmol/kgH₂O buffer (iso), 600-mosmol/kgH₂O buffer (hyper), or 600-mosmol/kgH₂O buffer with 10 μM chelerythrine (hyper + Chel) and then immunoprecipitated with anti-UT-A1 and analyzed by Western blot and autoradiography. A: autoradiogram of ³²P-UT-A1. B: Western blot of total UT-A1 for representative samples. Each lane provides results from the kidneys of a separate animal. Arrows indicate the 117- and 97-kDa glycoprotein forms of UT-A1. C: bar of the ratio of phosphorylated UT-A1 to total UT-A1 from all samples from 2 separate cohorts of animals, presented as means ± SE, n = 8/condition. *P < 0.05 vs. isotonic control.

Fig. 2.

Chel does not decrease phosphorylation under isotonic (Iso) conditions. Metabolically labeled (³²P) rat IMCDs were incubated for 30 min with 100 nM arginine vasopressin (AVP) or 10 μM Chel and then immunoprecipitated with anti-UT-A1 and analyzed by Western blot and autoradiography. A: autoradiogram of ³²P-UT-A1. B: Western blot of total UT-A1 for representative samples. Each lane provides results from the kidneys of a separate animal. Arrows indicate the 117- and 97-kDa glycoprotein forms of UT-A1. C: bar of the ratio of phosphorylated UT-A1 to total UT-A1 from all samples from 2 separate cohorts of animals, presented as means ± SE, n = 6/condition. *P < 0.05 vs. isotonic control.

Fig. 3.

Hypertonic stimulation of UT-A1 membrane accumulation is blocked by inhibition of PKC. Rat IMCD suspensions were incubated in either 450-mosmol/kgH₂O buffer (control), 900-mosmol/kgH₂O buffer (hyper), or 900-mosmol/kgH₂O buffer with 10 μM Chel (hyper + Chel), for 30 min, and then biotinylated to reveal membrane-associated UT-A1. The biotinylated protein population was subjected to Western blot analysis probed with anti-UT-A1. A: membrane-associated UT-A1. B: total UT-A1 in representative samples. Each lane provides results from the kidneys of a separate animal. Arrows indicate the 117- and 97-kDa glycoprotein forms of UT-A1. C: bar of the ratio of membrane UT-A1 to total UT-A1 from all samples presented as means ± SE, n = 6/condition. *P < 0.05 vs. isotonic control.

Fig. 4.

Stimulation of PKC results in increased UT-A1 phosphorylation. Metabolically labeled (³²P) rat IMCDs were incubated without (control, Ctrl) or with 2 μM phorbol dibutyrate (PDBu) for 30 min, and then UT-A1 was immunoprecipitated and analyzed by Western blot and autoradiography. A: autoradiogram of ³²P-UT-A1. B: Western blot of total UT-A1 for representative samples. Arrows indicate the 117- and 97-kDa glycoprotein forms of UT-A1. C: bar of the ratio of phosphorylated UT-A1 to total UT-A1 from all samples presented as means ± SE, n = 6/condition. *P < 0.05 vs. Ctrl.

Fig. 5.

Phosphorylation of UT-A1 by PKA+PKC exceeds PKC phosphorylation levels. Metabolically labeled (³²P) rat IMCDs were incubated with PDBu or both AVP with PDBu (AVP+PDBu) and then immunoprecipitated with anti-UT-A1 and analyzed by Western blot and autoradiography. A: autoradiogram of ³²P-UT-A1. B: Western blot of total UT-A1 for representative samples. Arrows indicate the 117- and 97-kDa glycoprotein forms of UT-A1. C: bar of the ratio of phosphorylated UT-A1 to total UT-A1 from all samples from 3 cohorts of animals, presented as means ± SE, n = 9/condition. *P < 0.05 vs. control.

Fig. 6.

PKC does not phosphorylate UT-A1 at serine 486. Rat IMCD suspensions were treated without (Ctrl) or with 100 nM AVP, 2 μM PDBu, or AVP+PDBu and then immunoprecipitated with anti-UT-A1 and analyzed by Western blot using an antibody specific to UT-A1 phosphorylated at serine 486. A: 3 representative samples per condition. B: ratio of Pser486-UT-A1 to total UT-A1 from all samples presented as means ± SE, n = 6/condition. *P < 0.05 vs. Ctrl. Arrows indicate the 117- and 97-kDa glycoprotein forms of UT-A1. The response to AVP+PDBu was not statistically different from vasopressin alone.

Fig. 7.

PKC-α is phosphorylated by hypertonicity in rat inner medulla. Rat IMCDs were treated with 290-mosmol/kgH₂O buffer (iso) or 600-mosmol/kgH₂O buffer (hyper) that was made hypertonic by the addition of sucrose. Whole cell lysates were analyzed by Western blot probed for phosphorylated PKC-α (A) or total PKC-α (B). C: bar graph of the ratio of phosphorylated PKC-α to total PKC-α from all samples presented as means ± SE, n = 11/condition. *P < 0.05 vs. Ctrl.

Fig. 8.

PKC-α-deficient (PKC-α^−/−) mice have dilute urine. A: normal wild-type (WT) and PKC-α^−/− mice were placed in metabolic cages and 24-h urines were collected and urine osmolality was measured. The bar graph provides means ± SE, n = 9 animals per mouse type. *P < 0.05. B: Western blot of IM tissue lysate from normal WT and PKC-α^−/− mice probed for UT-A1. C: bar graph showing means ± SE values for the combined 97- and 117-kDa band densities for 4 mice per group. *P < 0.05 vs. WT.

Fig. 9.

Hypertonicity stimulates UT-A1 phosphorylation in WT but not PKC-α^−/− mice. IMCDs from WT and PKC-α^−/− mice were metabolically labeled with ³²P and then incubated with 290-mosmol/kgH₂O buffer (iso) or 600-mosmol/kgH₂O buffer (hyper). UT-A1 was immunoprecipitated from each sample and analyzed by Western blot and autoradiography. The autoradiograms in A show the phosphorylated UT-A1 levels from representative IMCDs from WT (left) or PKC-α^−/− (right) mice. The Western blots in B show total UT-A1 protein levels in the same samples. C: bar graph showing the ratio of phosphorylated UT-A1 to total UT-A1 in hypertonically treated IMCDs as a percent of isotonic control levels (dashed line at 100%) for each mouse type. All data presented as means ± SE, n = 12/condition. *P < 0.05.