Mechanisms of connecting tubule glomerular feedback enhancement by aldosterone

Abstract

Connecting tubule glomerular feedback (CTGF) is a mechanism where an increase in sodium (Na) concentration in the connecting tubule (CNT) causes the afferent arteriole (Af-Art) to dilate. We recently reported that aldosterone within the CNT lumen enhances CTGF via a nongenomic effect involving GPR30 receptors and sodium/hydrogen exchanger (NHE), but the signaling pathways of this mechanism are unknown. We hypothesize that aldosterone enhances CTGF via cAMP/protein kinase A (PKA) pathway that activates protein kinase C (PKC) and stimulates superoxide (O₂^-) production. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing the CNT luminal NaCl. We found that the main effect of aldosterone was to sensitize CTGF and we analyzed data by comparing NaCl concentration in the CNT perfusate needed to achieve half of the maximal response (EC₅₀). During the control period, the NaCl concentration that elicited a half-maximal response (EC₅₀) was 37.0 ± 2.0 mmol/l; addition of aldosterone (10^-8 mol/l) to the CNT lumen decreased EC₅₀ to 19.3 ± 1.3 mmol/l (P ≤ 0.001 vs. Control). The specific adenylyl cyclase inhibitor 2',3'-dideoxyadenosine (ddA; 2 × 10^-4 mol/l) and the PKA inhibitor H-89 dihydrochloride hydrate (H-89; 2 × 10^-6 mol/l) prevented the aldosterone effect. The selective PKC inhibitor GF109203X (10^-8 mol/l) also prevented EC₅₀ reduction caused by aldosterone. CNT intraluminal addition of O₂^- scavenger tempol (10^-4 mol/l) blocked the aldosterone effect. We conclude that aldosterone inside the CNT lumen enhances CTGF via a cAMP/PKA/PKC pathway and stimulates O₂^- generation and this process may contribute to renal damage by increasing glomerular capillary pressure.

Keywords: PKA inhibitor; arterioles; distal kidney tubules; superoxide.

Fig. 1.

A: addition of 10⁻⁸ mol/l of aldosterone (Aldo) to the connecting tubule (CNT) perfusate enhanced connecting tubule glomerular feedback (CTGF; *P < 0.025; control vs. aldosterone). B: aldosterone enhanced CTGF; no differences were observed between the first and the second curve, indicating that the effect of aldosterone on CTGF is stable and reproducible over time.

Fig. 2.

A: protein kinase A (PKA) inhibitor (H-89) alone did not affect CTGF in the absence of the exogenous aldosterone. B: addition of 2 × 10⁻⁶ mol/l of H-89 to the CNT perfusate blocked the enhancement of CTGF by aldosterone, suggesting that the effect of aldosterone on CTGF is mediated by the activation of PKA (*P < 0.01; aldosterone vs. aldosterone + H-89).

Fig. 3.

A: addition of 2 × 10⁻⁴ mol/l of cAMP inhibitor 2′,3′-dideoxyadenosine (ddA) to the CNT perfusate blocked the enhancement of CTGF by aldosterone, indicating that the effect of aldosterone on CTGF is mediated by cAMP (*P < 0.025; aldosterone vs. aldosterone + ddA). B: ddA alone did not affect CTGF in the absence of the exogenous aldosterone.

Fig. 4.

A: addition of 10⁻⁸ mol/l of protein kinase C (PKC) inhibitor (GF109302X) to the CNT perfusate prevented the enhancement of CTGF by aldosterone, indicating that this effect is mediated by the activation of PKC (*P < 0.025; aldosterone vs. aldosterone + GF1-9302X). B: GF109302X alone did not affect CTGF in the absence of the exogenous aldosterone.

Fig. 5.

A: addition of 10⁻⁴ mol/l of superoxide dismutase mimetic (tempol) to the CNT perfusate attenuated the enhancement of CTGF by aldosterone, indicating that this effect is mediated by the stimulation of O₂⁻ (*P < 0.025; aldosterone vs. aldosterone + tempol). B: tempol alone did not affect CTGF in the absence of the exogenous aldosterone.

Fig. 6.

Addition of 2.5 × 10⁻⁵ mol/l of Ca²⁺ chelator (BAPTA-AM) to the CNT perfusate blocked the enhancement of CTGF by aldosterone and basal CTGF, indicating that intracellular Ca²⁺ is essential for CTGF. *P < 0.025; aldosterone vs. aldosterone + BAPTA-AM.

Fig. 7.

Aldosterone-enhanced CTGF signaling pathways. Aldosterone acts on GPR30 and increasing cAMP production as well as PKA activation. PKA activation leads to increased intracellular Ca²⁺ which results in the activation of PKC, increased O₂⁻ generation, that stimulate Na/H exchanger and enhanced CTGF.