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Afferent arteriole (Af-Art) diameter regulates pressure and flow into the glomerulus, which are the main determinants of the glomerular filtration rate. Thus, Af-Art resistance is crucial for Na + filtration. Af-Arts play a role as integrative centers, where systemic and local systems interact to determine the final degree of resistance. The tubule of a single nephron contacts an Af-Art of the same nephron at two locations: in the transition of the thick ascending limb to the distal tubule (macula densa) and again in the connecting tubule. These two sites are the anatomic basis of two intrinsic feedback mechanisms: tubule-glomerular feedback and connecting tubule-glomerular feedback. The cross communications between the tubules and Af-Arts integrate tubular Na + and water processing with the hemodynamic conditions of the kidneys. Tubule-glomerular feedback provides negative feedback that tends to avoid salt loss, and connecting tubule-glomerular feedback provides positive feedback that favors salt excretion by modulating tubule-glomerular feedback (resetting it) and increasing glomerular filtration rate. These feedback mechanisms are also exposed to systemic modulators (hormones and the nervous system); however, they can work in isolated kidneys or nephrons. The exaggerated activation or absence of any of these mechanisms may lead to disequilibrium in salt and water homeostasis, especially in extreme conditions (e.g., high-salt diet/low-salt diet) and may be part of the pathogenesis of some diseases. In this review, we focus on molecular signaling, feedback interactions, and the physiological roles of these two feedback mechanisms.

Purpose of Review: 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., Recent Findings: 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., Key Points: • 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.

Zucker obese rats (ZOR) have higher glomerular capillary pressure (P GC ) that can cause renal damage. P GC 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 P GC in ZOR. We performed a renal micropuncture experiment and measured stop-flow pressure (P SF ), which is an indirect measurement of P GC 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 P SF 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.

Unilaterally nephrectomized rats (UNx) have higher glomerular capillary pressure (P GC ) that can cause significant glomerular injury in the remnant kidney. P GC 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 (P SF ). P SF is an indirect measurement of P GC . 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.

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 (P SF ) 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 P SF 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 P SF. 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., (Copyright © 2017 the American Physiological Society.)

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., (Copyright © 2015 the American Physiological Society.)

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)., (Copyright © 2014 the American Physiological Society.)

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.

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 μm (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 μm; 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.

Increasing Na delivery to the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation, a process we call CNT glomerular feedback (CTGF). Angiotensin II (ANG II) in the CNT lumen enhances CTGF via PKC. We hypothesized that luminal ANG II stimulates CTGF via activation of protein kinase C (PKC), NADPH oxidase 2 (NOX2), and enhanced production of superoxide (O(2)(-)). Rabbit Af-Arts and adherent CNTs were microdissected and microperfused in vitro. Dilation of the Af-Art was induced by increasing luminal CNT NaCl from 0 to 5, 10, 30, 45, and 80 mM, and the concentration of NaCl that elicited a half-maximal response (EC(50)) was calculated. Compared with vehicle, adding ANG II (10(-9) M) to the CNT lumen reduced EC(50) from 37 ± 3 to 14 ± 1 mM (P < 0.001), indicating ANG II potentiates CTGF. In the presence of ANG II, the O(2)(-) scavenger tempol (10(-4) M) increased EC(50) from 20 ± 4 to 41 ± 3 mM (P < 0.01), the NOX inhibitor apocynin (10(-5) M) increased EC(50) from 17 ± 2 to 39 ± 4 mM (P < 0.01), and the specific NOX2 inhibitor gp91ds-tat (10(-5) M) increased EC(50) from 19 ± 2 to 34 ± 2 mM (P < 0.01). However, tempol, apocynin, and gp91ds-tat had no effect on CTGF in the absence of ANG II. Compared with vehicle, the PKC activator PMA (2 × 10(-7) M) decreased EC(50) from 35 ± 2 to 14 ± 1 (P < 0.001). In the presence of PMA, tempol increased EC(50) from 14 ± 2 to 35 ± 2 mM (P < 0.01). We conclude the PKC/NOX2/O(2)(-) pathway mediates the enhancement of CTGF by luminal ANG II but it does not participate in CTGF in the absence of ANG II.

Tubuloglomerular feedback (TGF) is a renal autoregulatory mechanism that constricts the afferent arteriole in response to increases in distal NaCl. Heme oxygenases (HO-1 and HO-2) release carbon monoxide (CO) and biliverdin, which may help control renal function. We showed in vitro that HO products inhibit TGF; however, we do not know whether this also occurs in vivo or the mechanism(s) involved. We hypothesized that in vivo HO-1 and HO-2 in the nephron inhibit TGF via release of CO and biliverdin. We first performed laser capture microdissection followed by real-time PCR and found that both HO-1 and HO-2 are expressed in the macula densa. We next performed micropuncture experiments in vivo on individual rat nephrons, adding different compounds to the perfusate, and found that an HO inhibitor, stannous mesoporphyrin (SnMP), potentiated TGF (P < 0.05, SnMP vs. control). The CO-releasing molecule (CORM)-3 partially inhibited TGF at 50 μmol/l (P < 0.01, CORM-3 vs. control) and blocked it completely at higher doses. A soluble guanylyl cyclase (sGC) inhibitor, LY83583, blocked the inhibitory effect of CORM-3 on TGF. Biliverdin also partially inhibited TGF (P < 0.01, biliverdin vs. control), most likely attributable to decreased superoxide (O(2)(-)) because biliverdin was rendered ineffective by tempol, a O(2)(-) dismutase mimetic. We concluded that HO-1 and HO-2 in the nephron inhibit TGF by releasing CO and biliverdin. The inhibitory effect of CO on TGF is mediated by the sGC/cGMP signaling pathway, whereas biliverdin probably acts by reducing O(2)(-).

In hypertension the pressure natriuresis set point is shifted to a higher pressure due to an increase in both renal vascular resistance and sodium (Na) reabsorption. The afferent arterioles (Af-Arts) and efferent arterioles (Ef-Arts) account for most renal vascular resistance; they control glomerular filtration rate (GFR) and peritubular pressure, and, consequently, renal function. Af-Art and Ef-Art resistance is regulated by factors similar to those in other arterioles and also by tubuloglomerular feedback (TGF). TGF operates via the macula densa, which senses increases in sodium chloride (NaCl) and sends a signal that constricts the Af-Art and dilates the Ef-Art. In the outer renal cortex, the connecting tubule (CNT) returns to the glomerular hilus and contacts the Af-Art. This morphology is compatible with cross-talk between the CNT and Af-Art, so-called connecting tubule glomerular feedback (CTGF). Our studies show that increasing NaCl delivery to the CNT results in Af-Art dilatation that can be blocked by inhibitors of Na transport. We believe cross-talk between the CNT and Af-Art is a novel mechanism that may contribute to regulation of renal blood flow and GFR.

Tubuloglomerular feedback (TGF) is the mechanism by which the macula densa (MD) senses increases in luminal NaCl concentration and sends a signal to constrict the afferent arteriole (Af-Art). The kidney expresses constitutively heme oxygenase-2 (HO-2) and low levels of HO-1. HOs release carbon monoxide (CO), biliverdin, and free iron. We hypothesized that renal HOs inhibit TGF via release of CO and biliverdin. Rabbit Af-Arts and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the MD perfusate. When HO activity was inhibited by adding stannous mesoporphyrin (SnMP) to the MD perfusate, the TGF response increased from 2.1+/-0.2 to 4.1+/-0.4 microm (P=0.003, control vs. SnMP, n=7). When a CO-releasing molecule, (CORM-3; 50 microM), was added to the MD perfusate, the TGF response decreased by 41%, from 3.6+/-0.3 to 2.1+/-0.2 microm (P<0.001, control vs. CORM-3, n=12). When CORM-3 at 100 microM was added to the perfusate, it completely blocked the TGF response, from 4.2+/-0.4 to -0.2+/-0.3 microm (P<0.001, control vs. CORM-3, n=6). When biliverdin was added to the perfusate, the TGF response decreased by 79%, from 3.4+/-0.3 to 0.7+/-0.4 microm (P=0.001, control vs. biliverdin, n=6). The effects of SnMP and CORM-3 were not blocked by inhibition of nitric oxide synthase. We concluded that renal HO inhibits TGF probably via release of CO and biliverdin. HO regulation of TGF is a novel mechanism that could lead to a better understanding of the control of renal microcirculation and function.

Superoxide (O(2)(-)) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O(2)(-) production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O(2)(-) production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O(2)(-) production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O(2)(-) production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O(2)(-), we used tempol (10(-4) M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O(2)(-), we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O(2)(-) production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O(2)(-) production was blocked. To test whether depolarization of the macula densa induces O(2)(-) production, we artificially induced depolarization by adding valinomycin (10(-6) M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O(2)(-) production. We conclude that increasing luminal NaCl induces O(2)(-) production during tubuloglomerular feedback. O(2)(-) generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.

Background: The macula densa senses increasing NaCl concentrations in tubular fluid and increases afferent arteriole tone by a process known as tubuloglomerular feedback (TGF). Nitric oxide (NO) production by macula densa neuronal nitric oxide synthase (nNOS) is enhanced by increasing NaCl in the macula densa lumen, and the NO thus formed inhibits TGF. Blocking apical Na(+)/H(+) exchange with amiloride augments TGF and mimics the effect of nNOS inhibition. We hypothesized that increasing NaCl in the macula densa lumen raises macula densa intracellular pH (pH(i)) and activates nNOS., Methods: The thick ascending limb and a portion of the distal tubule with intact macula densa plaque adherent to the glomerulus were microdissected and perfused. Macula densa perfusate was changed from a low (10 mmol/L) to high NaCl solution (80 mmol/L) to mimic the conditions that induce TGF. Osmolality of both solutions was 180 mOsm, so that changing the solutions did not alter cell volume., Results: Macula densa pH(i) increased significantly from 7.0 +/- 0.5 to 7.8 +/- 0.6 when the perfusate was changed from low to high (P < 0.05; N= 5). When amiloride was added to inhibit Na(+)/H(+) exchange, the increase in pH(i) during TGF was blocked (N= 5). Fluorescence intensity of DAF-2, an NO-sensitive dye, increased by 28.8 +/- 4.1% after increasing luminal NaCl (N= 5), indicating an increase in NO production. In the presence of the Na(+)/H(+) exchanger inhibitor amiloride or the nNOS inhibitor 7-NI, the increase in NO induced by switching the macula densa perfusate from low to high was blunted. To study whether changes in pH(i) can directly alter NO production, we used nigericin, a K(+)/H(+) ionophore, to equilibrate luminal and intracellular pH. When macula densa pH was raised from 7.3 to 7.8 in the presence of 10(-5) mol/L nigericin in the low NaCl solution, fluorescence of DAF-2 in the macula densa increased by 17.9 +/- 1.3% (P < 0.01; N= 5). In the presence of 7-NI, the increase in NO induced by raising pH(i) was blocked (N= 5)., Conclusion: We concluded that macula densa pH(i) increases during TGF, and this increase in pH(i) activates nNos.

Background: Superoxide (O(2) (-)) has been shown to augment tubuloglomerular feedback (TGF) both in vivo and in vitro by scavenging nitric oxide (NO) in the macula densa (MD). We hypothesized that in addition to this mechanism O(2) (-) potentiates TGF by acting directly on the afferent arteriole (Af-Art)., Methods: Microdissected Af-Arts and adherent tubular segments containing the MD were simultaneously microperfused in vitro, maintaining Af-Art pressure at 60 mm Hg. TGF response was determined by measuring changes in Af-Art diameter while increasing NaCl in the MD perfusate from 11/10 to 81/80 mmol/L Na/Cl., Results: To determine whether O(2) (-) acts at the MD in the absence of MD NO, we inhibited MD nNOS with 7-nitroindazole (7-NI) and added Tempol to the lumen. When 7-NI was added to the MD lumen, it increased TGF from 2.3 +/- 0.2 to 4.2 +/- 0.2 microm (P < 0.01). When Tempol was added to the MD lumen in the presence of 7-NI, it had no effect on TGF. To investigate whether O(2) (-) has any effect via the Af-Art in the absence of MD NO, we inhibited MD nNOS with 7-NI and added Tempol to the bath to scavenge O(2) (-) in the Af-Art. Adding Tempol to the bath with 7-NI in the MD lumen reduced TGF from 3.9 +/- 0.3 to 2.8 +/- 0.5 microm (P < 0.05 vs. 7-NI). To see if this effect was due to O(2) (-) scavenging NO production by the endothelium, we repeated the experiment in Af-Arts with damaged endothelium and found that adding Tempol to the bath lowered TGF from 3.4 +/- 0.9 to 1.2 +/- 0.6 microm (P < 0.01). When catalase was added to the bath together with Tempol, TGF response was not modified., Conclusion: We concluded that it is O(2) (-) rather than H(2)O(2) that enhances TGF response, both directly by constricting the Af-Art and indirectly by scavenging NO in the MD.

Background: Tubuloglomerular feedback is initiated by an increase in NaCl at the macula densa lumen, which in turn increases intracellular Ca++. In the present study, we examined the role of increased intracellular Ca++ in tubuloglomerular feedback and the source of the increased Ca++. We hypothesized that an increase in intracellular Ca++ at the macula densa via the basolateral Na+/Ca++ exchanger, caused by an increase in luminal NaCl, initiates Ca++-mediated Ca++ release from intracellular stores, which is essential for tubuloglomerular feedback., Methods: Rabbit afferent arterioles and attached macula densas were simultaneously microperfused in vitro. Tubuloglomerular feedback was induced by increasing macula densa Na+/Cl- from 11/10 mmol/L (low) to 81/80 mmol/L (high) and was measured before and after treatment., Results: To investigate whether elevations in intracellular Ca++ are required for tubuloglomerular feedback, the calcium ionophore A23187 or the Ca++ chelator BAPTA-AM was added to the macula densa lumen. During the control period, tubuloglomerular feedback decreased afferent arteriole diameter from 18.1 +/- 1.1 microm to 15.3 +/- 0.8 microm. Adding 2 x 10-6 mol/L A23187 to the low NaCl macula densa perfusate induced tubuloglomerular feedback; diameter decreased from 18.0 +/- 1.0 microm to 15.4 +/- 0.9 microm (N = 6; P < 0.01). After adding BAPTA-AM (25 micromol/L) to the macula densa lumen, tubuloglomerular feedback response was completely eliminated. We next studied the source of increased macula densa Ca++ in response to increased NaCl concentration. During the control period, tubuloglomerular feedback decreased afferent arteriole diameter from 18.5 +/- 1.6 microm to 15.3 +/- 1.2 microm (N = 6; P < 0.01). After adding the Na+/Ca++ exchanger inhibitor 2'4'-dichlorobenzamil (10 micromol/L) or KB-R7943 (30 micromol/L) to the bath, the tubuloglomerular feedback response was blocked; however, the afferent arteriole response to angiotensin II or adenosine was not altered. Next, we tested the Ca++-adenosine triphosphatase (ATPase) inhibitor thapsigargin (0.1 micromol/L), which has been reported to inhibit sarcoplasmic reticulum Ca++-ATPase activity and prevent restoration of intracellular Ca++ stores. When thapsigargin was added to the macula densa lumen, it reduced the first tubuloglomerular feedback response by 33% and completely eliminated the second and third tubuloglomerular feedback responses. In the absence of thapsigargin, there was no significant decrease in the tubuloglomerular feedback responses (N = 6). Neither the L-type Ca++ channel blocker nifedipine (25 micromol/L), nor the T-type Ca++ channel blocker pimozide (10 micromol/L), inhibited tubuloglomerular feedback when added to the macula densa lumen., Conclusion: We concluded that (1). increased intracellular Ca++ at the macula densa is required for the tubuloglomerular feedback response; (2). Na+/Ca++ exchange appears to initiate Ca++-mediated Ca++ release from intracellular stores; and (3). luminal L-type or T-type Ca++ channels are not involved in tubuloglomerular feedback.

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 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tubuloglomerular feedback (TGF) is a renal autoregulatory mechanism that constricts the afferent arteriole in response to increases in distal NaCl. Heme oxygenases (HO-1 and HO-2) release carbon monoxide (CO) and biliverdin, which may help control renal function. We showed in vitro that HO products inhibit TGF; however, we do not know whether this also occurs in vivo or the mechanism(s) involved. We hypothesized that in vivo HO-1 and HO-2 in the nephron inhibit TGF via release of CO and biliverdin. We first performed laser capture microdissection followed by real-time PCR and found that both HO-1 and HO-2 are expressed in the macula densa. We next performed micropuncture experiments in vivo on individual rat nephrons, adding different compounds to the perfusate, and found that an HO inhibitor, stannous mesoporphyrin (SnMP), potentiated TGF (P < 0.05, SnMP vs. control). The CO-releasing molecule (CORM)-3 partially inhibited TGF at 50 μmol/l (P < 0.01, CORM-3 vs. control) and blocked it completely at higher doses. A soluble guanylyl cyclase (sGC) inhibitor, LY83583, blocked the inhibitory effect of CORM-3 on TGF. Biliverdin also partially inhibited TGF (P < 0.01, biliverdin vs. control), most likely attributable to decreased superoxide (O2-) because biliverdin was rendered ineffective by tempol, a O2- dismutase mimetic. We concluded that HO-1 and HO-2 in the nephron inhibit TGF by releasing CO and biliverdin. The inhibitory effect of CO on TGF is mediated by the sGC/cGMP signaling pathway, whereas biliverdin probably acts by reducing O2-. [ABSTRACT FROM AUTHOR]

Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG II is taken up by proximal tubule cells (PTCs) through AT1 receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase-dependent mechanisms, and 3) AT1 receptor-mediated endocytosis of ANG II has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [125I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the AT1 receptor blocker losartan (16.5 ± 4.6%, P < 0.01 vs. ANG II, 78.3 ± 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 ± 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by ∼58% (basal, 229.8 ± 11.4 vs. ANG II, 361.3 ± 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan (P < 0.01), the cytoskeleton microtubule inhibitor colchicine (P < 0.05), and PAO (P < 0.01), whereas depletion of clathrin-coated pits with hyper-osmotic sucrose had no effect (356.1 ± 25.5 pg ANG II/mg protein, not significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 ± 2.8 vs. ANG II, 9.1 ± 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 ± 8.6 vs. forskolin + ANG II, 42.8 ± 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through AT1 receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling. [ABSTRACT FROM AUTHOR]

Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([Ca2+]i) and, if so, elucidated the cellular mechanisms involved. Changes in [Ca2+]i responses were studied by fluorescence imaging using the Ca2+ indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type I (AT1) receptors were blocked by losartan (10 μM). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [Ca2+]i that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [Ca2+]i that peaked at 30 s (basal: 2.2 ± 0.3 vs. ANG II: 14.9 ± 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular Ca2+ with EGTA (2 mM) did not alter microinjected ANG II-induced [Ca2+]i responses (Ca2+ free + ANG II:12.3 ± 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular Ca2+ stores or with U-73122 to inhibit phospholipase C (1 μM each) markedly attenuated microinjected ANG II-induced [Ca2+]i responses. Combined microinjection of ANG II and losartan abolished [Ca2+]i responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [Ca2+]i by stimulating intracellular AT1 receptors and releases Ca2+ from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function. [ABSTRACT FROM AUTHOR]

Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback. Background. The macula densa senses increasing NaCl concentrations in tubular fluid and increases afferent arteriole tone by a process known as tubuloglomerular feedback (TGF). Nitric oxide (NO) production by macula densa neuronal nitric oxide synthase (nNOS) is enhanced by increasing NaCl in the macula densa lumen, and the NO thus formed inhibits TGF. Blocking apical Na+/H+ exchange with amiloride augments TGF and mimics the effect of nNOS inhibition. We hypothesized that increasing NaCl in the macula densa lumen raises macula densa intracellular pH (pHi) and activates nNOS. Methods. The thick ascending limb and a portion of the distal tubule with intact macula densa plaque adherent to the glomerulus were microdissected and perfused. Macula densa perfusate was changed from a low (10 mmol/L) to high NaCl solution (80 mmol/L) to mimic the conditions that induce TGF. Osmolality of both solutions was 180 mOsm, so that changing the solutions did not alter cell volume. Results. Macula densa pHi increased significantly from 7.0± 0.5 to 7.8± 0.6 when the perfusate was changed from low to high ( P< 0.05; N= 5). When amiloride was added to inhibit Na+/H+ exchange, the increase in pHi during TGF was blocked ( N= 5). Fluorescence intensity of DAF-2, an NO-sensitive dye, increased by 28.8± 4.1% after increasing luminal NaCl ( N= 5), indicating an increase in NO production. In the presence of the Na+/H+ exchanger inhibitor amiloride or the nNOS inhibitor 7-NI, the increase in NO induced by switching the macula densa perfusate from low to high was blunted. To study whether changes in pHi can directly alter NO production, we used nigericin, a K+/H+ ionophore, to equilibrate luminal and intracellular pH. When macula densa pH was raised from 7.3 to 7.8 in the presence of 10−5 mol/L nigericin in the low NaCl solution, fluorescence of DAF-2 in the macula densa increased by 17.9± 1.3% ( P< 0.01; N= 5). In the presence of 7-NI, the increase in NO induced by raising pHi was blocked ( N= 5). Conclusion. We concluded that macula densa pHi increases during TGF, and this increase in pHi activates nNos. [ABSTRACT FROM AUTHOR]

Role of macula densa adenosine triphosphate (ATP) in tubuloglomerular feedback.Background.Recent studies have shown that adenosine triphosphate (ATP) is liberated from macula densa cells in response to increased tubular NaCl in vitro. We tested the hypothesis that increased NaCl in the macula densa stimulates the release of ATP, resulting in extracellular formation of adenosine which is involved in signal transmission of the tubuloglomerular feedback response.Methods.Rabbit afferent arterioles and attached macula densas were simultaneously microperfused in vitro.Tubuloglomerular feedback was induced by increasing macula densa Na/Cl from 11/10 to 81/80 mmol/L and was measured before and after treatment.Results.We first tested whether hydrolysis of ATP is required for tubuloglomerular feedback. When we enhanced conversion of ATP to adenosine by adding hexokinase or apyrase to the bath and arteriole lumen, the tubuloglomerular feedback response was augmented. During the control period, tubuloglomerular feedback decreased arteriole diameter by 2.2± 0.2μm. In the presence of hexokinase, tubuloglomerular feedback decreased diameter by 3.4± 0.3μm (N= 8) (P<0.05, with vs. without hexokinase). In the apyrase group, tubuloglomerular feedback decreased diameter by 2.7± 0.4μm during the control period. When apyrase was added, tubuloglomerular feedback decreased diameter by 4.7± 0.4μm (N= 8) (P<0.05, with vs. without apyrase). When hydrolysis of adenosine monophosphate (AMP) to adenosine was blocked by supplementing the bath with 100μmol/Lα,β-methylene adenosine 5′-diphosphate (MADP), an inhibitor of 5′-nucleotidase, tubuloglomerular feedback response was blocked and diameter remained unchanged. We next studied whether ATP released from the macula densa binds to P2 receptors and activates the tubuloglomerular feedback response. The P2 purinergic receptor inhibitor suramin was added to both arteriole lumen and bath. During the control period, tubuloglomerular feedback decreased diameter by 3.7± 0.5μm. Suramin (100μmol/L) did not significantly inhibit tubuloglomerular feedback, since in the presence of suramin diameter decreased by 3.8± 0.3μm (N= 7). Finally, we added the adenosine A1 receptor inhibitor FK838 to both bath and lumen and found that it completely blocked high NaCl-induced tubuloglomerular feedback.Conclusion.We concluded that ATP released from the macula densa is broken down to form AMP in the extracellular space. AMP in turn is degraded by ecto-5′-nucleotidases to adenosine, which mediates signal transmission of the tubuloglomerular feedback response. [ABSTRACT FROM AUTHOR]

Background: There is evidence that macula densa nitric oxide (NO) inhibits tubuloglomerular feedback (TGF). However, TGF response is not altered in mice deficient in neuronal nitric oxide synthase (nNOS) (-/-). Furthermore, nNOS expression in the macula densa is inversely related to salt intake, yet micropuncture studies have shown that NOS inhibition potentiates TGF in rats on high sodium intake but not in rats on a low-salt diet. These inconsistencies may be due to confounding systemic factors, such as changes in circulating renin. To further clarify the role of macula densa nNOS in TGF response, independent of systemic factors, we tested the hypothesis that (1) TGF response is inversely related to sodium intake, and (2) during low sodium intake, NO produced by macula densa nNOS tonically controls the basal diameter of the afferent arteriole (Af-Art).Methods: Af-Arts and attached macula densas were simultaneously microperfused in vitro. TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the macula densa perfusate. TGF response was studied in wild-type (+/+) and nNOS knockout mice (-/-), as well as in juxtaglomerular apparatuses (JGAs) from rabbits fed a low-, normal-, or high-NaCl diet.Results: TGF responses were similar in nNOS +/+ and -/- mice. However, in nNOS +/+ mice, 7-nitroindazole (7-NI) perfused into the macula densa significantly potentiated the TGF response (P = 0.001), while in nNOS -/- mice, this potentiation was absent. In rabbits on three different sodium diets, TGF responses were similar and were potentiated equally by 7-NI. However, in JGAs from rabbits on a low-NaCl diet, adding 7-NI to the macula densa while perfusing it with low-NaCl fluid caused Af-Art vasoconstriction, decreasing the diameter by 14% (from 21.7 +/- 1.3 to 18.6 +/- 1.5 microm; P < 0.001). This effect was not observed in JGAs from rabbits fed a normal- (19.0 +/- 0.5 vs. 19.3 +/- 0.8 microm after 7-NI) or high-NaCl diet (18.6 +/- 0.7 vs. 18.4 +/- 0.7 microm).Conclusions: First, in this in vitro preparation, chronic changes in macula densa nNOS do not play a major role in the regulation of TGF. Compensatory mechanisms may develop during chronic alteration of nNOS that keep TGF relatively constant. Second, nNOS regulates TGF response acutely. Third, the results obtained in the +/+ and -/- mice also confirm that the effect of 7-NI is due to inhibition of macula densa nNOS. Finally, during low sodium intake (without induction of TGF), the regulation of basal Af-Art resistance by macula densa nNOS suggests that NO in the macula densa helps maintain renal blood flow during the high renin secretion caused by low sodium intake. [ABSTRACT FROM AUTHOR]