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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.

The afferent arteriole (Af-Art) controls glomerular capillary pressure, an important determinant of glomerular injury. Af-Art myogenic response is mediated by ATP, and ATP signaling is in turn mediated by 20-HETE. Dahl salt-sensitive rats (Dahl SS) have decreased renal 20-HETE production. We hypothesized that Dahl SS have an impaired myogenic response and constrictor response to ATP, due to decreased 20-HETE. Af-Arts from Dahl SS or Dahl salt-resistant rats (Dahl SR) were microdissected and perfused. When myogenic response was induced by increasing Af-Art perfusion pressure from 60 to 140 mmHg, luminal Af-Art diameter decreased in Dahl SR but not in Dahl SS (−3.1 ± 0.8 vs. 0.5 ± 0.8 μm, P < 0.01). The 20-HETE antagonist 20-HEDE (10−6 M) blocked the myogenic response in Dahl SR but had no effect in Dahl SS. Addition of a subconstrictor concentration of 20-HETE (but not a subconstrictor concentration of norepinephrine) restored the myogenic response in Dahl SS. We then perfused Af-Arts at 60 mmHg and tested the effects of the ATP analog α,β-methylene-ATP (10−6 M). Maximum ATP-induced constriction was attenuated in Dahl SS compared with Dahl SR (1.5 ± 0.5 vs. 7.4 ± 0.8 μm, P < 0.001). 20-HEDE attenuated ATP-induced Af-Art constriction in Dahl SR but not in Dahl SS, and consequently, ATP-induced constriction was no longer different between strains. In conclusion, Dahl SS have an impaired myogenic response and ATP-induced Af-Art constriction due to a decrease in Af-Art 20-HETE. The impaired myogenic responses may contribute to the nephrosclerosis that develops in Dahl SS.

Possible role of adenosine in macula densa control of glomerular hemodynamics.BackgroundThe macula densa (MD), a plaque of specialized tubular epithelial cells, senses changes in tubular NaCl concentration and sends a signal(s) that controls the resistance of the glomerular afferent arteriole (Af-Art). This mechanism, called tubuloglomerular feedback (TGF), is thought to be important in the homeostasis of body fluids and electrolytes. Our aim was to determine the range of NaCl concentrations in tubular fluid at the MD that would elicit the Af-Art response. In addition, we examined the possible involvement of adenosine in transmitting the signal from the MD to the Af-Art.MethodsRabbit Af-Arts and attached MD were simultaneously microperfused in vitro, keeping pressure in the Af-Art at 60mm Hg.ResultsIncreasing the Na+/Cl- concentration of the MD perfusate from 26/7 to 41/22 mEq/L decreased the luminal diameter of the terminal Af-Art segment by 10 ± 4% (N = 9; P < 0.01). The response was maximal at 55/36 mEq/L (18 ± 6%), so that further elevation of NaCl concentration had no additional effect (20 ± 6% at 84/65 mEq/L). When FK838 (10-6 mol/L), a specific adenosine A1 receptor antagonist, was added to both Af-Art perfusate and bath, Af-Art constriction was completely abolished. The maximum response was 20 ± 3% before FK838 and 0.6 ± 1% afterward (N = 12). Adding adenosine at 10-8 mol/L to both bath and perfusate significantly augmented Af-Art constriction induced by increased NaCl at the MD (P < 0.01); however, adding 10-8 to 10-6 mol/L adenosine to the MD perfusate had no effect regardless of the NaCl concentration at the MD.ConclusionsThese results demonstrate that MD control of Af-Art resistance is induced by relatively low NaCl concentrations at the MD, and that activation of the adenosine A1 receptor in the vascular and interstitial space (but not the tubular lumen) may be essential for signal transmission from the MD to the Af-Art.

Afferent (Af-Art) and efferent arterioles resistance regulate glomerular capillary pressure. The nephron regulates Af-Art resistance via: 1) vasoconstrictor tubuloglomerular feedback (TGF), initiated in the macula densa via Na-K-2Cl cotransporters (NKCC2) and 2) vasodilator connecting tubuloglomerular feedback (CTGF), initiated in connecting tubules via epithelial Na channels (ENaC). Furosemide inhibits NKCC2 and TGF. Benzamil inhibits ENaC and CTGF. In vitro, CTGF dilates preconstricted Af-Arts. In vivo, benzamil decreases stop-flow pressure (PSF), suggesting that CTGF antagonizes TGF; however, even when TGF is blocked, CTGF does not increase PSF, suggesting there is another mechanism antagonizing CTGF. We hypothesize that in addition to NKCC2, activation of Na/H exchanger (NHE) antagonizes CTGF, and when both are blocked CTGF dilates Af-Arts and this effect is blocked by a CTGF inhibitor benzamil. Using micropuncture, we studied the effects of transport inhibitors on TGF responses by measuring PSF while increasing nephron perfusion from 0 to 40 nl/min. Control TGF response (−7.9 ± 0.2 mmHg) was blocked by furosemide (−0.4 ± 0.2 mmHg; P < 0.001). Benzamil restored TGF in the presence of furosemide (furosemide: −0.2 ± 0.1 vs. furosemide+benzamil: −4.3 ± 0.3 mmHg; P < 0.001). With furosemide and NHE inhibitor, dimethylamiloride (DMA), increase in tubular flow increased PSF (furosemide+DMA: 2.7 ± 0.5 mmHg, n = 6), and benzamil blocked this (furosemide+DMA+benzamil: −1.1 ± 0.2 mmHg; P < 0.01, n = 6). We conclude that NHE in the nephron decreases PSF (Af-Art constriction) when NKCC2 and ENaC are inhibited, suggesting that in the absence of NKCC2, NHE causes a TGF response and that CTGF dilates the Af-Art when TGF is blocked with NKCC2 and NHE inhibitors.

It has been reported that sensitivity to angiotensin II (Ang II) is higher in efferent (Ef) than afferent (Af) arterioles (Arts). We tested the hypothesis that this is due to arteriolar differences in the interaction between Ang II and endothelium-derived relaxing factor/nitric oxide (EDNO). Rabbit Af-Arts with glomerulus intact were microperfused in vitro at a constant pressure. Ef-Arts were perfused from the distal end of either the Af-Art (orthograde perfusion) or the Ef-Art (retrograde perfusion) to eliminate influences of the Af-Art or glomerulus, respectively. Ang II did not alter Af-Art luminal diameter until the concentration reached 10(-9) M, which decreased the diameter by 11 +/- 2.6% (n = 11; P < 0.002). In contrast, Ef-Arts became significantly constricted at concentrations as low as 10(-11) M with either perfusion. Surprisingly, the decrease in Ef-Art diameter at 10(-10), 10(-9), and 10(-8) M was significantly greater with retrograde perfusion (44 +/- 6.9%, 70 +/- 5.6%, and 74 +/- 4.1%, respectively; n = 5) than with orthograde perfusion (16 +/- 4.2%, 25 +/- 2.9%, and 35 +/- 3.5%; n = 9). ENDO synthesis inhibition with 10(-4) M nitro-L-arginine methyl ester (L-NAME) decreased the diameter to a greater extent in Af-Arts (22 +/- 3.0%; n = 11) compared to Ef-Arts with either orthograde (9.5 +/- 2.3%; n = 8) or retrograde perfusion (1.2 +/- 2.1%; n = 6). With L-NAME pretreatment, Af-Art constriction induced by 10(-10) M (14 +/- 4.0%, n = 9) and 10(-9) M Ang II (38 +/- 3.9%) was significantly greater compared to nontreated Af-Arts. In contrast, L-NAME pretreatment had no effect on Ang II-induced constriction in Ef-Arts with either perfusion. In conclusion, this study demonstrates higher sensitivity of Ef-Arts to Ang II, particularly with retrograde perfusion. Our results suggest that EDNO significantly modulates the vasoconstrictor action of Ang II in Af-Arts II but not Ef-Arts, contributing to the differential sensitivity to Ang II.

Glomerular prostaglandins modulate vascular reactivity of the downstream efferent arterioles. The balance of vascular resistance in afferent (Af–) and efferent arterioles (Ef-Arts) is a crucial factor that determines glomerular hemodynamics. We have recently reported that when Ef-Arts were perfused from the distal end of the Af-Art through the glomerulus (orthograde perfusion; OP), both angiotensin II (Ang II) and norepinephrine (NE) induced much weaker constriction than they did when Ef-Arts were perfused from the distal end (retrograde perfusion; RP). This difference was not affected by inhibiting synthesis of nitric oxide. In the present study, we tested the hypothesis that glomerular prostaglandins (PGs) may modulate vascular reactivity of the downstream Ef-Art. In addition, we examined the possible modulatory role of PGs in the Af-Art responses to Ang II or NE. Both Ang II and NE caused dose-dependent constriction of Ef-Arts with either OP or RP; however, the constriction was stronger in RP, At 10−8 M, Ang II decreased Ef-Art diameter by 35 ± 3.5% in OP (N = 9) compared to 73 ± 3.9% in RP (N = 5), while 10−6M NE decreased the diameter by 25 ± 3.6% in OP (N = 9) compared to 62 ± 7.2% in RP (N = 5). Pretreatment with 5 × 10−5M indomethacin (Indo) did not alter basal diameter with either method of perfusion. However, in OP it significantly augmented the constriction induced by Ang II and NE; 10−8M Ang II and 10−6M NE now decreased the diameter by 72 ± 4.7% (N = 8; P < 0.01 vs. non-treated) and 48 ± 3.3% (N = 7; P < 0.01), respectively. In contrast, Indo had no effect on either Ang II- or NE-induced constriction in Ef-Arts with RP. In vehicle-treated Af-Arts, Ang II had no effect until the concentration reached 10−9M, whereas it began to cause significant constriction at concentrations as low as 10−11M in Indo-treated Af-Arts. These results suggest that the resistance of the Ef-Art may be regulated by PGs released by the upstream glomerulus, while PGs synthesized within the Af-Art may modulate Ang II action in the Af-Art.

Despite evidence that platelet-activating factor (PAF) is produced by the glomerulus, its direct action on the glomerular microcirculation is poorly understood. It was recently reported that at picomolar concentrations, PAF dilates isolated microperfused afferent arterioles (Af-Art) via nitric oxide (NO). The present study tested the hypothesis that PAF acts on the glomerulus to release NO, which in turn controls the resistance of the efferent arteriole (Ef-Art). Rabbit Ef-Art were perfused from the distal end (retrograde perfusion [RP]) to eliminate the influence of the glomerulus, or through the glomerulus from the end of the Af-Art (orthograde perfusion [OP]) to maintain the influence of the glomerulus. Ef-Art were preconstricted by approximately 40% with norepinephrine and increasing doses of PAF were added to both the arteriolar perfusate and bath. Only with OP did PAF at picomolar concentrations cause significant dilation: at 400 pmol, the diameter increased by 64 +/- 11% from the preconstricted level (N = 6, P0.01). This dilation was completely abolished by pretreatment with an NO-synthesis inhibitor. To study its possible constrictor action, PAF was added to nonpreconstricted Ef-Art. At nanomolar concentrations, PAF constricted Ef-Art similarly in both RP and OP: at 40 nM, the diameter decreased by 24 +/- 4% (N = 6, P0.01) and 20 +/- 2% (N = 6, P0.01), respectively. This constriction was attenuated by pretreatment with indomethacin (Indo) in both RP (14 +/- 2%, N = 7; P0.02 versus without Indo) and OP (10 +/- 2%, N = 6; P0.02 versus without Indo).(1) at picomolar concentrations, PAF stimulates the glomerulus to release NO, which in turn dilates the Ef-Art; and (2) at nanomolar concentrations, PAF constricts the Ef-Art partly through release of cyclooxygenase metabolites. Thus, PAF may play a role in glomerular hemodynamics under various physiological and pathological conditions.