Ruisheng Liu

Changes of Cell Volume and Nitric Oxide Concentration in Macula Densa Cells Caused by Changes in Luminal NaCl Concentration

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.

Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa

Macula densa cells produce superoxide (O2−) 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 O2− 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 O2− production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O2− 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 O2− concentrations from 0.75±0.28 to 1.48±0.46 U/min per 105 cells in LS and HS, respectively (P<0.001). Inhibiting NOX2 blocked the HS-induced increase in O2− (0.62±0.39 versus 0.76±0.31 U/min per 105 cells in LS and HS groups, respectively). Blocking NOX4 did not affect HS-induced O2− levels. O2− levels in the control cells during LS and HS were 0.80±0.30 and 1.56±0.49 U/min per 105 cells, respectively (P<0.001); whereas O2− 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 105 cells, respectively (P<0.001). We conclude that, whereas macula densa cells express the NOX2 and NOX4 isoforms, NOX2 is primarily responsible for NaCl-induced O2− generation.

Enhanced myogenic response in the afferent arteriole of spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHRs) have normal glomerular capillary pressure even though renal perfusion pressure is higher, suggesting that preglomerular vessels exhibit abnormally high resistance. This may be due to increased superoxide (O(2)(-)) production, which contributes to the vasoconstriction in hypertension. We tested the hypothesis that the myogenic response of the afferent arteriole (Af-Art) is exaggerated in SHRs because of increased levels of reactive oxygen species (ROS). Single Af-Arts were microdissected from kidneys of SHRs and Wistar-Kyoto (WKY) rats and microperfused in vitro. When perfusion pressure in the Af-Art was increased stepwise from 60 to 140 mmHg, the luminal diameter decreased by 8.4 + or - 2.9% in WKY Af-Arts but fell by 29.3 + or - 5.6% in SHR Af-Arts. To test whether ROS production is enhanced during myogenic response in SHRs, we measured chloromethyl-dichlorodihydrofluorescein diacetate acetyl ester (CM-H(2)DCFDA) florescence before and after increasing intraluminal pressure from 60 to 140 mmHg. Pressure-induced increases in ROS were fourfold greater in SHR Af-Arts compared with WKY Af-Arts (SHR, 48.0 + or - 2.2%; and WKY, 12.2 + or - 0.3%). To test whether O(2)(-) contributes to the myogenic response in SHRs, either the membrane-permeant O(2)(-) scavenger Tempol or the nox2-based NADPH oxidase (NOX2) inhibitor gp91ds-tat were added to the Af-Art lumen and bath and the myogenic response was tested before and after treatment. Both Tempol (10(-4) M) and gp91ds-tat (10(-5) M) significantly attenuated the pressure-induced constriction in SHR Af-Arts but not in WKY Af-Arts. We conclude that 1) pressure-induced constriction is exaggerated in SHR Af-Arts, 2) NOX2-derived O(2)(-) may contribute to the enhanced myogenic response, and 3) O(2)(-) exerts little influence on the myogenic response under normotensive conditions.

Endothelin-1, Oxidative Stress, and Endogenous Angiotensin II Mechanisms of Angiotensin II Type I Receptor Autoantibody–Enhanced Renal and Blood Pressure Response During Pregnancy

Hypertension during preeclampsia is associated with increased maternal vascular sensitivity to angiotensin II (ANGII). This study was designed to determine mechanisms whereby agonistic autoantibodies to the ANGII type I receptor (AT1-AA) enhance blood pressure (mean arterial pressure [MAP]) and renal vascular sensitivity to ANGII during pregnancy. First, we examined MAP and renal artery resistance index in response to chronic administration of ANGII or AT1-AA or AT1-AA+ANGII in pregnant rats compared with control pregnant rats. To examine mechanisms of heightened sensitivity in response to AT1-AA during pregnancy, we examined the role of endogenous ANGII in AT1-AA–infused pregnant rats, and that of endothelin-1 and oxidative stress in AT1-AA+ANGII–treated rats. Chronic ANGII increased MAP from 95±2 in normal pregnant rats to 115±2 mm Hg; chronic AT1-AA increased MAP to 118±1 mm Hg in normal pregnant rats, which further increased to 123±2 mm Hg with AT1-AA+ANGII. Increasing ANGII from 10−11 to 10−8 decreased afferent arteriole diameter from 15% to 20% but sharply decreased afferent arteriole diameter to 60% in AT1-AA–pretreated vessels. Renal artery resistance index increased from 0.67 in normal pregnant rats to 0.70 with AT1-AA infusion, which was exacerbated to 0.74 in AT1-AA+ANGII–infused rats. AT1-AA–induced hypertension decreased with enalapril but was not attenuated. Both tissue endothelin-1 and reactive oxygen species increased with AT1-AA+ANGII compared with AT1-AA alone, and blockade of either of these pathways had significant effects on MAP or renal artery resistance index. These data support the hypothesis that AT1-AA, via activation of endothelin-1 and oxidative stress and interaction with endogenous ANGII, is an important mechanism whereby MAP and renal vascular responses are enhanced during preeclampsia. # Novelty and Significance {#article-title-24}Hypertension during preeclampsia is associated with increased maternal vascular sensitivity to angiotensin II (ANGII). This study was designed to determine mechanisms whereby agonistic autoantibodies to the ANGII type I receptor (AT1-AA) enhance blood pressure (mean arterial pressure [MAP]) and renal vascular sensitivity to ANGII during pregnancy. First, we examined MAP and renal artery resistance index in response to chronic administration of ANGII or AT1-AA or AT1-AA+ANGII in pregnant rats compared with control pregnant rats. To examine mechanisms of heightened sensitivity in response to AT1-AA during pregnancy, we examined the role of endogenous ANGII in AT1-AA–infused pregnant rats, and that of endothelin-1 and oxidative stress in AT1-AA+ANGII–treated rats. Chronic ANGII increased MAP from 95±2 in normal pregnant rats to 115±2 mm Hg; chronic AT1-AA increased MAP to 118±1 mm Hg in normal pregnant rats, which further increased to 123±2 mm Hg with AT1-AA+ANGII. Increasing ANGII from 10−11 to 10−8 decreased afferent arteriole diameter from 15% to 20% but sharply decreased afferent arteriole diameter to 60% in AT1-AA–pretreated vessels. Renal artery resistance index increased from 0.67 in normal pregnant rats to 0.70 with AT1-AA infusion, which was exacerbated to 0.74 in AT1-AA+ANGII–infused rats. AT1-AA–induced hypertension decreased with enalapril but was not attenuated. Both tissue endothelin-1 and reactive oxygen species increased with AT1-AA+ANGII compared with AT1-AA alone, and blockade of either of these pathways had significant effects on MAP or renal artery resistance index. These data support the hypothesis that AT1-AA, via activation of endothelin-1 and oxidative stress and interaction with endogenous ANGII, is an important mechanism whereby MAP and renal vascular responses are enhanced during preeclampsia.

Purinergic Receptor Signaling at the Basolateral Membrane of Macula Densa Cells

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 microM. 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+).

Salt-sensitive splice variant of nNOS expressed in the macula densa cells

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% (P < 0.05) 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.

NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O(2)(-)) and angiotensin II (ANG II). We have reported that NaCl-induced O(2)(-) in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O(2)(-) in MD is unknown. Thus we determined the pathways by which ANG II increased O(2)(-) in the MD by measuring O(2)(-) in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O(2)(-) levels to increase by more than twofold (P < 0.01). This increase was blocked by losartan (AT(1) receptor blocker) but not PD-123319 (AT(2) receptor antagonist). Apocynin (a NOX inhibitor) decreased O(2)(-) 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 O(2)(-) was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O(2)(-), whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O(2)(-) production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O(2)(-) production in the MD primarily via AT(1)-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.

Aldosterone Blunts Tubuloglomerular Feedback by Activating Macula Densa Mineralocorticoid 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 &mgr;m. 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 Psf when tubular perfusion rate was increased from 0 to 40 nL/min. Aldosterone (10−7 mol/L) decreased &Dgr;Psf 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.

Protective role of testosterone in ischemia-reperfusion-induced acute kidney injury

Men are at greater risk for renal injury and dysfunction after acute ischemia-reperfusion (I/R) than are women. Studies in animals suggest that the reason for the sex difference in renal injury and dysfunction after I/R is the protective effect of estrogens in females. However, a reduction in testosterone in men is thought to play an important role in mediating cardiovascular and renal disease, in general. In the present study, we tested the hypothesis that I/R of the kidney reduces serum testosterone, and that contributes to renal dysfunction and injury. Male rats that were subjected to renal ischemia of 40 min followed by reperfusion had a 90% reduction in serum testosterone by 3 h after reperfusion that remained at 24 h. Acute infusion of testosterone 3 h after reperfusion attenuated the increase in plasma creatinine and urinary kidney injury molecule-1 (KIM-1) at 24 h, prevented the reduction in outer medullary blood flow, and attenuated the increase in intrarenal TNF-α and the decrease in intrarenal VEGF at 48 h. Castration of males caused greater increases in plasma creatinine and KIM-1 at 24 h than in intact males with renal I/R, and treatment with anastrozole, an aromatase inhibitor, plus testosterone almost normalized plasma creatinine and KIM-1 in rats with renal I/R. These data show that renal I/R is associated with sustained reductions in testosterone, that testosterone repletion protects the kidney, whereas castration promotes renal dysfunction and injury, and that the testosterone-mediated protection is not conferred by conversion to estradiol.

Iodinated contrast media cause direct tubular cell damage, leading to oxidative stress, low nitric oxide, and impairment of tubuloglomerular feedback.

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 Henles 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.