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Females are protected against the development of angiotensin II (ANG II)-induced hypertension compared with males, but the mechanisms have not been completely elucidated. In the present study, we hypothesized that the effect of ANG II on the macula densa nitric oxide (NO) synthase 1β (NOS1β)-mediated tubuloglomerular feedback (TGF) mechanism is different between males and females, thereby contributing to the sexual dimorphism of ANG II-induced hypertension. We used microperfusion, micropuncture, clearance of FITC-inulin, and radio telemetry to examine the sex differences in the changes of macula densa NOS1β expression and activity, TGF response, natriuresis, and blood pressure (BP) after a 2-wk ANG II infusion in wild-type and macula densa-specific NOS1 knockout mice. In wild-type mice, ANG II induced higher expression of macula densa NOS1β, greater NO generation by the macula densa, and a lower TGF response in vitro and in vivo in females than in males; the increases of glomerular filtration rate, urine flow rate, and Na + excretion in response to an acute volume expansion were significantly greater and the BP responses to ANG II were significantly less in females than in males. In contrast, these sex differences in the effects of ANG II on TGF, natriuretic response, and BP were largely diminished in knockout mice. In addition, tissue culture of human kidney biopsies (renal cortex) with ANG II resulted in a greater increase in NOS1β expression in females than in males. In conclusion, macula densa NOS1β-mediated TGF is a novel and important mechanism for the sex differences in ANG II-induced hypertension.

Females are relatively resistant to salt-sensitive hypertension than males, but the mechanisms are not completely elucidated. We recently demonstrated a decisive role of macula densa neuronal NOS1β (nitric oxide synthase β)-mediated tubuloglomerular feedback (TGF) in the long-term control of glomerular filtration rate, sodium excretion, and blood pressure. In the present study, we hypothesized that the macula densa NOS1β-mediated TGF mechanism is different between male and female, thereby contributing to the sexual dimorphism of salt-sensitive hypertension. We used microperfusion, micropuncture, clearance of fluorescein isothiocyanate-inulin, and radio telemetry to examine the sex differences in the changes of macula densa NOS1β expression and activity, TGF response, natriuresis, and blood pressure after salt loading in wild-type and macula densa-specific NOS1 knockout mice. In wild-type mice, a high-salt diet induced greater increases in macula densa NOS1β expression and phosphorylation at Ser 1417, greater nitric oxide generation by the macula densa, and more inhibition in TGF response in vitro and in vivo in females than in males. Additionally, the increases of glomerular filtration rate, urine flow rate, and sodium excretion in response to an acute volume expansion were significantly greater in females than in males. The blood pressure responses to angiotensin II plus a high-salt diet were significantly less in females than in males. In contrast, these sex differences in TGF, natriuretic response, and blood pressure were largely diminished in knockout mice. In conclusion, macula densa NOS1β-mediated TGF is a novel and important mechanism for the sex differences in salt-sensitive hypertension.

Acute kidney injury (AKI) induced by clamping of renal vein or pedicle is more severe than clamping of artery, but the mechanism has not been clarified. In the present study, we tested our hypothesis that increased proximal tubular pressure (P t ) during the ischemic phase exacerbates kidney injury and promotes the development of AKI. We induced AKI by bilateral clamping of renal arteries, pedicles, or veins for 18 min at 37°C, respectively. P t during the ischemic phase was measured with micropuncture. We found that higher P t was associated with more severe AKI. To determine the role of P t during the ischemic phase on the development of AKI, we adjusted the P t by altering renal artery pressure. We induced AKI by bilateral clamping of renal veins, and the P t was changed by adjusting the renal artery pressure during the ischemic phase by constriction of aorta and mesenteric artery. When we decreased renal artery pressure from 85 ± 5 to 65 ± 8 mmHg, P t decreased from 53.3 ± 2.7 to 44.7 ± 2.0 mmHg. Plasma creatinine decreased from 2.48 ± 0.23 to 1.91 ± 0.21 mg/dl at 24 h after renal ischemia. When we raised renal artery pressure to 103 ± 7 mmHg, P t increased to 67.2 ± 5.1 mmHg. Plasma creatinine elevated to 3.17 ± 0.14 mg·dl·24 h after renal ischemia. Changes in KIM-1, NGAL, and histology were in the similar pattern as plasma creatinine. In summary, we found that higher P t during the ischemic phase promoted the development of AKI, while lower P t protected from kidney injury. P t may be a potential target for treatment of AKI., (Copyright © 2017 the American Physiological Society.)

The present study tested whether primary cilia on macula densa serve as a flow sensor to enhance nitric oxide synthase 1 (NOS1) activity and inhibit tubuloglomerular feedback (TGF). Isolated perfused macula densa was loaded with calcein red and 4,5-diaminofluorescein diacetate to monitor cell volume and nitric oxide (NO) generation. An increase in tubular flow rate from 0 to 40 nl/min enhanced NO production by 40.0 ± 1.2%. The flow-induced NO generation was blocked by an inhibitor of NOS1 but not by inhibition of the Na/K/2Cl cotransporter or the removal of electrolytes from the perfusate. NO generation increased from 174.8 ± 21 to 276.1 ± 24 units/min in cultured MMDD1 cells when shear stress was increased from 0.5 to 5.0 dynes/cm(2). The shear stress-induced NO generation was abolished in MMDD1 cells in which the cilia were disrupted using a siRNA to ift88. Increasing the NaCl concentration of the tubular perfusate from 10 to 80 mM NaCl in the isolated perfused juxtaglomerular preparation reduced the diameter of the afferent arteriole by 3.8 ± 0.1 μm. This response was significantly blunted to 2.5 ± 0.2 μm when dextran was added to the perfusate to increase the viscosity and shear stress. Inhibition of NOS1 blocked the effect of dextran on TGF response. In vitro, the effects of raising perfusate viscosity with dextran on tubular hydraulic pressure were minimized by reducing the outflow resistance to avoid stretching of tubular cells. These results suggest that shear stress stimulates primary cilia on the macula densa to enhance NO generation and inhibit TGF responsiveness., (Copyright © 2015 the American Physiological Society.)

Iodinated contrast media (CM) have adverse effects that may result in contrast-induced acute kidney injury. Oxidative stress is believed to play a role in CM-induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CM-induced direct cell damage and that increased local oxidative stress may increase tubuloglomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubuloglomerular feedback responsiveness were measured in isolated, perfused juxtaglomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats that received CM. CM increased superoxide concentration and reduced nitric oxide bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CM-perfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle's loop. SOD activity did not differ between CM and control groups. The tubuloglomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulovascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration.

Males have higher prevalence of hypertension and renal injury than females, which may be attributed in part to androgen-mediated effects on renal hemodynamics. Tubuloglomerular feedback (TGF) is an important mechanism in control of renal microcirculation. The present study examines the role of testosterone in the regulation of TGF responses. TGF was measured by micropuncture (change of stop-flow pressure, ΔPsf) in castrated Sprague-Dawley rats. The addition of testosterone (10(-7) mol/l) into the lumen increased the ΔPsf from 10.1 ± 1.2 to 12.2 ± 1.2 mmHg. To determine whether androgen receptors (AR) are involved, mRNA of AR was measured in the macula dense cells isolated by laser capture microdissection from kidneys, and a macula densa-like cell line (MMDD1). AR mRNA was expressed in the macula densa of rats and in MMDD1 cells. We next examined the effects of the AR blocker, flutamide (10(-5) mol/l) on the TGF response. The addition of flutamide blocked the effects of testosterone on TGF. The addition of Tempol (10(-4) mol/l) or polyethylene glycol-superoxide dismutase (100 U/ml) to scavenge superoxide blocked the effect of testosterone to augment TGF. We then applied apocynin to inhibit NAD(P)H oxidase and oxypurinol to inhibit xanthine oxidase and found the testosterone-induced augmentation of TGF was blocked. In additional experiments in MMDD1 cells, we found that testosterone increased O2(-) generation. Apocynin or oxypurinol blocked the testosterone-induced increases of O2(-), while blockade of COX-2 with NS-398 had no effect. These findings suggest that testosterone enhances TGF response by stimulating O2(-) production in macula densa via an AR-dependent pathway.

Previous studies have indicated that 20-hydroxyeicosatetraeonic acid (20-HETE) modulates vascular tone in large cerebral and renal arteries through inhibition of the large conductance, calcium sensitive potassium (BK) channel activity. However, the role of 20-HETE in modulating tubuloglomerular feedback (TGF) and the myogenic response in the afferent arteriole (Af-Art) is unknown. The present study examined the effects of inhibitors of the synthesis and action of 20-HETE on the myogenic and TGF responses of isolated rabbit and mouse Af-Arts. Luminal diameter decreased by 9.2±0.5% in mice and 8.9±1.3% in rabbit Af-Art when the perfusion pressure was increased from 60 to 120 mmHg. Administration of a 20-HETE synthesis inhibitor, HET0016 (1 μM), or a selective 20-HETE antagonist, 6, 15-20-hydroxyeicosadienoic acid (6, 15-20-HEDE, 10 μM) completely blocked the myogenic response of both rabbit and mouse Af-Art, while addition of 5, 14-20-HEDE (10 μM), a 20-HETE agonist, restored the myogenic response in vessels treated with HET0016. Increases in NaCl concentration from 10 to 80 mM of the solution perfusing the macula densa constricted the Af-Art of rabbits by 6.0±1.4 μm (n=5). Addition of a 20-HETE agonist to the tubular perfusate potentiated the TGF-mediated vasoconstrictor response. This response was blocked by addition of a 20-HETE antagonist (6, 15-20-HEDE, 10 μM) to the vascular perfusate. These studies indicate that locally produced 20-HETE plays an important role in modulating the myogenic and TGF responsiveness of the Af-Art and may help explain how deficiencies in the renal formation of 20-HETE could promote the development of hypertension induced glomerular injury., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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]