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. 2021 Mar 31;12:642274. doi: 10.3389/fphys.2021.642274

Figure 5.

Figure 5

Apelin-13 regulates PRR and AQP2 via the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. (A) Representative immunoblotting and densitometric analysis of kidney medullary p-CREB (Ser133) and CREB protein expression in the WD mice, β-actin as an internal control for equal loading of the samples. n = 4–5 per group. Data are mean ± SEM. *p < 0.05 vs. Control, #p < 0.05 vs. WD. (B) Effect of cAMP/PKA pathway on PRR and AQP2 expression in the primary rat inner medullary collecting duct (IMCD) cells. Cells were pretreated with H89 and then treated with 100 μM 8-Br-cAMP for 24 h. The expression of PRR and AQP2 protein was analyzed by immunoblotting and normalized by β-actin. n = 3 per group, repeat three times. Data are mean ± SEM. **p < 0.01 vs. Control; #p < 0.05 and ##p < 0.01 vs. 8-Br-cAMP. (C) Effect of apelin-13 on PRR and AQP2 expression in forskolin (FSK)/3-Isobutyl-1-methylxanthine (IBMX)-treated IMCD cells. Cells were pretreated with 100 nM apelin-13 and then treated with FSK (10 μM)/IBMX (100 μM) for 24 h. The expression of PRR and AQP2 protein was analyzed by immunoblotting and normalized by β-actin. n = 3 per group, repeat three times. Data are mean ± SEM. *p < 0.05 and **p < 0.01 vs. Control; #p < 0.05 vs. FSK/IBMX. (D) Effect of cAMP on AQP2 protein expression in FSK/IBMX + apelin-13-treated IMCD cells. Cells were pretreated with 100 nM apelin-13, or in combination with 100 μM 8-Br-cAMP, then treated with FSK (10 μM)/IBMX (100 μM) for 24 h. The expression of AQP2 protein was analyzed by immunoblotting and normalized by β-actin. n = 3 per group. Data are mean ± SEM. **p < 0.01 vs. Control; ##p < 0.01 vs. FSK/IBMX, &&p < 0.01 vs. FSK/IBMX + apelin-13.