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An increase of glomerular filtration rate (GFR) is a common observation in early diabetes and is considered a key risk factor for subsequent kidney injury. However, the mechanisms underlying diabetic hyperfiltration have not been fully clarified. Here, we tested the hypothesis that macula densa neuronal nitric oxide synthase (NOS1) is upregulated via sodium glucose cotransporter type 1 (SGLT1) in diabetes, which then inhibits tubuloglomerular feedback (TGF) promoting glomerular hyperfiltration. Therefore, we examined changes in cortical NOS1 expression and phosphorylation, nitric oxide production in the macula densa, TGF response, and GFR during the early stage of insulin-deficient (Akita) diabetes in wild-type and macula densa-specific NOS1 knockout mice. A set of sophisticated techniques including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of kidney tubules in vivo, and clearance kinetics of plasma fluorescent-sinistrin were employed. Complementary studies tested the role of SGLT1 in SGLT1 knockout mice and explored NOS1 expression and phosphorylation in kidney biopsies of cadaveric donors. Diabetic mice had upregulated macula densa NOS1, inhibited TGF and elevated GFR. Macula densa-selective NOS1 knockout attenuated the diabetes-induced TGF inhibition and GFR elevation. Additionally, deletion of SGLT1 prevented the upregulation of macula densa NOS1 and attenuated inhibition of TGF in diabetic mice. Furthermore, the expression and phosphorylation levels of NOS1 were increased in cadaveric kidneys of diabetics and positively correlated with blood glucose as well as estimated GFR in the donors. Thus, our findings demonstrate that the macula densa SGLT1-NOS1-TGF pathway plays a crucial role in the control of GFR in diabetes.

Hypertension is a common comorbid condition in patients with diabetes. The pathogenesis of hypertension in diabetes has not been fully clarified. Primary tubular hyperreabsorption may contribute, which may be counteracted by glomerular hyperfiltration in the early diabetic kidney. In this study, we hypothesize that in early diabetes, the macula densa neuronal nitric oxide synthase (NOS1)-derived nitric oxide (NO) production is enhanced, which blunts tubuloglomerular feedback (TGF) response, promotes glomerular hyperfiltration, and maintains normal blood pressure; conversely, insufficient NO generation by the macula densa induces hypertension by lowering glomerular filtration rate and thus inhibiting natriuresis. To test this hypothesis, we examined the changes of macula densa NOS1 expression and phosphorylation as well as NO production, TGF response, glomerular filtration rate, sodium excretion, and blood pressure in a murine model of leptin receptor-deficient (db/db) diabetes with or without macula densa-specific NOS1 deletion. We found that db/db mice presented reduced fractional renal sodium excretion and only a small increase in blood pressure, associated with upregulated expression and activity of macula densa NOS1, inhibited TGF response, and glomerular hyperfiltration. Genetic knockout of macula densa NOS1 restored the TGF response and attenuated glomerular hyperfiltration in db/db mice but also further reduced fractional renal sodium excretion and substantially increased blood pressure. In conclusion, the present study demonstrates that in the early stage of leptin receptor-deficient diabetes, the upregulation of macula densa NOS1 inhibits TGF and increases glomerular filtration rate, which counteracts renal sodium retention and limits the rise in blood pressure.

Background: Regulation of renal hemodynamics and blood pressure (BP) via tubuloglomerular feedback (TGF) may be an important adaptive mechanism during pregnancy. Because the β-splice variant of nitric oxide synthase 1 (NOS1β) in the macula densa is a primary modulator of TGF, we evaluated its role in normal pregnancy and gestational hypertension in a mouse model. We hypothesized that pregnancy upregulates NOS1β in the macula densa, thus blunting TGF, allowing glomerular filtration rate (GFR) to increase and BP to decrease. Methods: We employed sophisticated techniques, including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of renal tubules in vivo, clearance kinetics of plasma FITC-sinistrin, and radio-telemetry BP monitoring, to determine the effects of normal pregnancy or reduced uterine perfusion pressure (RUPP) on macula densa NOS1β/NO levels, TGF responsiveness, GFR, and BP in wild-type and macula densa-specific NOS1 knockout (MD-NOS1KO) mice. Results: Macula densa NOS1β was upregulated during pregnancy, resulting in blunted TGF, increased GFR, and decreased BP. These pregnancy-induced changes in TGF and GFR were largely diminished, with a significant rise in BP, in MD-NOS1KO mice. In addition, RUPP resulted in a downregulation in macula densa NOS1β, enhanced TGF, decreased GFR, and hypertension. The superimposition of RUPP into MD-NOS1KO mice only caused a modest further alteration in TGF and its associated changes in GFR and BP. Finally, in African green monkeys, renal cortical NOS1β expression increased in normotensive pregnancies but decreased in spontaneous gestational hypertensive pregnancies. Conclusions: Macula densa NOS1β plays a critical role in control of renal hemodynamics and BP during pregnancy.

Na+-glucose cotransporter (SGLT)1 mediates glucose reabsorption in late proximal tubules. SGLT1 also mediates macula densa (MD) sensing of an increase in luminal glucose, which increases nitric oxide (NO) synthase 1 (MD-NOS1)-mediated NO formation and potentially glomerular filtratrion rate (GFR). Here, the contribution of SGLT1 was tested by gene knockout (−/−) in type 1 diabetic Akita mice. A low-glucose diet was used to prevent intestinal malabsorption in Sglt1−/−mice and minimize the contribution of intestinal SGLT1. Hyperglycemia was modestly reduced in Sglt1−/−versus littermate wild-type Akita mice (480 vs. 550 mg/dl), associated with reduced diabetes-induced increases in GFR, kidney weight, glomerular size, and albuminuria. Blunted hyperfiltration was confirmed in streptozotocin-induced diabetic Sglt1−/−mice, associated with similar hyperglycemia versus wild-type mice (350 vs. 385 mg/dl). Absence of SGLT1 attenuated upregulation of MD-NOS1 protein expression in diabetic Akita mice and in response to SGLT2 inhibition in nondiabetic mice. During SGLT2 inhibition in Akita mice, Sglt1−/−mice had likewise reduced blood glucose (200 vs. 300 mg/dl), associated with lesser MD-NOS1 expression, GFR, kidney weight, glomerular size, and albuminuria. Absence of Sglt1 in Akita mice increased systolic blood pressure, associated with suppressed renal renin mRNA expression. This may reflect fluid retention due to blunted hyperfiltration. SGLT2 inhibition prevented the blood pressure increase in Sglt1−/−Akita mice, possibly due to additive glucosuric/diuretic effects. The data indicate that SGLT1 contributes to diabetic hyperfiltration and limits diabetic hypertension. Potential mechanisms include its role in glucose-driven upregulation of MD-NOS1 expression. This pathway may increase GFR to maintain volume balance when enhanced MD glucose delivery indicates upstream saturation of SGLTs and thus hyperreabsorption.

Background Glomerular hyperfiltration is common in early diabetes and is considered a risk factor for later diabetic nephropathy. We propose that sodium-glucose cotransporter 1 (SGLT1) senses increases in luminal glucose at the macula densa, enhancing generation of neuronal nitric oxide synthase 1 (NOS1)–dependent nitric oxide (NO) in the macula densa and blunting the tubuloglomerular feedback (TGF) response, thereby promoting the rise in GFR. Methods We used microperfusion, micropuncture, and renal clearance of FITC–inulin to examine the effects of tubular glucose on NO generation at the macula densa, TGF, and GFR in wild-type and macula densa–specific NOS1 knockout mice. Results Acute intravenous injection of glucose induced hyperglycemia and glucosuria with increased GFR in mice. We found that tubular glucose blunts the TGF response in vivo and in vitro and stimulates NO generation at the macula densa. We also showed that SGLT1 is expressed at the macula densa; in the presence of tubular glucose, SGLT1 inhibits TGF and NO generation, but this action is blocked when the SGLT1 inhibitor KGA-2727 is present. In addition, we demonstrated that glucose increases NOS1 expression and NOS1 phosphorylation at Ser1417 in mouse renal cortex and cultured human kidney tissue. In macula densa–specific NOS1 knockout mice, glucose had no effect on NO generation, TGF, and GFR. Conclusions We identified a novel mechanism of acute hyperglycemia–induced hyperfiltration wherein increases in luminal glucose at the macula densa upregulate the expression and activity of NOS1 via SGLT1, blunting the TGF response and promoting glomerular hyperfiltration.

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.

Supplemental Digital Content is available in the text., We investigated the significance of the primary cilia on the macula densa and thick ascending limb (TAL) in regulation of renal hemodynamics, sodium excretion, and blood pressure in this study. A tissue-specific primary cilia knock-out (KO) mouse line was generated by crossing NKCC2-Cre mice with IFT88-Δ/flox mice (NKCC2CRE; IFT88Δ/flox), in which the primary cilia were deleted from the macula densa and TAL. NO generation was measured with a fluorescent dye (4,5-diaminofluorescein diacetate) in isolated perfused juxtaglomerular apparatus. Deletion of the cilia reduced NO production by 56% and 42% in the macula densa and TAL, respectively. NO generation by the macula densa was inhibited by both a nonselective and a selective nitric oxide synthesis inhibitors, whereas TAL-produced NO was inhibited by a nonselective and not by a selective NO synthesis 1 inhibitor. The tubuloglomerular feedback response was enhanced in the KO mice both in vitro measured with isolated perfused juxtaglomerular apparatuses and in vivo measured with micropuncture. In response to an acute volume expansion, the KO mice exhibited limited glomerular filtration rate elevation and impaired sodium excretion compared with the wild-type mice. The mean arterial pressure measured with telemetry was the same for wild-type and KO mice fed a normal salt diet. After a high salt diet, the mean arterial pressure increased by 17.4±1.6 mm Hg in the KO mice. On the basis of these findings, we concluded that the primary cilia on the macula densa and TAL play an essential role in the control of sodium excretion and blood pressure.

Females are resistant to salt-sensitive hypertension than males, but the mechanisms are not completely elucidated. We recently demonstrated that a decisive role of macula densa neuronal nitric oxide synthase β (NOS1β) in the tubuloglomerular feedback (TGF) response, long-term control of glomerular filtration rate (GFR), sodium excretion and blood pressure. Thus, in this study, we tested a hypothesis that high salt diet-induced upregulation of NOS1β in the macula densa is exaggerated in females than males, which induces more inhibition in TGF response, thereby facilitating sodium excretion and contributing to the resistance to salt-sensitive hypertension. We used microperfusion, micropuncture, clearance of FITC–insulin and radio telemetry to examine the sex differences in macula densa NOS1β expression, nitric oxide (NO) generation, TGF response, kidney clearance function and mean arterial pressure (MAP) in response to a high salt diet in C57BL/6 and macula densa–specific NOS1 knockout (KO) mice. In C57BL/6 mice, males and females had comparable basal levels in macula densa NOS1β expression, NO generation, TGF response and MAP. An 2-week high salt diet resulted in more increases of NOS1β expression in female mice than male mice (3.4±0.3-fold vs. 1.7±0.1-fold), greater increases of NO generation by the macula densa in female mice than male mice (99.1±19.8 % vs. 55.6±11.6 %), and more attenuation in TGF responses in vivo (48.7±7.2 % vs. 25.5±2.9 %) as well as TGF response in vitro (58.5±5.6 % vs. 33.7±9.3 % )in female mice than male mice. Additionally, in response to a 2-week high salt diet along with low dose Ang II, the MAP increased by 13.7±4.2 mmHg in females and 35.4±5.6 mmHg in males with a sex difference of 21.7±4.7 mmHg. In the KO mice, the high salt diet had no significant effects on the macula densa NOS1β expression, NO generation, TGF response in vivo or in vitro in both females and males. Moreover, in response to the high salt diet along with low dose Ang II, the MAP increased by 41.7±4.1 mmHg in KO females and 52.6±3.8 mmHg in KO males with a sex difference of 10.9±3.1 mmHg, which was significantly reduced compared with C57BL/6 mice. In conclusion, macula densa NOS1β-mediated TGF response plays an important role in the sex difference in salt-sensitive hypertension.

It is well-known that high protein intake increases glomerular filtration rate. Evidence from several studies indicated that nitric oxide and tubuloglomerular feedback mediate the effect. However, a recent study with a neuronal nitric oxide synthase α knockout model refuted this mechanism and concluded that neither neuronal nitric oxide synthase nor tubuloglomerular feedback response is involved in the protein-induced hyperfiltration. To examine the discrepancy, this study tested a hypothesis that neuronal nitric oxide synthase β in the macula densa mediates the high protein diet-induced glomerular hyperfiltration via tubuloglomerular feedback mechanism. We examined the effects of high protein intake on nitric oxide generation at the macula densa, tubuloglomerular feedback response, and glomerular filtration rate in wild-type and macula densa–specific neuronal nitric oxide synthase knockout mice. In wild-type mice, high protein diet increased kidney weight, glomerular filtration rate and renal blood flow, while reduced renal vascular resistance. Tubuloglomerular feedback response in vivo and in vitro were blunted and nitric oxide generation in the macula densa was increased following high protein diet, associated with upregulations of neuronal nitric oxide synthase β expression and phosphorylation at Ser1417. In contrast, these high protein diet-induced changes in nitric oxide generation at the macula densa, tubuloglomerular feedback response, renal blood flow and glomerular filtration rate in wild-type mice were largely attenuated in macula densa–specific neuronal nitric oxide synthase knockout mice. In conclusion, we demonstrated that high protein diet-induced glomerular hyperfiltration is dependent on neuronal nitric oxide synthase β in the macula densa via tubuloglomerular feedback response.

We recently showed that α, β, and γ splice variants of neuronal nitric oxide synthase (NOS1) expressed in the macula densa and NOS1β accounts for most of the NO generation. We have also demonstrated that the mice with deletion of NOS1 specifically from the macula densa developed salt-sensitive hypertension. However, the global NOS1 knockout (NOS1KO) strain is neither hypertensive nor salt sensitive. This global NOS1KO strain is actually an NOS1αKO model. Consequently, we hypothesized that inhibition of NOS1β in NOS1αKO mice induces salt-sensitive hypertension. NOS1αKO and C57BL/6 wild-type (WT) mice were implanted with telemetry transmitters and divided into 7-nitroindazole (10 mg/kg/d)–treated and nontreated groups. All of the mice were fed a normal salt (0.4% NaCl) diet for 5 days, followed by a high-salt diet (4% NaCl). NO generation by the macula densa was inhibited by >90% in WT and NOS1αKO mice treated with 7-nitroindazole. Glomerular filtration rate in conscious mice was increased by ≈40% after a high-salt diet in both NOS1αKO and WT mice. In response to acute volume expansion, glomerular filtration rate, diuretic and natriuretic response were significantly blunted in the WT and knockout mice treated with 7-nitroindazole. Mean arterial pressure had no significant changes in mice fed a high-salt diet, but increased ≈15 mm Hg similarly in NOS1αKO and WT mice treated with 7-nitroindazole. We conclude that NOS1β, but not NOS1α, plays an important role in control of sodium excretion and hemodynamics in response to either an acute or a chronic salt loading.

Nitric oxide (NO) is an important negative modulator of tubuloglomerular feedback responsiveness. We recently found that macula densa expresses a-, b-, and g-splice variants of neuronal nitric oxide synthase 1 (NOS1), and NOS1b expression in the macula densa increases on a high-salt diet. This study tested whether upregulation of NOS1b expression in the macula densa affects sodium excretion and saltsensitive hypertension by decreasing tubuloglomerular feedback responsiveness. Expression levels of NOS1b mRNA and protein were 30-and five-fold higher, respectively, than those of NOS1a in the renal cortex of C57BL/6 mice. Furthermore, macula densa NO production was similar in the isolated perfused juxtaglomerular apparatus of wild-type (WT) and nitric oxide synthase 1a-knockout (NOS1aKO) mice. Compared with control mice, mice with macula densa-specific knockout of all nitric oxide synthase 1 isoforms (MD-NOS1KO) had a significantly enhanced tubuloglomerular feedback response and after acute volume expansion, significantly reduced GFR, urine flow, and sodium excretion. Mean arterial pressure increased significantly in MD-NOS1KO mice (P,0.01) but not NOS1flox/flox mice fed a high-salt diet. After infusion of angiotensin II, mean arterial pressure increased by 61.6 mmHg in MD-NOS1KO mice versus 32.0mmHg in WT mice (P,0.01) fed a high-salt diet. These results indicate that NOS1b is a primary NOS1 isoformexpressed in themacula densa and regulates the tubuloglomerular feedback response, the natriuretic response to acute volume expansion, and the development of salt-sensitive hypertension. These findings show a novel mechanism for salt sensitivity of BP and the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP.

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/cm2. 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.

Glomerular hyperfiltration is a common observation in early diabetes and considered as risk factor for diabetic nephropathy. The mechanisms underlying glomerular hyperfiltration have not been fully clarified. In the present study, we tested a novel SGLT1-NOS1 -TGF (sodium-glucose cotransporter 1 - neuronal nitric oxide synthase – tubuloglomerular feedback) pathway with a hypothesis that tubular glucose is sensed by SGLT1 of the macula densa, which enhances macula densa NOS1 activity and blunts TGF response, therefore increasing glomerular filtration rate (GFR). NO generation by the macula densa, measured by DAF-2 DA in isolated perfused juxtaglomerular apparatus (JGA), increased by 78.4±9.1% when tubular glucose (glucose) was increased from 100 mg/dl to 300 mg/dl (n=4, p-6 M) blocked this glucose-induced NO generation. Human kidney biopsy tissues were cultured in normal glucose (100 mg/dl) and high glucose (300 mg/dl) media for 30 minutes with hanging drop technique. Protein levels of P-NOS1 ser1417 increased by 63.1±9.2% and P-NOS1 ser847 decreased by 47.4±5.9% (n=4, psf was from 7.8±1.3 to 7.3±2.1 mmHg) and GFR (from 223±15.7 to 240±6.9 μl/min) (n=5) were blocked in the KO mice. We conclude that increase of tubular glucose concentration activates NOS1 by phosphorylation of NOS1 ser1417 via SGLT1 in the macula densa, which increases GFR by inhibiting TGF response.

Aim Tubuloglomerular feedback (TGF) is an important mechanism in control of signal nephron glomerular filtration rate. The oxidative stress in the macula densa, primarily determined by the interactions between nitric oxide (NO) and superoxide (O2−), is essential in maintaining the TGF responsiveness. However, few studies examining the interactions between and amount of NO and O2− generated by the macula densa during normal and hypertensive states. Methods In this study, we used isolated perfused juxtaglomerular apparatus to directly measure the amount and also studied the interactions between NO and O2− in macula densa in both physiological and slow pressor Angiotensin II (Ang II)-induced hypertensive mice. Results We found that slow pressor Ang II at a dose of 600 ng kg−1 min−1 for two weeks increased mean arterial pressure by 26.1 ± 5.7 mmHg. TGF response increased from 3.4 ± 0.2 μm in control to 5.2 ± 0.2 μm in hypertensive mice. We first measured O2− generation by the macula densa and found it was undetectable in control mice. However, O2− generation by the macula densa increased to 21.4 ± 2.5 unit min−1 in Ang II-induced hypertensive mice. We then measured NO generation and found that NO generation by the macula densa was 138.5 ± 9.3 unit min−1 in control mice. The NO was undetectable in the macula densa in hypertensive mice infused with Ang II. Conclusions Under physiological conditions, TGF response is mainly controlled by the NO generated in the macula densa; in Ang II induced hypertension, the TGF response is mainly controlled by the O2− generated by the macula densa.

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.

Hyperfiltration is common in early diabetes and considered a risk factor for diabetic nephropathy. Inhibited tubuloglomerular feedback (TGF) mediated by less NaCl delivery at the macula densa contributes to the diabetic hyperfiltration. Nitric oxide (NO) released from macula densa via neuronal nitric oxide synthase (NOS1) inhibits TGF response. We recently demonstrated the significance of TGF response in the long-term control of GFR, sodium excretion and blood pressure, mediated by macula densa NOS1β. However, whether the NOS1β-mediated TGF response play an important role in the diabetic hyperfiltration is unknown. We hypothesized that macula densa NOS1β is upregulated in diabetes, which blunts TGF response and contributes to the diabetic hyperfiltration. The mice with deletion of NOS1 specifically from the macula densa (MD-NOS1KO) and wild-type C57BL/6 (WT) mice were used. Diabetes was induced by alloxan (55 mg/kg i.v.) with blood glucose from 350 to 450 mg/dl. Expression of NOS1 splice variants was measured with real-time PCR and Western blot. GFR was measured by plasma FITC-inulin clearance following a single bolus intravenous injection in conscious mice. GFR increased by 24.7% in diabetic WT mice (from 241.60±19.73 to 301.35±21.76 μl/min) and only by 16.5% in diabetic MD-NOS1KO mice (from 236.61±16.12 to 275.61±11.73 μl/min) (n=6/group, p

Two major factors which regulate tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole are release of superoxide (O2−) and nitric oxide (NO) by macula densa (MD) cells. MD O2− inactivates NO; however, among the factors that increase MD O2− release, the role of aldosterone is unclear. We hypothesize that aldosterone activates the mineralocorticoid receptor (MR) on MD cells, resulting in increased O2− production due to upregulation of cyclooxygenase-1 (COX-2) and NOX-2, and NOX-4, isoforms of NAD(P)H oxidase. Studies were performed on MMDD1 cells, a renal epithelial cell line with properties of MD cells. RT-PCR and Western blotting confirmed the expression of MR. Aldosterone (10−8 mol/l for 30 min) doubled MMDD1 cell O2− production, and this was completely blocked by MR inhibition with 10−5 mol/l eplerenone. RT-PCR, real-time PCR, and Western blotting demonstrated aldosterone-induced increases in COX-2, NOX-2, and NOX-4 expression. Inhibition of COX-2 (NS398), NADPH oxidase (apocynin), or a combination blocked aldosterone-induced O2− production to the same degree. These data suggest that aldosterone-stimulated MD O2− production is mediated by COX-2 and NADPH oxidase. Next, COX-2 small-interfering RNA (siRNA) specifically decreased COX-2 mRNA without affecting NOX-2 or NOX-4 mRNAs. In the presence of the COX-2 siRNA, the aldosterone-induced increases in COX-2, NOX-2, and NOX-4 mRNAs and O2− production were completely blocked, suggesting that COX-2 causes increased expression of NOX-2 and NOX-4. In conclusion 1) MD cells express MR; 2) aldosterone increases O2− production by activating MR; and 3) aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O2−. The resulting balance between O2− and NO in the MD is important in modulating TGF.

Neuronal nitric oxide synthase (nNOS), which is abundantly expressed in the macula densa cells, attenuates tubuloglomerular feedback (TGF). We hypothesize that splice variants of nNOS are expressed in the macula densa, and nNOS-beta is a salt-sensitive isoform that modulates TGF. Sprague-Dawley rats received a low-, normal-, or high-salt diet for 10 days and levels of the nNOS-alpha, nNOS-beta, and nNOS-gamma were measured in the macula densa cells isolated with laser capture microdissection. Three splice variants of nNOS, alpha-, beta-, and gamma-mRNAs, were detected in the macula densa cells. After 10 days of high-salt intake, nNOS-alpha decreased markedly, whereas nNOS-beta increased two- to threefold in the macula densa measured with real-time PCR and in the renal cortex measured with Western blot. NO production in the macula densa was measured in the perfused thick ascending limb with an intact macula densa plaque with a fluorescent dye DAF-FM. When the tubular perfusate was switched from 10 to 80 mM NaCl, a maneuver to induce TGF, NO production by the macula densa was increased by 38 +/- 3% in normal-salt rats and 52 +/- 6% (P0.05) in the high-salt group. We found 1) macula densa cells express nNOS-alpha, nNOS-beta, and nNOS-gamma, 2) a high-salt diet enhances nNOS-beta, and 3) TGF-induced NO generation from macula densa is enhanced in high-salt diet possibly from nNOS-beta. In conclusion, we found that the splice variants of nNOS expressed in macula densa cells were alpha-, beta-, and gamma-isoforms and propose that enhanced level of nNOS-beta during high-salt intake may contribute to macula densa NO production and help attenuate TGF.

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O2−) and angiotensin II (ANG II). We have reported that NaCl-induced O2−in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O2−in MD is unknown. Thus we determined the pathways by which ANG II increased O2−in the MD by measuring O2−in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O2−levels to increase by more than twofold ( P < 0.01). This increase was blocked by losartan (AT1receptor blocker) but not PD-123319 (AT2receptor antagonist). Apocynin (a NOX inhibitor) decreased O2−by 86% ( P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O2−was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O2−, whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O2−production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O2−production in the MD primarily via AT1-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

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 μm ( P = 0.003, control vs. SnMP, n = 7). When a CO-releasing molecule, (CORM-3; 50 μM), was added to the MD perfusate, the TGF response decreased by 41%, from 3.6 ± 0.3 to 2.1 ± 0.2 μm ( P < 0.001, control vs. CORM-3, n = 12). When CORM-3 at 100 μM was added to the perfusate, it completely blocked the TGF response, from 4.2 ± 0.4 to −0.2 ± 0.3 μm ( 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 μm ( 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.

The renal afferent arterioles (Af-Arts) account for most of the renal vascular resistance, which is controlled similar to other arterioles and by tubuloglomerular feedback (TGF). The latter signal is generated by sensing sodium chloride concentrations in the macula densa; this in turn results in a signal which acts through the extraglomerular mesangium leading to constriction of the Af-Art. In the outer renal cortex, the connecting tubule (CNT) returns to the glomerular hilus and contacts the Af-Art suggesting that crosstalk may exist here as well. To investigate this, we simultaneously perfused a microdissected Af-Art and adherent CNT. Increasing the sodium chloride concentration perfusing the CNT significantly dilated preconstricted Af-Arts. We called this crosstalk 'connecting tubule glomerular feedback' (CTGF) to differentiate it from TGF. We tested whether entry of Na þ and/or CIinto the CNT is required to induce CTGF by replacing Na þ with choline þ . Increasing choline chloride concentration did not dilate the Af-Art. To test whether epithelial Na channels (ENaCs) mediate CTGF, we blocked ENaC with amiloride and found that the dilatation induced by CTGF was completely blocked. Inhibiting sodium chloride cotransporters with hydrochlorothiazide failed to prevent Af-Art dilatation. Finally, we tested whether nitric oxide released by the CNT mediates CTGF by the addition of a non-selective nitric oxide synthase inhibitor to the CNT. This potentiated CTGF rather than blocking it. We suggest that crosstalk exists between CNTs and attached Af-Arts, which is initiated by sodium reabsorption through amiloride-sensitive channels and this can contribute to the regulation of renal blood flow and glomerular filtration. The renal afferent arterioles (Af-Arts) account for most renal vascular resistance; they control glomerular filtration rate (GFR) and peritubular pressure, and thus renal function. In addition, Af-Art and efferent arteriole resistance control intraglomerular pressure, which is important not only for filtration pressure but also the development of glomerulo- sclerosis in hypertension and diabetes. Af-Art resistance is regulated by factors similar to other arterioles, and in addition is controlled in part by tubuloglomerular feedback (TGF). TGF operates via the macula densa: when concentra- tions of sodium and chloride in the macula densa are increased, a signal is transmitted through the extraglomerular mesangium that constricts the Af-Art. 1 In humans and other mammals, there is a transitional region of the nephron between the distal convoluted tubule and the cortical collecting duct, called the connecting tubule (CNT). This segment of the nephron plays a significant role in the

Superoxide (O2−) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O2−production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O2−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 O2−production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O2−production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O2−, we used tempol (10−4M), 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 O2−, we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O2−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, O2−production was blocked. To test whether depolarization of the macula densa induces O2−production, we artificially induced depolarization by adding valinomycin (10−6M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O2−production. We conclude that increasing luminal NaCl induces O2−production during tubuloglomerular feedback. O2−generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.

Adenosine triphosphate (ATP) is liberated from macula densa cells in response to increased tubular NaCl delivery. However, it is not known whether ATP from the macula densa is broken down to adenosine, or whether this adenosine mediates efferent arteriole (Ef-Art) tubuloglomerular feedback (TGF). We hypothesized that increased macula densa Ca(2+), release of ATP and degradation of ATP to adenosine are necessary for Ef-Art TGF. Rabbit Ef-Arts and adherent tubular segments (with the macula densa) were simultaneously microperfused in vitro while changing the NaCl concentration at the macula densa. The Ef-Art was perfused orthograde through the end of the afferent arteriole (Af-Art). In Ef-Arts preconstricted with norepinephrine (NE), increasing NaCl concentration from 10 to 80 mM at the macula densa dilated Ef-Arts from 7.5+/-0.7 to 11.1+/-0.3 microm. Buffering increases in macula densa Ca(2+) with the cell-permeant Ca(2+) chelator BAPTA-AM diminished Ef-Art TGF from 3.1+/-0.3 to 0.1+/-0.2 microm. Blocking adenosine formation by adding alpha-beta-methyleneadenosine 5'-diphosphate (MADP) blocked Ef-Art TGF from 2.9+/-0.5 to 0.1+/-0.2 microm. Increasing luminal NaCl at the macula densa from 10 to 45 mM caused a moderate Ef-Art TGF response, 1.3+/-0.1 microm. It was potentiated to 4.0+/-0.3 microm by adding hexokinase, which enhances conversion of ATP into adenosine. Our data show that in vitro changes in macula densa Ca(2+) and ATP release are necessary for Ef-Art TGF. ATP is broken down to form adenosine, which mediates signal transmission of Ef-Art TGF.

Renal function measured by glomerular filtration rate (GFR) declines with aging. But the mechanism has not been clarified. Nitric oxide synthase 1 β (NOS1β) is highly expressed in the macula densa (MD) and modulates tubuloglomerular feedback (TGF), which is an important mechanism for GFR regulation. We hypothesized that expression of NOS1β in MD decreases with aging, which enhances TGF response and contributes to the decline of GFR. We further hypothesized that NOS1β expression in superficial (SF) MD is lower than that of juxtamedullary (JM) MD, which contribute to more severe renal injury in renal cortex with aging. First, GFR was measured by plasma FITC-inulin clearance after a single bolus injection in conscious 12-week young mice and 20-month-old C57/BL6 mice. GFR was reduced by 36.4% in aged mice (236±9.7 vs 150±7.6 μl/min). Then we compared the glomerular injury in 20-month-old C57/BL6 mice with 12-week mice. The aged mice have significant severed injury compared to the young mice as indicated by injury scores (3.6) and expanded fractional mesangial area (FMA) per glomerulus (26.4%). In addition, injuries were more evident in SF glomeruli with larger FMA (29.3%) than that in JM glomeruli (16.9%). Next we measured NOS1β protein abundance and found that NOS1β protein in aged mice was reduced by 73% compared to young mice. In addition, we compared the NOS1β abundance between SF and JM glomeruli in aged and young mice. NOS1β protein level decreased by more than 90% in SF nephrons in aged mice compared with the young mice. In contrast, there was no significant changes in NOS1β expression in JM nephron between aged and young mice. To determine whether NOS1β mediates the changes in GFR and renal injury, we repeated the experiments in age matched MD specific NOS1β knock out (MD-NOS1KO) mice. In MD-NOS1KO mice, GFR in aged mice was reduced by 29.5% (203±15.7 vs 143±13.2 μl/min) compared young mice. Aged MD-NOS1KO mice had more severer renal injury compared with age-match wild type mice with higher injury score (4.7 vs 3.6), increased FMA (40.9% vs 26.4%) and occurrence of glomerulosclerosis (GS 30.8% ), but diminished difference between JM glomerulus (FMA37.4%, GS34.1%) and SF glomerulus (FMA42.2%, GS29.7%).

Cell volume and cytosolic Ca2+ concentration ([Ca2+]i) were measured in rabbit macula densa (MD) cells loaded with calcein and Fura Red using confocal microscopy. [Ca2+]i was also analysed with Indo-1 and fura-2. We used isolated microperfused thick ascending limbs with attached glomerulus. The results showed that when the luminal NaCl concentration (‘NaCl’) was decreased from 35 to 10 mm, the cell volume decreased by 10.4%, and [Ca2+]i increased by 9.5%. This increase was inhibited in Ca2+-free solution. When luminal [NaCl] was changed from 35 to 135 mm, the cell volume increased by 15.1%, and [Ca2+]i did not change. The cell volume alterations were not different in Ca2+-free solutions. Using Indo-1, basal [Ca2+]i in MD cells was 107.8 nm. When luminal [NaCl] was changed from 135 to 10 mm, [Ca2]i increased by 23.5 nm. Using fura-2, the basal [Ca2+]i in MD cells was 115.3 nm, and when luminal [NaCl] was changed from 135 or 35 to 10 mm, [Ca2+]i change was 30.1 or 10.6 nm, respectively. An increase in [NaCl] caused no change in [Ca2+]i. In Ca2+-free solution, no change in [Ca2+]i occurred. A stepwise decrease in luminal [NaCl] resulted in a sigmoid increase in [Ca2+]i in MD cells. The steepest part of the curve was between 70 and 10 mm. In conclusion, we found that MD cells have cell volume regulation, and that [Ca2+]i elevation caused by decreased luminal [NaCl] is independent of the cell volume.

Superoxide enhances tubuloglomerular feedback by constricting the afferent arteriole.BackgroundSuperoxide (O2−) 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 O2− potentiates TGF by acting directly on the afferent arteriole (Af-Art).MethodsMicrodissected 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.ResultsTo determine whether O2− 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 μm (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 O2− 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 O2− 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 μm (P < 0.05 vs. 7-NI). To see if this effect was due to O2− 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 μm (P < 0.01). When catalase was added to the bath together with Tempol, TGF response was not modified.ConclusionWe concluded that it is O2− rather than H2O2 that enhances TGF response, both directly by constricting the Af-Art and indirectly by scavenging NO in the MD.

Increased intracellular Ca++ in the macula densa regulates tubuloglomerular feedback.BackgroundTubuloglomerular 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.MethodsRabbit afferent arterioles and attached macula densas were simultaneously microperfused in vitro. Tubuloglomerular feedback was induced by increasing macula densa Na+/Cl- from 11/10mmol/L (low) to 81/80mmol/L (high) and was measured before and after treatment.ResultsTo 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 μm to 15.3 ± 0.8 μm. Adding 2 × 10-6 mol/L A23187 to the low NaCl macula densa perfusate induced tubuloglomerular feedback; diameter decreased from 18.0 ± 1.0 μm to 15.4 ± 0.9 μm (N = 6; P < 0.01). After adding BAPTA-AM (25 μmol/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 μm to 15.3 ± 1.2 μm (N = 6; P < 0.01). After adding the Na+/Ca++ exchanger inhibitor 2′4′-dichlorobenzamil (10 μmol/L) or KB-R7943 (30 μmol/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 μmol/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 μmol/L), nor the T-type Ca++ channel blocker pimozide (10 μmol/L), inhibited tubuloglomerular feedback when added to the macula densa lumen.ConclusionWe 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.

The tubuloglomerular feedback (TGF) mechanism is an important regulator of the glomerular filtration rate (GFR) and urine excretion rate. It operates by sensing the distal delivery of fluid at the macula densa site and adjusting the tone of the glomerular arterioles to control GFR. We found evidence that nitric oxide is an important modulator of the setting of the sensitivity of the TGF mechanism. Studies on adenosine A1 receptor deficient mice have shown that these animals lack the TGF response and have an increased renin release. These findings show the important role of adenosine as a mediator of the signal for the TGF mechanism and as an inhibitor of renin release.

Purinergic receptors are important in the regulation of renal hemodynamics; therefore, this study sought to determine if such receptors influence macula densa cell function. Isolated glomeruli containing macula densa cells, with and without the cortical thick ascending limb, were loaded with the Ca 2+ sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flow-through perfusion system. Changes in cytosolic calcium concentration ([Ca 2+ ] i ) from exposed macula densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2- MeS-ATP) for 2 min added to the bathing solution. There was no change in [Ca 2+ ] i with addition of adenosine (10 −7 to 10 −3 M). UTP and ATP (10 −4 M) caused [Ca 2+ ] i to increase by 268 ± 40 nM ( n = 21) and 295 ± 53 nM ( n = 21), respectively, whereas in response to 2MesATP and ADP, [Ca 2+ ] i increased by only 67 ± 13 nM ( n = 8) and 93 ± 36 nM ( n = 14), respectively. Dose response curve for ATP (10 −7 to 10 −3 M) added in bath showed an EC 50 of 15 μM. No effect on macula densa [Ca 2+ ] i was seen when ATP was added from the lumen. ATP caused similar increases in macula densa [Ca 2+ ] i in the presence or absence of bath Ca 2+ and addition of 5 mM ethyleneglycotetraacetic acid (EGTA). Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca 2+ ] i dynamics. Also, ATP-Ca 2+ responsiveness was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. These results suggest that macula densa cells possess P2Y 2 purinergic receptors on basolateral but not apical membranes and that activation of these receptors results in the mobilization of Ca 2+ .

NAD(P)H oxidase (Nox)2 and Nox4 are the isoforms of Nox expressed in the macula densa (MD). MD-derived superoxide (O2−), primarily generated by Nox2, is enhanced by acute ANG II stimulation. However, the effects of chronic elevations in ANG II during ANG II-induced hypertension on MD-derived O2− are unknown. We infused a slow pressor dose of ANG II (600 ng·min−1·kg−1) for 2 wk in C57BL/6 mice and found that mean arterial pressure was elevated by 22.3 ± 3.4 mmHg ( P < 0.01). We measured O2− generation in isolated and perfused MDs and found that O2− generation by the MD was increased from 9.4 ± 0.9 U/min in control mice to 34.7 ± 1.8 U/min in ANG II-induced hypertensive mice ( P < 0.01). We stimulated MMDD1 cells, a MD-like cell line, with ANG II and found that O2− generation increased from 921 ± 91 to 3,687 ± 183 U·min−1·105 cells−1, which was inhibited with apocynin, oxypurinol, or NS-398 by 46%, 14%, and 12%, respectively. We isolated MD cells using laser capture microdissection and measured mRNA levels of Nox. Nox2 and Nox4 levels increased by 3.7 ± 0.17- and 2.6 ± 0.15-fold in ANG II-infused mice compared with control mice. In MMDD1 cells treated with Nox2 or Nox4 small interfering (si)RNAs, ANG II-stimulated O2− generation was blunted by 50% and 41%, respectively. In cells treated with p22 phox siRNA, ANG II-stimulated O2− generation was completely blocked. In conclusion, we found that a subpressor dose of ANG II enhances O2− generation in the MD and that the sources of this O2− are primarily Nox2 and Nox4.

The juxtaglomerular apparatus (JGA) has the very important functions of detecting the fluid flow rate to the distal tubule and thus controlling the glomerular filtration rate (GFR) (tubuloglomerular feedback mechanism [TGF]) and renin release from the afferent arteriole. In studies of the TGF it has been evident that the sensitivity of this mechanism can be reset. Volume expansion will reset it to a low sensitivity leading to a high GFR and urine excretion rate, while dehydration will sensitize the TGF mechanism, giving rise to a low GFR and low urine excretion rate. Furthermore, we have found that in animals that spontaneously develop hypertension there is initially a sensitization of the TGF, leading to a reduced GFR and urine excretion rate, with fluid volume retention in the body and a consequent rise in blood pressure. When the pressure is raised, the TGF characteristics are normalized. In the macula densa (MD) cells in the JGA, there is a large production of NO from neuronal NOS. This production continuously reduces TGF sensitivity and is apparently impaired in animals that spontaneously develop hypertension. When we added an nNOS inhibitor to the drinking water for several weeks while measuring blood pressure, we found an increase in blood pressure after 3-4 weeks of treatment This effect was abolished by a high salt diet From these investigations, it also appeared as if nNOS-derived NO inhibited renin release. Experiments have also indicated that NO may resensitize inhibited G-protein coupled purinergic receptors.

Chronic aldosterone administration increases glomerular filtration rate, whereas inhibition of mineralocorticoid receptors (MRs) markedly attenuates glomerular hyperfiltration and hypertension associated with primary aldosteronism or obesity. However, the mechanisms by which aldosterone alters glomerular filtration rate regulation are poorly understood. In the present study, we hypothesized that aldosterone suppresses tubuloglomerular feedback (TGF) via activation of macula densa MR. First, we observed the expression of MR in macula densa cells isolated by laser capture microdissection and by immunofluorescence in rat kidneys. Second, to investigate the effects of aldosterone on TGF in vitro, we microdissected the juxtaglomerular apparatus from rabbit kidneys and perfused the afferent arteriole and distal tubule simultaneously. Under control conditions, TGF was 2.8±0.2 μm. In the presence of aldosterone (10 −8 mol/L), TGF was reduced by 50%. The effect of aldosterone to attenuate TGF was blocked by the MR antagonist eplerenone (10 −5 mol/L). Third, to investigate the effect of aldosterone on TGF in vivo, we performed micropuncture, and TGF was determined by maximal changes in stop-flow pressure P sf when tubular perfusion rate was increased from 0 to 40 nL/min. Aldosterone (10 −7 mol/L) decreased ΔP sf from 10.1±1.4 to 7.7±1.2 mm Hg. In the presence of l -NG-monomethyl arginine citrate (10 −3 mol/L), this effect was blocked. We conclude that MRs are expressed in macula densa cells and can be activated by aldosterone, which increases nitric oxide production in the macula densa and blunts the TGF response.

Superoxide (O2− ) enhances tubuloglomerular feedback (TGF) by scavenging nitric oxide at the macula densa (MD). The primary source of O2− in the MD during TGF is NADPH oxidase, which is activated by membrane depolarization. While Rac, a small GTP-binding protein, has been shown to enhance NADPH oxidase activity, its role in O2− generation by the MD is unknown. We hypothesized that depolarization of the MD leads to translocation of Rac to the apical membrane, and its activation, in turn, augments O2− generation during TGF. We tested this by measuring membrane potential and increased O2− levels during TGF responses in isolated, perfused tubules containing the intact MD plaque. Switching tubular NaCl from 10 to 80 mM, which induces TGF, depolarized membrane potential by 28.4 ± 4.5% from control ( P < 0.05) and O2− levels from 124 ± 19 to 361 ± 27 U/min. This NaCl-induced depolarization and O2− generation were blocked by a Cl− channel blocker, 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB; 10−6 M). Inhibition of Rac blunted NaCl-induced O2− generation by 47%. When the NaCl content of the MD perfusate was increased from 10 to 80 mM, immunointensity of Rac on the apical side increased from 32 ± 3.1 to 46 ± 2.5% of the total immunofluorescence in the MD, indicating that high NaCl induces the translocation of Rac to the apical membrane. This NaCl-induced Rac translocation was blocked by a Cl− channel blocker, NPPB, indicating that depolarization of the MD induced Rac translocation. In conclusion, we found that depolarization of the MD during TGF leads to translocation of Rac to the apical membrane, which enhances O2− generation by the MD.

Tubuloglomerular feedback (TGF) is one of the main mechanisms that regulate the renal microcirculation, NaCl excretion, and ultimately blood pressure. Superoxide (O-2) in the macula densa (MD) enha...

Macula densa cells produce superoxide (O 2 − ) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa and the role of each one in NaCl-induced O 2 − production. To identify which isoforms are expressed, we applied single-cell RT-PCR to macula densa cells isolated by laser capture microdissection and to MMDD1 cells (a macula densa-like cell line). The captured cells expressed neuronal NOS (marker of macula densa), NOX2, and NOX4 but not NOX1. Expression of the NOXs and neuronal NOS was essentially identical in the MMDD1 cells. Thus, we used MMDD1 cells to investigate which isoform is responsible for NaCl-induced O 2 − production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O 2 − exposed to low-salt solution (LS; 70 mmol/L of NaCl) or high-salt solution (HS; 140 mmol/L of NaCl). Exposing control cells (scrambled small-interfering RNA) to HS increased O 2 − concentrations from 0.75±0.28 to 1.48±0.46 U/min per 10 5 cells in LS and HS, respectively ( P 2 − (0.62±0.39 versus 0.76±0.31 U/min per 10 5 cells in LS and HS groups, respectively). Blocking NOX4 did not affect HS-induced O 2 − levels. O 2 − levels in the control cells during LS and HS were 0.80±0.30 and 1.56±0.49 U/min per 10 5 cells, respectively ( P 2 − levels in NOX4-small-interfering RNA–treated cells during LS and HS were 0.40±0.25 and 1.26±0.51 U/min per 10 5 cells, respectively ( P 2 − generation.

We hypothesized that elevated macula densa intracellular pH (pHi) during tubuloglomerular feedback enhances O2−production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, O2−production increased from 0.53 ± 0.09 to 2.62 ± 0.54 U/min ( P < 0.01). To determine whether inhibiting the Na/H exchanger blocks O2−production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, O2−production induced by NaCl was blunted by 40%. To study the effect of pHion O2−in intact macula densa cells, we measured O2−while pHiwas changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, O2−production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of O2−, we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin (10−5M), O2−production induced by elevating pHiwas blocked. Finally, we measured the optimum pH for O2−production by the macula densa and found optimum extracellular pH is at 7.7 and optimum pHiis ∼8 for O2−production. We found that elevated pHienhances O2−production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and O2−production is pH sensitive, with an optimum pHiof 8.

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 (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 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 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.

The juxtaglomerular apparatus in the kidney is important in controlling extracellular fluid volume and renin release. The fluid load to the distal tubule is first sensed at the macula densa site via the entry of NaCl, through a Na, K, 2Cl co-transport mechanism. The next step is unclear, but there is recent evidence of an increased macula densa cell calcium concentration with a reduction in fluid load to the macula densa. An increase in macula densa cell calcium could activate phospholipase A2 to release arachidonic acid, the rate-limiting step in the formation of prostaglandins. Recent evidence suggests that the prostaglandin formed is PGE2, a potent stimulator for renin release. Recent evidence has also shown that adenosine has an important function in the juxtaglomerular apparatus. It stimulates calcium release in afferent arteriolar smooth muscle cells, leading to contraction of the afferent arteriole as part of the tubuloglomerular feedback mechanism, and inhibits renin release. Thus, renin release from the afferent arteriole is mediated partly through formation of PGE2, and partly through the reduction of adenosine formation that inhibits renin production.

A fluorescent nitric oxide (NO) indicator, 4,5-diaminofluorescein diacetate, and the calcium indicator, indo-1, with 488 nm and 364 nm UV confocal laser scanning microscopy were used to detect NO and calcium concentration in rabbit macula densa (MD) cells challenged by angiotensin II (Ang II). Glomeruli with attached thick ascending limbs with the MD plaque were isolated and perfused. Ang II concentration from 10 −9 to 10 −5 progressively increased MD cell calcium and NO to peak values at 10 −6 and 10 −7 , respectively. Ang II (10 −6 M) caused the cytosolic calcium concentration ([Ca 2+ ] i ) to increase by 125.8±16.3 nM (n=17) from the bath and by 52.3±11.5 nM (n=18) from the lumen. AT 1 antagonist CV-11974 (10 −6 M) blocked the Ang II-induced calcium responses from bath and lumen, but AT 2 antagonist PD-123319 (10 −6 M) did not. AT 2 agonist CGP-42112A (10 −6 M) did not affect [Ca 2+ ] i in MD cells from either side. Ang II (10 −6 M) increased the NO production by 16%±3.4% (n=26) from the bath and by 18%±3.1% (n=24) from the lumen. CV-11974 (10 −6 M) blocked the NO responses from both sides, but PD-123319 (10 −6 M) did not on either side. CGP-42112A (10 −6 M) had no effect on NO in MD cells. In calcium-free experiments there was no difference from the result in normal calcium solutions. In conclusion, we found that Ang II increased [Ca 2+ ] i and stimulated NO production in MD cells from the basolateral and luminal sides through AT 1 receptors.

The luminal NaCl concentration ([NaCl]) at the macula densa (MD) controls both tubuloglomerular feedback (TGF) and renin release. Nitric oxide (NO) inhibits TGF sensitivity to a great extent. The NO concentration in the MD cells is not known. This study measured this concentration in MD cells with confocal microscopy in the isolated perfused thick ascending limb using a NO-sensitive fluorophore 4,5-diaminofluorescein (DAF-2). Calcein was used to measure cell volume changes. The loop perfusion fluid was a modified Ringer solution containing 10, 35, or 135 mM NaCl with a constant total osmolarity (290 mOsm), and the bath was perfused with the 135 mM NaCl solution. The results show that MD cell volume and NO concentration measured with DAF-2 DA increased considerably with increasing luminal [NaCl] and with calcium-free solutions in the lumen and bath. l-arginine (5 mM) increased NO concentration in the MD cells by 30%. 7-nitroindazole could totally inhibit the NO production caused by l-arginine and by increased luminal [NaCl]. In conclusion, the MD cell volume changes caused by the changes of luminal [NaCl] were quantitatively measured, and it was found that increasing the luminal [NaCl] resulted in an increase in cell volume. It was also found that NO formation in MD cells could be measured with DAF-2 and that NO production was increased through neuronal NO synthase activation with an increased luminal [NaCl]. An increased NO production will inhibit the vasoconstriction induced by the TGF and at the same time will reduce TGF sensitivity.