Connecting Tubule Glomerular Feedback Research Articles

Luminal Flow in the Connecting Tubule induces Afferent Arteriole Vasodilation.

Renal autoregulatory mechanisms modulate renal blood flow. Connecting tubule glomerular feedback (CNTGF) is a vasodilator mechanism in the connecting tubule (CNT), triggered paracrinally when high sodium levels are detected via the epithelial sodium channel (ENaC). The primary activation factor of CNTGF-whether NaCl concentration, independent luminal flow, or the combined total sodium delivery-is still unclear. We hypothesized that increasing luminal flow in the CNT induces CNTGF via O2- generation and ENaC activation. Rabbit afferent arterioles (Af-Arts) with adjacent CNTs were microperfused ex-vivo with variable flow rates and sodium concentrations ranging from <1 mM to 80 mM and from 5 to 40 nL/min flow rates. Perfusion of the CNT with 5 mM NaCl and increasing flow rates from 5 to 10, 20, and 40 nL/min caused a flow rate-dependent dilation of the Af-Art (p<0.001). Adding the ENaC blocker benzamil inhibited flow-induced Af-Art dilation, indicating a CNTGF response. In contrast, perfusion of the CNT with <1 mM NaCl did not result in flow-induced CNTGF vasodilation (p>0.05). Multiple linear regression modeling (R2=0.51;p<0.001) demonstrated that tubular flow (β=0.163 ± 0.04;p<0.001) and sodium concentration (β=0.14 ± 0.03;p<0.001) are independent variables that induce afferent arteriole vasodilation. Tempol reduced flow-induced CNTGF, and L-NAME did not influence this effect. Increased luminal flow in the CNT induces CNTGF activation via ENaC, partially due to flow-stimulated O2- production and independent of nitric oxide synthase (NOS) activity.

Luminal flow-stimulated superoxide participates in connecting tubule-glomerular feedback

Increasing NaCl concentration in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art) through the connecting tubule-glomerular feedback (CNTGF). CNTGF is mediated by Na+ transport via epithelial Na+ channels (ENaC) in the CNT. It is unknown if CNTGF is mainly activated by NaCl concentration or sodium delivery or if there is an independent luminal flow effect. Previous studies showed that luminal flow stimulates superoxide (O2−), and O2− can activate ENaC. Thus we hypothesized that increasing luminal flow in the CNT induces CNTGF via O2− generation and ENaC activation. Rabbit Af-Arts with the adjacent CNTs were microdissected and perfused in vitro. Protocols consist of two consecutive dose-response curves to flow rate in the same preparation. When the CNT was perfused with 5mM NaCl, increasing flow rates from 5 to 10, 20, and 40 nL/min caused flow rate-dependent Af-Art dilatation (p&lt;0.01). Adding an ENaC blocker benzamil to the perfusate blocked flow-induced Af-Art dilatation, indicating a CNTGF response. Using mathematical modeling, an increased tubular flow controlled by NaCl delivery into the CNT independently increases CNTGF vasodilation. However, when CNT was perfused with 0 mM NaCl, increasing the flow rate did not induce CNTGF vasodilation. The O2− dismutase mimetic tempol reduced flow-induced CNTGF. In the presence of L-NAME (NOS inhibitor), the reduced CNTGF mediated by tempol was unaffected. We concluded that increasing luminal flow in CNT induces CNTGF activation via ENaC, partly due to flow-stimulated O2− production and independently of NOS. 1K01HL155235. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PS-B09-8: N-METHYL-D-ASPARTATE (NMDA) RECEPTORS INDUCE RENAL VASODILATION AND REGULATES BLOOD PRESSURE.

Introduction: N-methyl-D-aspartate receptors (NMDAr) are glutamate/glycine receptors expressed in the kidney. NMDAr induce renal vasodilation through an unknown mechanism. Connecting tubule-glomerular feedback (CNTGF) is a vasodilator feedback mechanism, initiated by ENaC in the connecting tubule producing afferent arteriole (AffA) vasodilatation and promoting sodium excretion. We hypothesized that NMDAr activation stimulate CNTGF and that the inhibition of NMDAr decrease CNTGF vasodilation inducing hypertension. Methods: We explore the presence of NMDAr receptors in mouse by transcriptome analysis of microdisseted tubules segments, total kidney western blot and immunofluorescence. We used mpkCCD cells (mouse principal cells) to explore the interaction between NMDAr and ENaC in PC using trasnwell current analysis and split-open tubule using patch clamp method. To determine the effect of NMDAr on CNTGF, we measured CNTGF-mediated AffA dilation in the presence or absence of NMDAr agonists glutamate and glycine with and without NMDAr antagonist (MK801), using a double-microperfusion method in rabbits, and in-vivo by micropunture technique in rats. To evaluate the effect of NMDAr on blood pressure (BP), we measure BP in NMDA 2C global knock out mice. BP was measure using tail cuff methods. To confirm the effect of NMDAr on BP we infused subcutaneously NMDAr inhibitor MK801 for 7 days in a gain of function β ENaC mutated allele mice on129/Sv background on normal salt diet. Results: NMDAr was found to be expressed in connecting tubule and co-localized with AQP2 and ENaC. In mpkCCD cells and open split tubules, NMDAr activation increase ENaC activity, while NMDAr inhibition decrease ENaC activity (p &lt; 00.1). In-vitro, tubular microperfusion of NMDAr agonist increased AffA dilation (p &lt; 0.001). NMDAr agonist-induced dilation was blunted with MK-801 (NMDAr blocker) and totally blocked with benzamil (CNTGF inhibitor). In vivo, NMDAr agonist increase CNTGF mediated vasodilation (P &lt; 0.01). NMDAr 2C knock out mice showed high BP in comparison with wildtype (SBP 114.6 ± 7.3 vs 100.4 ± 2.2 mmHg, respectively, p = 0.01). In β ENaC mutated mice, the infusion of NMDAr inhibitor (Mk-801) induces hypertension (SBP 146 ± 8 mmHg) while the vehicle infused mice remained normotensives (110.7 ± 7.7 mmHg; p &lt; 0.01). Conclusions: NMDAr in the connecting tubule increase renal vasodilation by activating CNTGF. The genetic ablation of NMDAr 2C or the pharmacological inhibition of NMDAr increase blood pressure. Future studies need to confirm the role of NMDAr in the pathogenesis of hypertension.

ENaC and N‐methyl‐D‐aspartate (NMDA) receptors interact in the connecting tubule inducing renal vasodilation and blood pressure changes

Introduction N-methyl-D-aspartate (NMDA) receptors are expressed throughout the nephron, including distal nephron principal cells (PC), and induce renal vasodilation through an unknown mechanism. Connecting tubule-glomerular feedback (CNTGF) is a vasodilator feedback mechanism mediated by PC's ENaC producing afferent arteriole (AfA) vasodilatation and promote sodium excretion. We hypothesized that NMDA receptors activation stimulate ENaC and consequently CNTGF, and that the inhibition of NMDA receptors decrease CNTGF inducing hypertension. Methods To determine the effect of NMDA receptor on CNTGF-mediated AfA dilation, we measured CNTGF-mediated AfA dilation in the presence or absence of NMDA receptor antagonists with and without NMDA antagonist (MK801), using a double-perfusion method of the AfA and CNT ex vivo in rabbits and stop-flow pressure (SFP) technique in vivo in rats. To investigate NMDAr-ENsC interaction we measured ENaC open probability (NPo) on split open tubules (CCD) from mice at baseline and with the NMDA agonist. To evaluate the effect of the NMDA receptor in blood pressure regulation, we use the NMDA inhibitor MK801 infused by osmotic minipumps for 7 days in liddle syndrome mouse (β ENaC mutated allele on129/Sv background) on normal salt diet. Under normal salt diet, blood pressure (BP) is not different from wild type despite evidence for increased sodium reabsorption in distal colon and low plasma aldosterone, suggesting chronic hypervolemia. BP was measure using tail cuff methods. Results In pre-constricted AfA, in vitro NMDA agonist application to the connecting tubule lumen increased AfA dilation (p<0.001). This amino-acid-induced dilation was blocked with the CNTGF inhibitor benzamil and blunted with the NMDA receptor blocker MK-801. In vivo, NMDA agonist increase CNTGF (P<0.01). In split open tubules, NMDA activation increased ENaC NPo (p=0.04), suggesting an interaction between those channels. Basal BP was similar in both mice group (p=0.4). The infusion of NMDA inhibitor (Mk-801) during 7 days induces hypertension (SBP 146±8 mmHg) while the vehicle infused mice remained normotensives (110.7±7.7 mmHg; p<0.01). Conclusions NMDA receptor activation induced renal vasodilation by increasing the ENaC dependent CNTGF responses . NMDA inhibition decrease ENaC activation and consequently inhibit CNTGF induced-vasodilation and promote hypertension. These results provide evidence on the role of NMDA receptors on renal hemodynamic and blood pressure regulation.

N-METHYL-D-ASPARTATE (NMDA) RECEPTORS IN THE CONNECTING TUBULE INDUCE RENAL VASODILATION AND REGULATES BLOOD PRESSURE

Objective: N-methyl-D-aspartate (NMDA) receptors are expressed throughout the nephron, including distal nephron principal cells (PC), and induce renal vasodilation through an unknown mechanism. Connecting tubule-glomerular feedback (CNTGF) is a vasodilator feedback mechanism producing afferent arteriole (AfA) vasodilatation and promote sodium excretion. We hypothesized that NMDA receptors activation stimulate CNTGF and that the inhibition of NMDA receptors decrease CNTGF inducing hypertension. Design and method: To determine the effect of NMDA receptor on CNTGF-mediated AfA dilation, we measured CNTGF-mediated AfA dilation in the presence or absence of NMDA receptor antagonists with and without NMDA antagonist (MK801), using a double-perfusion method of the AfA and CNT ex vivo in rabbits and stop-flow pressure (SFP) technique in vivo in rats. To evaluate the effect of the NMDA receptor in blood pressure regulation, we use the NMDA inhibitor MK801 infused by osmotic minipumps for 7 days in liddle syndrome mouse (βENaC mutated allele on129/Sv background) on normal salt diet. Under normal salt diet, blood pressure (BP) is not different from wild type despite evidence for increased sodium reabsorption in distal colon and low plasma aldosterone, suggesting chronic hypervolemia. BP was measure using tail cuff methods. Results: In pre-constricted AfA, in vitro NMDA agonist application to the connecting tubule lumen increased AfA dilation (p &lt; 0.001). This amino-acid-induced dilation was blocked with the CNTGF inhibitor benzamil and blunted with the NMDA receptor blocker MK-801. In vivo, NMDA agonist increase CNTGF (P &lt; 0.01). Basal BP was similar in both mice group (p = 0.4). The infusion of NMDA inhibitor (Mk-801) during 7 days induces hypertension (SBP 146 ± 8 mmHg) while the vehicle infused mice remained normotensives (110.7 ± 7.7 mmHg; p &lt; 0.01). Conclusions: NMDA receptor activation induced renal vasodilation by increasing CNTGF responses. NMDA inhibition decrease CNTGF induced-vasodilation and promote hypertension. These results provide evidence on the role of NMDA receptors on renal hemodynamic and blood pressure regulation.

Abstract P025: Hyperinsulinemia Causes Renal Hyperperfusion And Increases Glomerular Basement Membrane Thickness Independent Of Renal Perfusion Pressure And Glycemic Status: Potential Role Of Connecting Tubule Glomerular Feedback?

Obesity is often associated with hyperinsulinemia (HI) and renal damage. However, the role of HI in Obesity related Renal Damage (ORD) is unclear. Renal hyperperfusion/hyperfiltration plays a major role in ORD. Normally, the kidneys autoregulate blood flow by two feedback mechanisms: 1.) Tubuloglomerular feedback (TGF), a vasoconstrictor mechanism and 2.) Connecting Tubule Glomerular Feedback (CTGF), a vasodilator mechanism. Previously, we found that Zucker obese rats had higher CTGF (TGF was unchanged) and were hyperinsulinemic before the development of ORD. Epithelial sodium channel (ENaC) initiates CTGF and Insulin is a known ENaC activator. Hypothesis: HI increases renal cortical blood flow (CBF) by increasing CTGF and causes renal damage. To isolate the effect of HI from the blood glucose (BG) level, HI-euglycemic clamp was created in normal anesthetized Sprague Dawley (SD) rats by simultaneous intravenous (IV) infusion of 10% glucose and insulin. Average baseline BG in non-fasted anesthetized SD rats was 199.0±31.6 mmol/L. Insulin infusion increased CBF significantly by 12.2 ± 1.3% (n=3, p&lt;0.05) from the baseline even before the BG level starts decreasing. Insulin was further infused to attain normoglycemic condition (96.0±2.3 mmol/L) and this was associated with additional increase in CBF by 19.2 ± 4.5% (p&lt;0.05) from the baseline. Subsequent ENaC inhibition by benzamil (BZ) (400 μg/kg, IV) completely reversed the insulin-induced increase in CBF. Neither Insulin nor BZ treatment altered the renal perfusion pressure (RPP) suggesting insulin-induced increase in CBF was independent of RPP. In a separate group of SD rats, renal-HI (1.8 IU/kg/day) was created in only one of the two kidneys for 6 weeks using renal subcapsular catheter to measure glomerular basement membrane (GBM) thickness (a marker of renal damage). GBM was significantly thickened in insulin-infused kidney compared to vehicle-infused kidney (199.4±17 vs. 145.5±3.6nm, n=3, p&lt;0.05). Conclusion: Acute HI increased CBF, that was completely reversed by ENaC inhibition implying a possible role of enhanced CTGF. Chronic renal-HI caused GBM thickening. Perspective: HI observed in obesity or type-2 diabetes may cause renal hyperperfusion by increasing CTGF and contribute to the ORD.

A Novel Mechanism of Renal Microcirculation Regulation: Connecting Tubule-Glomerular Feedback.

In this review, we summarized the current knowledge of connecting tubule-glomerular feedback (CTGF), a novel mechanism of renal microcirculation regulation that integrates sodium handling in the connecting tubule (CNT) with kidney hemodynamics. Connecting tubule-glomerular feedback is a crosstalk communication between the CNT and the afferent arteriole (Af-Art), initiated by sodium chloride through the epithelial sodium channel (ENaC). High sodium in the CNT induces Af-Art vasodilation, increasing glomerular pressure and the glomerular filtration rate and favoring sodium excretion. CTGF antagonized and reset tubuloglomerular feedback and thus increased sodium excretion. CTGF is absent in spontaneous hypertensive rats and is overactivated in Dahl salt-sensitive rats. CTGF is also modulated by angiotensin II and aldosterone. CTGF is a feedback mechanism that integrates sodium handling in the CNT with glomerular hemodynamics. Lack of CTGF could promote hypertension, and CTGF overactivation may favor glomerular damage and proteinuria. More studies are needed to explore the alterations in renal microcirculation and the role of these alterations in the genesis of hypertension and glomerular damage in animals and humans. • CTGF is a vasodilator mechanism that regulates afferent arteriole resistance. • CTGF is absent in spontaneous hypertensive rats and overactivated in Dahl salt-sensitive rats. • CTGF in excess may promote glomerular damage and proteinuria, while the absence may participate in sodium retention and hypertension.

Role of connecting tubule glomerular feedback in obesity related renal damage.

Zucker obese rats (ZOR) have higher glomerular capillary pressure (PGC) that can cause renal damage. PGC is controlled by afferent (Af-Art) and efferent arteriole (Ef-Art) resistance. Af-Art resistance is regulated by factors that regulate other arterioles, such as myogenic response. In addition, it is also regulated by 2 intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that increased CTGF contributes to TGF attenuation, which in turn increases PGC in ZOR. We performed a renal micropuncture experiment and measured stop-flow pressure (PSF), which is an indirect measurement of PGC in ZOR. Maximal TGF response at 40 nl/min was attenuated in ZOR (4.47 ± 0.60 mmHg) in comparison to the Zucker lean rats (ZLR; 8.54 ± 0.73 mmHg, P < 0.05), and CTGF was elevated in ZOR (5.34 ± 0.87 mmHg) compared with ZLR (1.12 ± 1.28 mmHg, P < 0.05). CTGF inhibition with epithelial sodium channel blocker normalized the maximum PSF change in ZOR indicating that CTGF plays a significant role in TGF attenuation (ZOR, 10.67 ± 1.07 mmHg vs. ZLR, 9.5 ± 1.53 mmHg). We conclude that enhanced CTGF contributes to TGF attenuation in ZOR and potentially contribute to progressive renal damage.

Abstract 066: Regulation of Nephron Afferent Arteriole Resistance in Obesity: Role of Insulin and Connecting Tubule Glomerular Feedback

Introduction: In obesity, increased glomerular capillary pressure (P GC ) may participate in renal damage.P GC is controlled in part by afferent arteriolar ( Af-Art ) resistance that in turn is regulated by two renal intrinsic feedback mechanisms, the vasoconstrictor Tubulo-Glomerular Feedback (TGF), and vasodilator Connecting Tubule Glomerular Feedback (CTGF). CTGF is initiated by an increase in NaCl transport by the epithelial sodium channel (ENaC) in the connecting tubule (CNT). Interestingly, obesity is strongly associated with hyperinsulinemia and insulin is a potent ENaC activator. Hypothesis: In obesity, hyperinsulinemia increases CTGF via activation of the ENaC, this increase, in turn, contributes to TGF attenuation leading to increased P GC and renal damage. Methods: In vivo : In zucker obese rats (ZOR) and zucker lean rats (ZLR), we measured TGF and CTGF using renal micropuncture at 9-10 weeks of age. We quantify stop-flow pressure (P SF ) as an index of P GC . We measured proteinuria as a marker of renal damage in both ZOR and ZLR. In vitro: Microdissected rabbit Af-Arts and their adherent CNTs were perfused with NaCl, insulin or ENaC inhibitor (Benzamil; BZ) to investigate the role of insulin on TGF and CTGF. Results: In-vivo : Maximal TGF response was significantly less in ZOR (6.11 ± 0.75 mmHg) in comparison to the ZLR (9.5 ± 1.1 mmHg, p&lt;0.05). CTGF inhibition by BZ normalized the TGF response in ZOR similar to ZLR (ZOR, 14.01±1.70 mmHg vs ZLR, 11.46± 2.25 mmHg) suggesting CTGF playing a key role in TGF resetting in ZOR. Additionally, ZOR develops proteinuria (mg/24h) at 12 weeks of age (ZOR; 24.85±3.02 vs ZLR; 7.21±1.09, p&lt;0.05 ). In-vitro microperfusion of NaCl in the CNT that elicited a half-maximal response (EC 50 , mmol/L) of Af-Art dilation was 25.0±0.8; an addition of insulin 10 -7 mol/l to the CNT lumen decreased the EC 50 to 8.1±0.8 ( P &lt;0.05) suggesting insulin potentiates CTGF. BZ blocked the insulin-mediated CTGF (Insulin EC 50 : 7.8±0.9 vs . Insulin+BZ, EC 50 : 19.7±5.5; P &lt;0.05). Conclusion: In-vivo : TGF is reset in ZOR due to enhanced CTGF before they develop proteinuria. In-vitro : Insulin increased CTGF during microperfusion experiments. Perspective: Insulin-induced increased in CTGF may explain higher P GC and renal damage in obesity.

Effect of salt intake on afferent arteriolar dilatation: role of connecting tubule glomerular feedback (CTGF).

Afferent arteriole (Af-Art) resistance is modulated by two intrinsic nephron feedbacks: 1) the vasoconstrictor tubuloglomerular feedback (TGF) mediated by Na+-K+-2Cl- cotransporters (NKCC2) in the macula densa and blocked by furosemide and 2) the vasodilator connecting tubule glomerular feedback (CTGF), mediated by epithelial Na+ channels (ENaC) in the connecting tubule and blocked by benzamil. High salt intake reduces Af-Art vasoconstrictor ability in Dahl salt-sensitive rats (Dahl SS). Previously, we measured CTGF indirectly, by differences between TGF responses with and without CTGF inhibition. We recently developed a new method to measure CTGF more directly by simultaneously inhibiting NKCC2 and the Na+/H+ exchanger (NHE). We hypothesize that in vivo during simultaneous inhibition of NKCC2 and NHE, CTGF causes an Af-Art dilatation revealed by an increase in stop-flow pressure (PSF) in Dahl SS and that is enhanced with a high salt intake. In the presence of furosemide alone, increasing nephron perfusion did not change the PSF in either Dahl salt-resistant rats (Dahl SR) or Dahl SS. When furosemide and an NHE inhibitor, dimethylamiloride, were perfused simultaneously, an increase in tubular flow caused Af-Art dilatation that was demonstrated by an increase in PSF. This increase was greater in Dahl SS [4.5 ± 0.4 (SE) mmHg] than in Dahl SR (2.5 ± 0.3 mmHg; P < 0.01). We confirmed that CTGF causes this vasodilation, since benzamil completely blocked this effect. However, a high salt intake did not augment the Af-Art dilatation. We conclude that during simultaneous inhibition of NKCC2 and NHE in the nephron, CTGF induces Af-Art dilatation and a high salt intake failed to enhance this effect.

Connecting tubule glomerular feedback mediates tubuloglomerular feedback resetting after unilateral nephrectomy.

Unilaterally nephrectomized rats (UNx) have higher glomerular capillary pressure (PGC) that can cause significant glomerular injury in the remnant kidney. PGC is controlled by the ratio of afferent (Af-Art) and efferent arteriole resistance. Af-Art resistance in turn is regulated by two intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa; and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel (ENaC). Resetting of TGF post-UNx can allow systemic pressure to be transmitted to the glomerulus and cause renal damage, but the mechanism behind this resetting is unclear. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that CTGF is increased after UNx and contributes to TGF resetting. To test this hypothesis, we performed UNx in Sprague-Dawley (8) rats. Twenty-four hours after surgery, we performed micropuncture of individual nephrons and measured stop-flow pressure (PSF). PSF is an indirect measurement of PGC. Maximal TGF response at 40 nl/min was 8.9 ± 1.24 mmHg in sham-UNx rats and 1.39 ± 1.02 mmHg in UNx rats, indicating TGF resetting after UNx. When CTGF was inhibited with the ENaC blocker benzamil (1 μM/l), the TGF response was 12.29 ± 2.01 mmHg in UNx rats and 13.03 ± 1.25 mmHg in sham-UNx rats, indicating restoration of the TGF responses in UNx. We conclude that enhanced CTGF contributes to TGF resetting after UNx.

Abstract 062: Regulation of Nephron Afferent Arteriole Resistance in Obesity: Role of Connecting Tubule Glomerular Feedback

In obesity, renal damage is caused by increase in renal blood flow (RBF), glomerular capillary pressure (P GC ), and single nephron glomerular filtration rate but the mechanism behind this alteration in renal hemodynamics is unclear. P GC is controlled mainly by the afferent arteriole (Af-Art) resistance. Af-Art resistance is regulated by mechanism similar to that in other arterioles and in addition, it is regulated by two intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to an increase in sodium chloride (NaCl) in the macula densa, via sodium–potassium-2-chloride cotransporter-2 (NKCC2) and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation and is mediated by connecting tubule via epithelial sodium channel (ENaC). CTGF is blocked by the ENaC inhibitor benzamil. Attenuation of TGF reduces Af-Art resistance and allows systemic pressure to get transmitted to the glomerulus that causes glomerular barotrauma/damage. In the current study, we tested the hypothesis that TGF is attenuated in obesity and that CTGF contributes to this effect. We used Zucker obese rats (ZOR) while Zucker lean rats (ZLR) served as controls. We performed in-vivo renal micropuncture of individual rat nephrons while measuring stop-flow pressure (P SF ), an index of P GC. TGF response was measured as a decrease in P SF induced by changing the rate of late proximal perfusion from 0 to 40nl/min in stepwise manner.CTGF was calculated as the difference of P SF value between vehicle and benzamil treatment, at each perfusion rate. Maximal TGF response was significantly less in ZOR (6.16 ± 0.52 mmHg) when compared to the ZLR (8.35 ± 1.00mmHg), p&lt;0.05 , indicating TGF resetting in the ZOR. CTGF was significantly higher in ZOR (6.33±1.95 mmHg) when compared to ZLR (1.38±0.89 mmHg), p&lt;0.05 . When CTGF was inhibited with the ENaC blocker Benzamil (1μM), maximum P SF decrease was 12.30±1.72 mmHg in ZOR and 10.60 ± 1.73 mmHg in ZLR, indicating that blockade of CTGF restored TGF response in ZOR. These observations led us to conclude that TGF is reset in ZOR and that enhanced CTGF contributes to this effect. Increase in CTGF may explain higher renal blood flow, increased P GC and higher glomerular damage in obesity.

Abstract P148: Connecting Tubule-glomerular Feedback (ctgf) in Renal Hemodynamics and Blood Pressure (bp) After Unilateral Nephrectomy (unx)

Afferent arteriole resistance is regulated in part by myogenic response, tubuloglomerular feedback (TGF) and connecting tubule-glomerular feedback (CTGF). CTGF dilates afferent arteriole in response to high sodium in connecting tubule (CNT) counteracting and shifting to the right the TGF response (resetting); CTGF increases renal blood flow and glomerular pressure, both favoring glomerular filtration and sodium excretion. CTGF is initiated by epithelial sodium channel (ENaC) in CNT and inhibited by Benzamil. Unilateral nephrectomy (UNx) is accompanied by TGF resetting, increase in renal blood flow (RBF) and single nephron GFR in the remnant kidney, without any changes in systemic BP. We evaluated the effects of CTGF in TGF resetting, RBF and BP after UNx. UNx was performed on Sprague-Dawley rats and 24h later TGF was evaluated in vivo by renal micropuncture techniques by measure stop flow pressure (Psf). CTGF was evaluated by the differences between TGF maximal responses (TGFmax) with or without tubular benzamil perfusion. Another set of animals received chronic infusion of benzamil directly into the kidney, that started 1 week before UNx. Renal blood flow (RBF) was measured by arterial spin labeling-MRI 24h before and 24h after the UNx. Direct BP measurement was performed before and 3 weeks after the UNx. After UNx, TGF resetting was observed in UNx rats (TGFmax 8±1 vs. 1±1 mmHg, Sham vs. UNx; p&lt;0.05). This TGF resetting was inhibited by benzamil. RBF increased after the UNx in comparison to sham and this increase was prevented by chronic infusion of Benzamil into the kidney (Sham: 3±0.6; UNx: 4.6±0.3; UNx+Benzamil 3.5±0.6 ml/min/g tissue p&lt;0.002). Basal mean BP values were not different between the vehicle or treated rats before the UNx; however 3 weeks after the UNx, rats receiving benzamil into the kidney showed higher mean BP values than vehicle (88±0.3 vs. 97±4 mmHg, p&lt;0.01). We conclude that CTGF participates in TGF resetting and RBF regulation after UNx, and that could participate in BP regulation after UNx.

Mechanisms of connecting tubule glomerular feedback enhancement by aldosterone.

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.

Modeling the effects of positive and negative feedback in kidney blood flow control

Blood flow in the mammalian kidney is tightly autoregulated. One of the important autoregulation mechanisms is the myogenic response, which is activated by perturbations in blood pressure along the afferent arteriole. Another is the tubuloglomerular feedback, which is a negative feedback that responds to variations in tubular fluid [Cl−] at the macula densa.11The macula densa is a cluster of specialized cells lining the wall of the cortical thick ascending limb at the transition to the distal convoluted tubule. When initiated, both the myogenic response and the tubuloglomerular feedback adjust the afferent arteriole muscle tone. A third mechanism is the connecting tubule glomerular feedback, which is a positive feedback mechanism located at the connecting tubule, downstream of the macula densa. The connecting tubule glomerular feedback is much less well studied. The goal of this study is to investigate the interactions among these feedback mechanisms and to better understand the effects of their interactions. To that end, we have developed a mathematical model of solute transport and blood flow control in the rat kidney. The model represents the myogenic response, tubuloglomerular feedback, and connecting tubule glomerular feedback. By conducting a bifurcation analysis, we studied the stability of the system under a range of physiologically-relevant parameters. The bifurcation results were confirmed by means of a comparison with numerical simulations. Additionally, we conducted numerical simulations to test the hypothesis that the interactions between the tubuloglomerular feedback and the connecting tubule glomerular feedback may give rise to a yet-to-be-explained low-frequency oscillation that has been observed in experimental records.

Abstract 118: Role of Connecting Tubule Glomerular Feedback in Tubuloglomerular Feedback Resetting After Unilateral Nephrectomy

Tubuloglomerular feedback (TGF) and connecting tubule glomerular feedback (CTGF) autoregulate nephronal afferent arteriolar resistance. In TGF, the macula densa signals the afferent arteriole to constrict when NaCl transport is enhanced by increased luminal NaCl, via sodium[[Unable to Display Character: &amp;#8211;]]potassium-2-chloride cotransporter-2 (NKCC2). CTGF is mediated by connecting tubule sodium transport via epithelial sodium channel (ENaC) and dilates the afferent arteriole. Attenuation or resetting of TGF occurs after unilateral nephrectomy (UNX), but the mechanism behind this resetting remains unclear. This TGF resetting after UNX has been implicated in progressive glomerular damage due to sustained increase in glomerular capillary pressure. Since TGF is attenuated after UNX, we sought to test the hypothesis that CTGF is enhanced and that it contributes to TGF resetting after UNX. To test this hypothesis, we performed right side UNX in Sprague Dawley (SD) rats. 24 hours after surgery, we performed micropuncture of individual rat nephrons while measuring stop-flow pressure (PSF), which is an index of glomerular capillary pressure and afferent arteriolar tone. PSF decreases with an increase in afferent arteriolar tone. TGF response was measured as a decrease in PSF induced by switching late proximal perfusion from 0,10,20,30 and 40nl/min. Maximal TGF response was 1.3 ± 1.7 mmHg in UNX rats while 8.2 ± 0.9 mmHg in sham-UNX rats indicating a TGF resetting in UNX rats. When CTGF was inhibited with the ENaC blocker Benzamil (1μM), TGF response was 10±1.2 mmHg in UNX rats and 14.8± 1.3 mmHg in sham-UNX rats, indicating the restoration of TGF responses in UNX. We conclude that enhanced CTGF contributes to the TGF resetting after 24 hours of UNX. Enhanced CTGF may be responsible for glomerular damage post UNX.

Renal autoregulation in health and disease.

Intrarenal autoregulatory mechanisms maintain renal blood flow (RBF) and glomerular filtration rate (GFR) independent of renal perfusion pressure (RPP) over a defined range (80-180 mmHg). Such autoregulation is mediated largely by the myogenic and the macula densa-tubuloglomerular feedback (MD-TGF) responses that regulate preglomerular vasomotor tone primarily of the afferent arteriole. Differences in response times allow separation of these mechanisms in the time and frequency domains. Mechanotransduction initiating the myogenic response requires a sensing mechanism activated by stretch of vascular smooth muscle cells (VSMCs) and coupled to intracellular signaling pathways eliciting plasma membrane depolarization and a rise in cytosolic free calcium concentration ([Ca(2+)]i). Proposed mechanosensors include epithelial sodium channels (ENaC), integrins, and/or transient receptor potential (TRP) channels. Increased [Ca(2+)]i occurs predominantly by Ca(2+) influx through L-type voltage-operated Ca(2+) channels (VOCC). Increased [Ca(2+)]i activates inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) to mobilize Ca(2+) from sarcoplasmic reticular stores. Myogenic vasoconstriction is sustained by increased Ca(2+) sensitivity, mediated by protein kinase C and Rho/Rho-kinase that favors a positive balance between myosin light-chain kinase and phosphatase. Increased RPP activates MD-TGF by transducing a signal of epithelial MD salt reabsorption to adjust afferent arteriolar vasoconstriction. A combination of vascular and tubular mechanisms, novel to the kidney, provides for high autoregulatory efficiency that maintains RBF and GFR, stabilizes sodium excretion, and buffers transmission of RPP to sensitive glomerular capillaries, thereby protecting against hypertensive barotrauma. A unique aspect of the myogenic response in the renal vasculature is modulation of its strength and speed by the MD-TGF and by a connecting tubule glomerular feedback (CT-GF) mechanism. Reactive oxygen species and nitric oxide are modulators of myogenic and MD-TGF mechanisms. Attenuated renal autoregulation contributes to renal damage in many, but not all, models of renal, diabetic, and hypertensive diseases. This review provides a summary of our current knowledge regarding underlying mechanisms enabling renal autoregulation in health and disease and methods used for its study.

Abstract 520: Na/H Exchanger Participates in Tubuloglomerular Feedback

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&lt;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&lt;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&lt;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.

Aldosterone sensitizes connecting tubule glomerular feedback via the aldosterone receptor GPR30

Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone sensitizes CTGF via a nongenomic mechanism that stimulates CNT ENaC via the aldosterone receptor GPR30. 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. During the control period, the concentration of NaCl 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 caused a left-shift (decrease) in EC50 to 19.3 ± 1.3 mmol/l (P = 0.001 vs. control; n = 6). Neither the transcription inhibitor actinomycin D nor the translation inhibitor cycloheximide prevented the effect of aldosterone (control EC50 = 34.7 ± 1.9 mmol/l; aldosterone+actinomycin D EC50 = 22.6 ± 1.6 mmol/l; P < 0.001 and control EC50 = 32.4 ± 4.3 mmol/l; aldosterone+cycloheximide EC50 = 17.4 ± 3.3 mmol/l; P < 0.001). The aldosterone antagonist eplerenone prevented the sensitization of CTGF by aldosterone (control EC50 = 33.2 ± 1.7 mmol/l; aldosterone+eplerenone EC50 = 33.5 ± 1.3 mmol/l; n = 7). The GPR30 receptor blocker G-36 blocked the sensitization of CTGF by aldosterone (aldosterone EC50 = 16.5 ± 1.9 mmol/l; aldosterone+G-36 EC50 = 29.0 ± 2.1 mmol/l; n = 7; P < 0.001). Finally, we found that the sensitization of CTGF by aldosterone was mediated, at least in part, by the sodium/hydrogen exchanger (NHE). We conclude that aldosterone in the CNT lumen sensitizes CTGF via a nongenomic effect involving GPR30 receptors and NHE. Sensitized CTGF induced by aldosterone may contribute to renal damage by increasing Af-Art dilation and glomerular capillary pressure (glomerular barotrauma).

Response to Prostaglandin E 2 Mediates Connecting Tubule Glomerular Feedback

We thank Elijovich and Laffer1 for their interest in our article. First, we clarify that prostaglandin E2 is not the only mediator of connecting tubule glomerular feedback (CTGF). We had previously reported that about half of the CTGF response is mediated by epoxyeicosatrienoic acids, with the other half attributable to a prostaglandin,2 which …