Mechanisms of PKC-Dependent Na⁺ K⁺ ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure

Figures & data

Table 1 Body weight, plasma and urine parameters in control and CRF rats

Group	Control rats (n = 12)	CRF rats (n = 11)
Body weight (g)	300.4 ± 11.6	287.7 ± 12.7
Inuline clearance (ml.min^−1.100 g BW^−1)	1.1 ± 1.2	0.4 ± 0.03
Urinary osmolality (mosmol.kg H20^−1)	2601 ± 178.4	899.9 ± 55.6
Water intake (ml.d^−1)	18.2 ± 2.0	36.41 ± 7.0
Plasmatic osmolality (mosmol.kg H20^−1)	292.1 ± 4.0	312.6 ± 3.6
Plasmatic sodium (mM)	139.3 ± 1.3	139.9 ± 2.3
UNaV (μmol.min^−1.100 g BW^−1)	0.31 ± 0.03	0.26 ± 0.05

Data represent mean ± SE. *p < 0.05 compared to control values.

Figure 1. Differences in Na⁺ K⁺ ATPase activity and total Na⁺ K⁺ ATPase expression in outer renal medulla of control and CRF rats: (a) Na⁺ K⁺ ATPase activity was measured in outer renal medulla membranes by hydrolysis of [γ-³²P]ATP; values are means ± SEM and expressed as μmol Pi / mg. prot . hr, and (b) Na⁺ K⁺ ATPase expression was measured in outer renal medulla homogenates using a common monoclonal antibody that recognizes total Na⁺ K⁺ ATPase α1-subunit independently of its phosphorylation degree; values are means ± SEM and were expressed as normalized arbitrary units. Normalized arbitrary units were obtained as total Na⁺ K⁺ ATPase α1-subunit expression with common antibody per μg protein. Na⁺ K⁺ ATPase activity and total Na⁺ K⁺ ATPase expression were increased in CRF rats as compared with control rats. *p < 0.05, as compared with control rats.

Figure 2. Differences in α1-subunit Na⁺ K⁺ ATPase expression at Ser-23 in mTAL microdissected segments of control and CRF rats. The immunoblot was performed with a monoclonal antibody (McK-1) against dephosphorylated PKC site, Ser-23, of the Na⁺ K⁺ ATPase α1-subunit. Values are means ± SEM and were expressed as normalized arbitrary units. Normalized arbitrary units were obtained as (a) Na⁺ K⁺ ATPase α1-subunit expression with McK-1 antibody per μg protein, and (b) Na⁺ K⁺ ATPase α1-subunit expression with McK-1 antibody / total Na⁺ K⁺ ATPase α1-subunit expression with common antibody per μg protein in microdissected tubules. (see Table 2). Densitometric analysis of all samples revealed a higher immunosignal (decreased phosphorylation) of Na⁺ K⁺ ATPase α1-subunit expression in CRF rats, as compared with control rats. *p < 0.05, as compared with control rats.

Table 2 Expression of α1-subunit Na⁺ K⁺ ATPase phosphorylation degree at Ser-23 in microdissected mTAL in control and CRF rats

	Control	CRF
Basal	2.26 ± 0.18	2.11 ± 0.12
RO-318220, 10^−6M	2.16 ± 0.086	2.72 ± 0.13^†
PMA, 10^−6M	0.90 ± 0.28	0.17 ± 0.24^†
DA, 10^−6M	0.94 ± 0.23	1.13 ± 0.28^†
FK-506, 10^−6M	1.41 ± 0.02	2.12 ± 0.20

Expression of phosphorylation degree was measured using McK-1 antibody. Data are expressed as normalized arbitrary units ± SEM. Normalized arbitrary units (NU) were obtained as Na⁺ K⁺ ATPase α1-subunit expression with McK-1 antibody / total Na⁺ K⁺ ATPase α1-subunit expression with common antibody per μg protein in microdissected tubules.

*p < 0.05, as compared with basal condition of control rats.

^†p < 0.05, as compared with basal condition of CRF rats.

Figure 3. Effect of PKC inhibition on α1-subunit Na⁺ K⁺ ATPase phosphorylation degree at Ser-23 in mTAL microdissected from control and CRF rats. Na⁺ K⁺ ATPase α1-subunit phosphorylation degree at Ser-23 in immunoblots of mTAL segments from control and CRF rats were treated with RO-318220 10⁻⁶ M, a specific PKC inhibitor. The immunoblot was performed with a monoclonal antibody (McK-1) against dephosphorylated PKC site, Ser-23, of the Na⁺ K⁺ ATPase α1-subunit. Values are means ± SEM and were expressed as percentage of normalized arbitrary units over basal. Normalized arbitrary units were obtained as Na⁺ K⁺ ATPase α1-subunit expression with McK-1 antibody / total Na⁺ K⁺ ATPase α1-subunit expression with common antibody per μg protein in microdissected tubules (see Table 2). Densitometric analysis of all samples revealed an increase in immunosignal of Na⁺ K⁺ ATPase α1-subunit (decreased phosphorylation) under RO-318220 in mTAL segments of CRF rats. No changes were observed with RO-318220 in mTAL segments of control rats (p = NS). *p < 0.05, as compared with basal in CRF rats.

Figure 4. Effect of PKC stimulation on α1-subunit Na⁺ K⁺ ATPase phosphorylation degree at Ser-23 in mTAL microdissected segments from CRF rats. Na⁺ K⁺ ATPase α1-subunit phosphorylation degree at Ser-23 in immunoblots mTAL segments in CRF rats and under (a) phorbol 12-myristate 13-acetate (PMA) 10⁻⁶ M, a specific PKC agonist, or (b) dopamine (DA) 10⁻⁶ M. The immunoblot was performed with a monoclonal antibody (McK-1) against dephosphorylated PKC site, Ser-23, of the Na⁺ K⁺ ATPase α1-subunit. Values are means ± SEM and were expressed as percentage of normalized arbitrary units over basal. Normalized arbitrary units were obtained as Na⁺ K⁺ ATPase α1-subunit expression with McK-1 antibody / total Na⁺ K⁺ ATPase α1-subunit expression with common antibody per μg protein in microdissected tubule (see Table 2). Densitometric analysis of all samples revealed a decrease in immunosignal (higher phosphorylation) of Na⁺ K⁺ ATPase α1-subunit under PMA and DA in mTAL segments of CRF rats. *p < 0.05, as compared with basal in CRF rats.

Figure 5. Effect of calcineurin inhibition on α1-subunit Na⁺ K⁺ ATPase phosphorylation degree at Ser-23 in mTAL microdissected segments of control and CRF rats. Na⁺ K⁺ ATPase α1-subunit phosphorylation degree at Ser-23 in immunoblots of mTAL segments from control and CRF rats were treated with FK-506 10⁻⁶ M, a specific calcineurin inhibitor. The immunoblot was performed with a monoclonal antibody (McK-1) against dephosphorylated PKC site, Ser-23, of the Na⁺ K⁺ ATPase α1-subunit. Values are means ± SEM and were expressed as percentage of normalized arbitrary units over basal. Normalized arbitrary units were obtained as Na⁺ K⁺ ATPase α1-subunit expression with McK-1 antibody / total Na⁺ K⁺ ATPase α1-subunit expression with common antibody per μg protein in microdissected tubules (see Table 2). Densitometric analysis of all samples revealed no changes in immunosignal of α1-subunit Na⁺ K⁺ ATPase at Ser-23 under FK-506 in mTAL segments of CRF rats (p = NS). The calcineurin inhibitor FK-506 decreased immunosignal (higher phosphorylation) in mTAL of control rats. *p < 0.05, as compared with basal in control rats.