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N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Its effect on salt-sensitive (SS) hypertension is unknown. We hypothesized that in Dahl SS rats on high-salt (HS) diet, Ac-SDKP prevents loss of nephrin expression and renal immune cell infiltration, leading to a decrease in albuminuria, renal inflammation, fibrosis, and glomerulosclerosis. To test this, Dahl SS rats and consomic SS13BN controls were fed either a low-salt (0.23% NaCl) or HS (4% NaCl) diet and treated for 6 weeks with vehicle or Ac-SDKP at either low or high dose (800 or 1600 μg/kg per day, respectively). HS increased systolic blood pressure in SS rats (HS+vehicle, 186±5 versus low salt+vehicle, 141±3 mm Hg; P<0.005) but not in SS13BN rats. Ac-SDKP did not affect blood pressure. Compared with low salt, HS-induced albuminuria, renal inflammation, fibrosis, and glomerulosclerosis in both strains, but the damages were higher in SS than in SS13BN. Interestingly, in SS13BN rats, Ac-SDKP prevented albuminuria induced by HS (HS+vehicle, 44±8 versus HS+low Ac-SDKP, 24±3 or HS+high Ac-SDKP, 8±1 mg/24 h; P<0.05), whereas in SS rats, only high Ac-SDKP dose significantly attenuated albuminuria (HS+vehicle, 94±10 versus HS+high Ac-SDKP, 57±7 mg/24 h; P<0.05). In both strains, Ac-SDKP prevented HS-induced inflammation, interstitial fibrosis, and glomerulosclerosis. In summary, in SS rats on HS diet, at low and high doses, Ac-SDKP prevented renal damage without affecting the blood pressure. Only the high dose of Ac-SDKP attenuated HS-induced albuminuria. Conversely, in SS13BN rats, both doses of Ac-SDKP prevented HS-induced renal damage and albuminuria. [ABSTRACT FROM AUTHOR]

Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation. CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids; however, their exact nature and source remain unknown. We hypothesized that during CTGF, the CNT releases prostaglandin E2, which binds its type 4 receptor (EP4) and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected, perfused, and preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mmol/L. We induced CTGF with or without the EP4 receptor blocker ONO-AE3-208 added to the bath in the presence of the epoxyeicosatrienoic acid synthesis inhibitor MS-PPOH. ONO-AE3-208 abolished CTGF (control, 9.4 ± 0.5; MS-PPOH+ONO-AE3-208, -0.6 ± 0.2 μm; P<0.001; n=6). To confirm these results, we used a different, specific EP4 blocker, L161982 (10(-5) mol/L), that also abolished CTGF (control, 8.5 ± 0.9; MS-PPOH+L161982, 0.8 ± 0.4 μm; P<0.001; n=6). To confirm that the eicosanoids that mediate CTGF are released from the CNT rather than the Af-Art, we first disrupted the Af-Art endothelium with an antibody and complement. Endothelial disruption did not affect CTGF (7.9 ± 0.9 versus 8.6 ± 0.6 μm; P=NS; n=7). We then added arachidonic acid to the lumen of the CNT while maintaining zero NaCl in the perfusate. Arachidonic acid caused dose-dependent dilation of the attached Af-Art (from 8.6 ± 1.2 to 15.3 ± 0.7 μm; P<0.001; n=6), and this effect was blocked by ONO-AE3-208 (10(-7) mol/L). We conclude that during CTGF, the CNT releases prostaglandin E2, which acts on EP4 on the Af-Art inducing endothelium-independent dilation. [ABSTRACT FROM AUTHOR]

Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation. CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids; however, their exact nature and source remain unknown. We hypothesized that during CTGF, the CNT releases prostaglandin E2, which binds its type 4 receptor (EP4) and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected, perfused, and preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mmol/L. We induced CTGF with or without the EP4 receptor blocker ONO-AE3-208 added to the bath in the presence of the epoxyeicosatrienoic acid synthesis inhibitor MS-PPOH. ONO- AE3-208 abolished CTGF (control, 9.4±0.5; MS-PPOH+ONO-AE3-208, -0.6±0.2 μm; P<0.001; n=6). To confirm these results, we used a different, specific EP4 blocker, L161982 (10-5 mmol/L), that also abolished CTGF (control, 8.5±0.9; MS-PPOH+L161982, 0.8±0.4 μm; P<0.001; n=6). To confirm that the eicosanoids that mediate CTGF are released from the CNT rather than the Af-Art, we first disrupted the Af-Art endothelium with an antibody and complement. Endothelial disruption did not affect CTGF (7.9±0.9 versus 8.6±0.6 μm; P=NS; n=7). We then added arachidonic acid to the lumen of the CNT while maintaining zero NaCl in the perfiisate. Arachidonic acid caused dose-dependent dilation of the attached Af-Art (from 8.6±1.2 to 15.3±0.7 μm; P<0.001; n=6), and this effect was blocked by ONO-AE3-208 (10-7 mol/L). We conclude that during CTGF, the CNT releases prostaglandin E2, which acts on EP4 on the Af-Art inducing endothelium- independent dilation. [ABSTRACT FROM AUTHOR]

In Dahl salt-sensitive rats (Dahl SS), glomerular capillary pressure increases in response to high salt intake and this is accompanied by significant glomerular injury compared with spontaneously hypertensive rats with similar blood pressure. Glomerular capillary pressure is controlled mainly by afferent arteriolar resistance, which is regulated by the vasoconstrictor tubule glomerular feedback (TGF) and the vasodilator connecting TGF (CTGF). We hypothesized that Dahl SS have a decreased TGF response and enhanced TGF resetting compared with spontaneously hypertensive rats, and that these differences are attributable in part to an increase in CTGE In vivo, using micropuncture we measured stop-flow pressure (a surrogate of glomerular capillary pressure). TGF was calculated as the maximal decrease in stop-flow pressure caused by increasing nephron perfusion, TGF resetting as the attenuation in TGF induced by high salt diet, and CTGF as the difference in TGF response before and during CTGF inhibition with benzamil. Compared with spontaneously hypertensive rats, Dahl SS had (1) lower TGF responses in normal (6.6±0.1 versus 11.0±0.2 mmHg; P<0.001) and high-salt diets (3.3±0.1 versus 10.1±0.3 mmHg; P<0.001), (2) greater TGF resetting (3.3±0.1 versus 1.0±0.3 mmHg; P<0.001), and (3) greater CTGF (3.4±0.4 versus 1.2±0.1 mmHg; P<0.001). We conclude that Dahl SS have lower TGF and greater CTGF than spontaneously hypertensive rats, and that CTGF antagonizes TGE Furthermore, CTGF is enhanced by a high-salt diet and contributes significantly to TGF resetting. Our findings may explain in part the increase in vasodilatation, glomerular capillary pressure, and glomerular damage in SS hypertension during high salt intake. [ABSTRACT FROM AUTHOR]

Tubuloglomerular feedback (TGF) is a mechanism that senses NaCl in the macula densa (MD) and causes constriction of the afferent arteriole. CO, either endogenous or exogenous, inhibits TGF at least in part via cGMP. We hypothesize that CO in the MD, acting via both cGMP-dependent and -independent mechanisms, attenuates TGF by acting downstream from depolarization and calcium entry into the MD cells. In vitro, microdissected rabbit afferent arterioles and their MD were simultaneously perfused and TGF was measured as the decrease in afferent arteriole diameter. MD depolarization was induced with ionophores, while adding the CO-releasing molecule-3 to the MD perfusate at nontoxic concentrations. CO-releasing molecule-3 blunted depolarization-induced TGF at 50 µmol/L, from 3.6±0.4 to 2.5±0.4 µm (P<0.01), and abolished it at 100 µmol/L, to 0.1±0.1 urn (P<0.001; n=6). When cGMP generation was blocked by guanylyl cyclase inhibitor LY83583 added to the MD, CO-releasing molecule-3 no longer affected depolarization-induced TGF at 50 µmol/L (2.9±0.4 versus 3.0±0.4 µm) but partially inhibited TGF at 100 µmol/L (to 1.3±0.2 urn; P<0.05; n=9). Experiments using eicosatetraynoic acid and indomethacin suggest arachidonic acid metabolites do not mediate the cGMP-independent effect of CO. We then added the calcium ionophore A23187 to the MD, which caused TGF (4.1+0.6 µmol/L); A23187-induced TGF was inhibited by CO-releasing molecule-3 at 50 µmol/L (1.9+0.6 µmol/L; P<0.01) and 100 µmol/L (0.2±0.5 µmol/L; P<0.001; n=6). We conclude that CO inhibits TGF acting downstream from depolarization and calcium entry, acting via cGMP at low concentrations, but additional mechanisms of action may be involved at higher concentrations. [ABSTRACT FROM AUTHOR]

The article presents a study that deals with the mechanisms underlying the role of carbon monoxide (CO) in attenuating tubuloglomerular feedback. The study investigated the influence of CO and cGMP-stimulated phosphodiesterase 2 in reducing cAMP in the macula densa, leading to TGF attenuation. It shows that TGF is attenuated by CO through reduction of cAMP via a cGMP-dependent pathway mediated by cGMP-dependent protein kinase.

Increasing Na delivery to epithelial Na channels (ENaCs) in the connecting tubule (CNT) causes dilation of the afferent arteriole (Af-Art), a process we call CNT glomerular feedback (CTGF). Angiotensin II (Ang II) stimulates ENaC in the collecting duct via Ang II type 1 receptors. We hypothesized that Ang II in the CNT lumen enhances CTGF by activation of Ang II type 1 receptors, protein kinase C and ENaC. Rabbit afferent arterioles and their adherent CNT were microperfused and preconstricted with norepinephrine. Each experiment involved generating 2 consecutive concentration-response curves by increasing NaCl in the CNT lumen. During the control period, the maximum dilation of the afferent arteriole was 7.9±0.4 μm, and the concentration of NaCl in the CNT needed to achieve half maximal response (EC(50)) was 34.7±5.2 mmol/L. After adding Ang II (10(-9) mol/L) to the CNT lumen, the maximal response was 9.5±0.7 μm and the EC(50) was 11.6±1.3 mmol/L (P=0.01 versus control). Losartan, an Ang II type 1 antagonist (10(-6) mol/L) blocked the stimulatory effect of Ang II; PD123319, an Ang II type 2 antagonist (10(-6) mol/L), did not. The protein kinase C inhibitor staurosporine (10(-8) mol/L) added to the CNT inhibited the stimulatory effect of Ang II. The ENaC inhibitor benzamil (10(-6) mol/L) prevented both CTGF and its stimulation by Ang II. We concluded that Ang II in the CNT lumen enhances CTGF via activation of Ang II type 1 and that this effect requires activation of protein kinase C and ENaC. Potentiation of CTGF by Ang II could help preserve glomerular filtration rate in the presence of renal vasoconstriction. [ABSTRACT FROM AUTHOR]

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring peptide of which the plasma concentration is increased 4- to 5-fold by angiotensin-converting enzyme inhibitors. We reported previously that, in models of both hypertension and postmyocardial infarction, Ac-SDKP reduces cardiac inflammation and fibrosis. However, it is unknown whether Ac-SDKP can prevent or reverse renal injury and dysfunction in hypertension. In the present study, we tested the hypothesis that, in rats with 5/6 nephrectomy (5/6Nx)-induced hypertension, Ac-SDKP reduces renal damage, albuminuria, and dysfunction by decreasing inflammatory cell infiltration and renal fibrosis and by increasing nephrin protein. Ac-SDKP (800 microg/kg per day, SC via osmotic minipump) or vehicle was either started 7 days before 5/6Nx (prevention) and continued for 3 weeks or started 3 weeks after 5/6Nx (reversal) and continued for another 3 weeks. Rats with 5/6Nx developed high blood pressure, left ventricular hypertrophy, albuminuria, decreased glomerular filtration rate, and increased macrophage infiltration (inflammation) and renal collagen content (fibrosis). Ac-SDKP did not affect blood pressure or left ventricular hypertrophy in either group; however, it significantly reduced albuminuria, renal inflammation, and fibrosis and improved glomerular filtration rate in both prevention and reversal groups. Moreover, slit diaphragm nephrin protein expression in the glomerular filtration barrier was significantly decreased in hypertensive rats. This effect was partially prevented or reversed by Ac-SDKP. We concluded that Ac-SDKP greatly attenuates albuminuria and renal fibrosis and improves renal function in rats with 5/6Nx. These effects may be related to decreased inflammation (macrophages) and increased nephrin protein. [ABSTRACT FROM AUTHOR]

This article discusses research into the possible role of arachidonic acid metabolites in the process of connecting tubule glomerular feedback (CTGF) in the renal cortex. The authors found that increasing salt (NaCl) concentrations in the connecting tubule led to the release of prostaglandins and the arachidonic acid form epoxyeicosatrienoic acid, which help to mediate CTGF.

In the renal cortex, the connecting tubule (CNT) returns to the glomerular hilum and contacts the afferent arteriole (Af-Art). Increasing Na delivery to the CNT dilates the Af-Art by activating epithelial Na channels, a process that we call connecting tubule glomerular feedback (CTGF). However, the mediator(s) of CTGF are unknown. We tested the hypothesis that Na reabsorption by the CNT induces release of arachidonic acid metabolites that diffuse to and dilate the Af-Art. Microdissected rabbit Af-Arts and adherent CNTs were simultaneously microperfused. CTGF was measured as the increase in diameter of norepinephrine-preconstricted Af-Arts in response to switching NaCl concentration in the lumen of the CNT from 10 to 80 mmol/L. Under control conditions, CTGF was repeatable and completely reversed norepinephrine-induced vasoconstriction. In the presence of 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, CTGF was completely blocked (-0.7+/-0.3 versus 7.3+/-0.5 microm), suggesting that arachidonic acid metabolites mediate CTGF. Because both cyclooxygenase-derived prostaglandins and epoxygenase-derived epoxyeicosatrienoic acids are known vasodilatory arachidonic acid metabolites, we tested whether indomethacin or MS-PPOH (a cyclooxygenase and an epoxygenase inhibitor) could block CTGF. Both indomethacin and MS-PPOH partially blocked CTGF (2.3+/-0.8 versus 6.5+/-0.5 microm, and 2.9+/-0.8 versus 6.6+/-1.1 microm, respectively). When combined, they completely blocked CTGF (-0.4+/-0.3 versus 6.6+/-1.1 microm). We confirmed these findings by using the epoxyeicosatrienoic acid antagonist 14,15-EEZE. The combination of indomethacin plus 14,15-EEZE completely abolished CTGF (-0.3+/-0.2 versus 8.0+/-1.0 microm). We conclude that increasing Na concentrations in the CNT stimulate release of prostaglandins and epoxyeicosatrienoic acids, which mediate CTGF. [ABSTRACT FROM AUTHOR]

The afferent arteriole (Af-Art) accounts for most renal vasculature resistance, thus controlling glomerular filtration and renal function. The nephron regulates Af-Art resistance via two mechanisms, the vasoconstrictor tubuloglomerular feedback (TGF), initiated in the macula densa via Na/K/2Cl cotransporters (NKCC2), and the vasodilator connecting tubule glomerular feedback (CTGF), initiated in the connecting tubule via the epithelial Na channels (ENaC). However, when TGF and CTGF are inhibited by furosemide and benzamil, a novel form of TGF is observed.We hypothesize that in addition to NKCC2, TGF can be initiated by Na/H exchangers (NHE). Furthermore, we hypothesize that when NKCC2 and NHE are blocked, CTGF causes Af-Art dilation.In vivo, using the nephron micropuncture technique, we performed two consecutive stop-flow pressure (PSF) curves (an index of glomerular capillary pressure) by increasing the perfusion of the nephron from 0 to 40 nL/min while adding drugs to the tubular perfusate.TGF was blocked by furosemide (vehicle -7.9±0.2 mmHg at 40 nL/min, furosemide -0.4±0.2; P<0.001). An NKCC2-independent form of TGF was revealed by blocking TGF with furosemide and CTGF with the ENaC inhibitor benzamil (furosemide: -0.2±0.1 mmHg, furosemide+benzamil: -4.3±0.3 mmHg, P<0.001). TGF was completely blocked by the combination of furosemide and the NHE inhibitor dimethylamiloride (DMA), resulting in vasodilatation (furosemide+DMA: 2.7±0.5 mmHg, n=6), that was blocked by CTGF inhibition (furosemide+DMA+benzamil: -1.1±0.2 mmHg, P<0.01, n=6).We conclude that NHE in the nephron causes TGF when NKCC2 and ENaC are inhibited and that CTGF causes dilation of the Af-Art when TGF is completely blocked with NKCC2 and NHE inhibitors. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by development of auto antibodies against various self-antigens and deposition of immune complexes leading to inflammation, fibrosis and end organ damage. Ac-SDKP is a naturally occurring tetrapeptide that in hypertension and other diseases such as autoimmune myocarditis prevents inflammation and fibrosis in heart, kidney and vasculature. Here we tested the hypothesis that Ac-SDKP in a mouse model of SLE decreases autoimmune responses, thus preventing inflammation and renal damage. To test this, lupus mice were treated with Ac-SDKP for 20 weeks. Ac-SDKP treated lupus mice exhibited significant decrease in 1) macrophage and T cell infiltration, 2) expression of pro-inflammatory cytokines, 3) complement system activation and 4) expression of cell adhesion protein ICAM-1. In addition, the lupus mice had a significant improvement in renal function (GFR), decrease in glomerular damage and interstitial collagen deposition. Ac-SDKP also reduced the facial skin lesion. Ac-SDKP did not decrease the production of autoantibodies nor blood pressure. Therefore the protective effect of Ac-SDKP is most likely achieved by modulation of the activation of the complement system, expression of chemokines and ICAM-1, leading to a reduction of innate and adaptive immunity cell infiltration in the kidney [ABSTRACT FROM AUTHOR]

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide hydrolyzed by angiotensin-converting enzyme. We previously reported that Ac-SDKP protects the kidney from inflammation, fibrosis, and dysfunction caused by hypertension. Salt-sensitivity is highly prevalent in African-Americans and elderly patients, and is associated with a high rate of hypertensive renal disease.The effect of Ac-SDKP on Salt-Sensitive (SS) hypertension is unknown; here, we hypothesized that Ac-SDKP prevents renal fibrosis, glomerulosclerosis and albuminuria in Dahl SS rats on high salt diet without affecting blood pressure.Dahl SS rats and their consomic control, SS-13BN were fed either 0.23% NaCl (low salt, LS) or 4% NaCl (high salt, HS) diet and infused with Ac-SDKP (800 μg/kg/day, subcutaneously via osmotic minipump) or vehicle (0.01 N acetic acid) for 6 weeks. Animals were divided into following groups: LS+vehicle, HS+vehicle, and HS+Ac-SDKP.High salt increased systolic blood pressure (SBP) in SS rats (HS+vehicle 186±5 vs. LS+vehicle 141±3 mmHg), but not in SS-13BN (HS+vehicle 140±3 vs. LS+vehicle 140±2 mmHg). Ac-SDKP did not affect SBP. In both SS and SS-13BN, HS induced renal and cardiac hypertrophy, which were not prevented by Ac-SDKP. However, in SS rats, Ac-SDKP prevented HS-induced renal interstitial fibrosis (HS+vehicle 12±0.5 vs. HS+Ac-SDKP 7±1 % of renal interstitial area) and glomeruloslerosis (HS+vehicle 18±1 vs. HS+Ac-SDKP 9±1 % of glomerular tuft area). Compared to LS, HS increased albuminuria in both strains, from 13±1 to 94±10 mg/24h in SS and from 7±1 to 44±8 mg/24h in SS-13BN. HS also decreased nephrin protein expression in both strains. Interestingly, in SS-13BN, Ac-SDKP attenuated albuminuria (24±3.18 mg/24h) and partially attenuated the decrease in nephrin expression, but in SS, Ac-SDKP had no effect on either albuminuria (126±34 mg/24h) or nephrin expression.In summary, Ac-SDKP prevents renal fibrosis, glomerulosclerosis but not albuminuria in Dahl SS rats on high salt diet. On the other hand, the consomic controls fed high salt developed albuminuria and decreased nephrin, and these effects were prevented by Ac-SDKP. [ABSTRACT FROM AUTHOR]

Increasing Na delivery to the connecting tubule (CNT) stimulates epithelial Na channels (ENaC) and dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone (aldo) enhances CTGF via a nongenomic mechanism that stimulates CNT ENaC via GPR30 and/or mineralocorticoid receptors (MR). Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. Addition of aldo 10-8 M to the CNT potentiated CTGF, seen as a left-shift in the concentration of NaCl that elicited a half-maximal response (EC50), see Figure. The MR blocker eplerenone (10-5 M) prevented the enhancement of CTGF by aldo (control EC50 = 32.4 ± 2.3 mM; aldo + eplerenone EC50 = 35.4 ± 1.7 mM; n = 7). Neither the transcription inhibitor actinomycin D (5x10-6M) nor the translation inhibitor cycloheximide (10-5M) prevented the effect of aldo (control EC50 = 33.0 ± 2.0 mM; aldo + actinomycin D EC50 = 15.4 ± 1.5 mM; n = 6; P < 0.001, control EC50 = 33.2 ± 2.4 mM; aldo + cycloheximide EC50 = 11.2 ± 1.3 mM; n = 6; P < 0.001). We conclude that aldo in the lumen of the CNT enhances CTGF via a nongenomic effect possibly involving MR and/or GPR30 receptors. Enhanced CTGF induced by aldosterone may contribute to renal damage by causing increases in Af-Art dilation and glomerular capillary pressure (glomerular barotrauma).Figure. Control CTGF (○) seen as dilation of norepinephrine-preconstricted Af-Arts induced by increasing NaCl in the CNT. Aldo 10-8M (•) enhanced CTGF (n = 6; ∗P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; vs. control). Vertical dashed lines indicate EC50. [ABSTRACT FROM AUTHOR]

The afferent arteriole (Af-Art) accounts for most of renal vasculature resistance, thus controlling glomerular filtration rate and renal function. Two mechanisms have been described in which the nephron helps control Af-Art resistance, namely tubuloglomerular feedback (TGF), and connecting tubule glomerular feedback (CTGF). TGF is a constrictor mechanism initiated at the macula densa by the Na/K/2Cl cotransporter (NKCC2), while CTGF is a dilator mechanism initiated at the connecting tubule by the epithelial Na channel (ENaC). However, when NKCC2 is blocked by furosemide, CTGF-induced Af-Art dilation is not evident, thus we hypothesize that there is a constrictor mechanism that is furosemide-insensitive and counters CTGF. To test this, we used in vivo micropuncture of single nephrons. Stop-flow pressure (PSF) was measured as an index of glomerular capillary pressure (which decreases when the Af-Art constricts). Two consecutive PSF curves were generated by raising nephron perfusion from 0 to 40 nL/min while adding drugs to the tubular perfusate. The decrease in PSF induced by increasing nephron perfusion was blocked by furosemide 10-4M (control: 7.9±0.2 mmHg, furosemide: 0.4±0.2 mmHg, P<0.001, n=6), but was partially restored when blocking CTGF with the ENaC inhibitor benzamil 10-6M (furosemide: 0.2±0.1 mmHg, furosemide+benzamil: 4.3±0.3 mmHg, P<0.001, n=6). A possible explanation for these observations is that TGF may be activated to some degree by Na/H exchanger (NHE)-mediated Na entry at the macula densa when NKCC2 is blocked. When we added the NHE blocker dimethylamiloride (DMA, 10-4M) in the presence of furosemide and benzamil, the decrease in PSF was significantly prevented (furosemide+benzamil: 4.6±0.3 mmHg, furosemide+benzamil+DMA: 1.1±0.2 mmHg, P<0.001, n=6). Therefore, we conclude that a constrictor mechanism of the Af-Art is initiated by NHE in the nephron and is observable when NKCC2-mediated TGF and ENaC-mediated CTGF have been blocked. [ABSTRACT FROM AUTHOR]

The afferent arteriole (Af-Art) accounts for most of renal vasculature resistance, thus controlling glomerular filtration rate. The connecting tubule (CNT) is in close contact with the Af-Art. Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the CNT causes Af-Art dilation. We have shown that CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids (EETs), however their exact source and nature remain unknown. Here we tested the hypothesis that during CTGF the CNT releases prostaglandin E2 (PGE2), which binds EP4 receptors and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected and perfused. The Af-Arts were preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mM. To identify the source of the vasodilator prostaglandins we first disrupted the vascular endothelium by adding anti-Factor-VIII antibody to the Af-Art lumen. This did not affect CTGF (control: 7.9±0.9, endothelium disrupted: 8.6±0.6 μm, n=7). We then added arachidonic acid (AA) to the lumen of the CNT while maintaining zero NaCl in the perfusate, and found that AA caused dose-dependent dilation of the attached Af-Art (from 7.9±2.2 to 15.5±1.3 μm, P<0.01, n=4). This cannot be explained by AA diffusing to the Af-Art because AA is a vasoconstrictor for the Af-Art. Taken together, these data indicate that CTGF-induced vasorelaxation is endothelium-independent, and that prostaglandins originate from the CNT. To study which specific prostaglandin and receptor mediate CTGF, we added the EP4 receptor blocker L161982 (10 μM) to the bath in the presence of the EET synthesis inhibitor MS-PPOH. EP4 receptor blockade abolished CTGF (control: 8.5±0.9, MS-PPOH+L161982: 0.8±0.4μm, P<0.001, n=6). To confirm these results we used a different, specific EP4 blocker, ONO-AE-208 (100 nM), which also abolished CTGF (control: 9.4±0.5, MS-PPOH+ONO-AE-208: -0.6±0.2 μm, P<0.001, n=6). We conclude that increasing luminal Na stimulates release of prostaglandin E2 from the CNT, which acts on EP4 on the Af-Art inducing endothelium-independent dilation during CTGF. Our studies provide a better understanding of the mechanisms by which CTGF controls Af-Art resistance. [ABSTRACT FROM AUTHOR]

Tubuloglomerular feedback (TGF) is an autoregulatory mechanism of the renal microcirculation in which the macula densa (MD) senses NaCl concentration in the lumen of the nephron and sends a signal that controls glomerular filtration rate by constricting the afferent arteriole (Af-Art). We have shown that MD depolarization is sufficient for inducing TGF. Carbon monoxide (CO), either endogenous or exogenous, is known to inhibit TGF, at least in part via cGMP. However, whether cGMP-independent mechanisms are involved, and where in the TGF cascade CO exerts its inhibitory effect, remain unknown. Thus we hypothesize that CO, acting via both cGMP-dependent and -independent mechanisms, attenuates TGF by acting downstream from MD cell depolarization. In vitro, microdissected rabbit Af-Arts and their attached MD were simultaneously perfused and TGF was measured as the decrease in Af-Art diameter. Depolarization of the MD was induced by switching luminal KCl from 4 to 50 mM in the presence of the potassium ionophore valinomycin, while adding the CO-releasing molecule CORM-3 to the MD perfusate at non-toxic concentrations. CORM-3 blunted depolarization-induced TGF at a concentration of 50 μM, from 3.6±0.4 to 2.5±0.4 μm (P<0.01), and completely abolished it at a concentration of 100 μM, to 0.1±0.1 μm (P<0.001, n=6). Similar results were found with 100 μM CORM-3 when depolarization was induced by nystatin (3.0±0.2 vs. 0.4±0.2 μm, P<0.001, n=6). This indicates that CO inhibits TGF acting downstream from depolarization. When cGMP generation was blocked with the guanylate cyclase inhibitor LY-83583 (1 μM) added to the MD, CORM-3 no longer had an effect on depolarization-induced TGF at 50 μM (2.9±0.4 vs. 3.0±0.4 μm), but retained partial inhibitory effect on TGF at 100 μM (1.3±0.2 μm, P=0.02, n=9). This suggests that CO acts via cGMP at low concentrations, but additional mechanisms of action may be involved at higher concentrations. Finally, we confirmed that cGMP inhibits TGF downstream from MD depolarization by adding the degradation-resistant cGMP analog dibutyryl-cGMP (500 μM), which attenuated depolarization-induced TGF (from 3.9±0.5 to 0.6±0.2 μm, P<0.01, n=6). Our results could help explain the physiological role of CO in controlling the renal microcirculation. [ABSTRACT FROM AUTHOR]