The macula densa prorenin receptor is essential in renin release and blood pressure control.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

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 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.

Role of the SNARE protein SNAP23 on cAMP-stimulated renin release in mouse juxtaglomerular cells.

Renin, the rate-limiting enzyme in the formation of angiotensin II, is synthesized and stored in granules in juxtaglomerular (JG) cells. Therefore, the controlled mechanism involved in renin release is essential for the regulation of blood pressure. Exocytosis of renin-containing granules is likely involved in renin release; a process stimulated by cAMP. We found that the "soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor" (SNARE) protein VAMP2 mediates cAMP-stimulated renin release and exocytosis in JG cells. To mediate exocytosis, VAMP2 must interact with a synaptosome-associated protein (SNAP). In the renal cortex, the isoform SNAP23 is abundantly expressed. We hypothesized that SNAP23 mediates cAMP-stimulated renin release from primary cultures of mouse JG cells. We found that SNAP23 protein is expressed and colocalized with renin-containing granules in primary cultures of mouse JG cell lysates. Thus, we then tested the involvement of SNAP23 in cAMP-stimulated renin release by transducing JG cells with a dominant-negative SNAP23 construct. In control JG cells transduced with a scrambled sequence, increasing cAMP stimulated renin release from 1.3 ± 0.3 to 5.3 ± 1.2% of renin content. In cells transduced with dominant-negative SNAP23, cAMP increased renin from 1.0 ± 0.1 to 3.0 ± 0.6% of renin content, a 50% blockade. Botulinum toxin E, which cleaves and inactivates SNAP23, reduced cAMP-stimulated renin release by 42 ± 17%. Finally, adenovirus-mediated silencing of SNAP23 significantly blocked cAMP-stimulated renin release by 50 ± 13%. We concluded that the SNARE protein SNAP23 mediates cAMP-stimulated renin release. These data show that renin release is a SNARE-dependent process.

Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptor.

The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P < 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P < 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 ± 8 to 118 ± 22% of control (P < 0.01). Combining inhibition of IP(3) and RyR was not additive. G(i) inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 ± 12 to 41 ± 8% of control, P < 0.001). We conclude stimulating JG cell CaSR activates G(q), initiating the PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

Endogenously produced 20-HETE modulates myogenic and TGF response in microperfused afferent arterioles.

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.

Parathyroid hormone stimulates juxtaglomerular cell cAMP accumulation without stimulating renin release.

Parathyroid hormone (PTH) is positively coupled to the generation of cAMP via its actions on the PTH1R and PTH2R receptors. Renin secretion from juxtaglomerular (JG) cells is stimulated by elevated intracellular cAMP, and every stimulus that increases renin secretion is thought to do so via increasing cAMP. Thus we hypothesized that PTH increases renin release from primary cultures of mouse JG cells by elevating intracellular cAMP via the PTH1R receptor. We found PTH1R, but not PTH2R, mRNA expressed in JG cells. While PTH increased JG cell cAMP content from (log(10) means ± SE) 3.27 ± 0.06 to 3.92 ± 0.12 fmol/mg protein (P < 0.001), it did not affect renin release. The PTH1R-specific agonist, parathyroid hormone-related protein (PTHrP), also increased JG cell cAMP from 3.13 ± 0.09 to 3.93 ± 0.09 fmol/mg protein (P < 0.001), again without effect on renin release. PTH2R receptor agonists had no effect on cAMP or renin release. PTHrP increased cAMP in the presence of both low and high extracellular calcium from 3.31 ± 0.17 to 3.83 ± 0.20 fmol/mg protein (P < 0.01) and from 3.29 ± 0.18 to 3.63 ± 0.22 fmol/mg protein (P < 0.05), respectively, with no effect on renin release. PTHrP increased JG cell cAMP in the presence of adenylyl cyclase-V inhibition from 2.85 ± 0.17 to 3.44 ± 0.14 fmol/mg protein (P < 0.001) without affecting renin release. As a positive control, forskolin increased JG cell cAMP from 3.39 ± 0.13 to 4.48 ± 0.07 fmol/mg protein (P < 0.01) and renin release from 2.96 ± 0.10 to 3.29 ± 0.08 ng ANG I·mg prot(-1)·h(-1) (P < 0.01). Thus PTH increases JG cell cAMP via non-calcium-sensitive adenylate cyclases without affecting renin release. These data suggest compartmentalization of cAMP signaling in JG cells.

Renoprotective effect of pioglitazone by the prevention of glomerular hyperfiltration through the possible restoration of altered macula densa signaling in rats with type 2 diabetic nephropathy.

BACKGROUND/AIMS: Pioglitazone (PGZ), one of the thiazolidinediones, has been known to show renoprotective effects. In this study, we focused on the effect of PGZ on glomerular hyperfiltration (GHF), resultant glomerular injury and altered macula densa signaling as a cause of sustained GHF through modified tubuloglomerular feedback in rats with diabetic nephropathy. METHODS: Kidneys from 24-week-old male OLETF rats and LET rats, nondiabetic controls, were used for the experiment. PGZ was administered (10 mg/kg/day, p.o.) for 2 weeks from 22 to 24 weeks of age in some of the OLETF rats (OLETF+PGZ). RESULTS: Parameters relating GHF, kidney weight, creatinine clearance, urine albumin/creatinine ratio and glomerular surface were all increased in OLETF rats and partially restored in OLETF+PGZ rats. Expressions of desmin and TGF-ß were also increased in OLETF rats and restored in OLETF+PGZ rats. The changes in TGF-ß expression were confirmed to be independent of podocyte number. Finally, the immunoreactivity of neuronal nitric oxide synthase (nNOS) and cyclooxygenase 2 (COX-2) in the macula densa was assessed for the evaluation of macula densa signaling. Altered intensities of nNOS and COX-2 in OLETF rats were restored in OLETF+PGZ rats, which agreed with the gene expression analysis (nNOS: 100.2 ± 2.9% in LET, 64.2 ± 2.7% in OLETF, 87.4 ± 12.1% in OLETF+PGZ; COX-2: 100.8 ± 7.4% in LET, 249.2 ± 19.4% in OLETF, 179.9 ± 13.5% in OLETF+PGZ; n = 5) and the semiquantitative analysis of nNOS/COX-2-positive cells. CONCLUSION: PGZ effectively attenuated the GHF and hyperfiltration-associated glomerular injury in diabetic nephropathy. The restoration of altered macula densa signaling might be involved in the renoprotective effect of PGZ.

Juxtaglomerular apparatus hyperplasia under dual angiotensin blockade. A footprint of adequate RAS inhibition or a concern for renal fibrosis?

BACKGROUND: Dual renin-angiotensin system blockade with angiotensin-converting enzyme inhibitors and angiotensin receptor blockers has been advocated to minimize proteinuria. However, recent trials have questioned the renal safety of this approach. Our understanding on the molecular effects of dual blockade in humans is incomplete. CASE PRESENTATION: We present a patient with corticoid resistant nephrotic syndrome who developed marked juxtaglomerular apparatus hyperplasia and renin expression in the context of dual angiotensin system blockade. CONCLUSIONS: Although renin may have profibrotic effects mediated by (pro)renin receptor activation, this report raises questions on the potential consequences of local renin activation on chronic kidney disease in patients with dual angiotensin blockade.

Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney.

Diabetes mellitus is the most common and rapidly growing cause of end-stage renal disease in developed countries. A classic hallmark of early diabetes mellitus includes activation of the renin-angiotensin system (RAS), which may lead to hypertension and renal tissue injury, but the mechanism of RAS activation is elusive. Here we identified a paracrine signaling pathway in the kidney in which high levels of glucose directly triggered the release of the prohypertensive hormone renin. The signaling cascade involved the local accumulation of succinate and activation of the kidney-specific G protein-coupled metabolic receptor, GPR91, in the glomerular endothelium as observed in rat, mouse, and rabbit kidney sections. Elements of signal transduction included endothelial Ca2+, the production of NO and prostaglandin (PGE2), and their paracrine actions on adjacent renin-producing cells. This GPR91 signaling cascade may serve to modulate kidney function and help remove metabolic waste products through renal hyperfiltration, and it could also link metabolic diseases, such as diabetes, or metabolic syndrome with RAS overactivation, systemic hypertension, and organ injury.

Stimulation of renin secretion by angiotensin II blockade is Gsalpha-dependent.

Angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) presumably stimulate renin secretion by interrupting angiotensin II feedback inhibition. The increase in cytosolic calcium caused by activation of Gq-coupled AT1 receptors may mediate the renin-inhibitory effect of angiotensin II at the cellular level, implying that ACEI and ARB may work by reducing intracellular calcium. Here, we investigated whether angiotensin II blockade acts predominantly through Gs-mediated stimulation of adenylyl cyclase (AC) by testing the effect of ACEI and ARB in mice with juxtaglomerular cell-specific deficiency of the AC-stimulatory Gsalpha. The ACEI captopril and quinaprilate and the ARB candesartan significantly increased plasma renin concentration (PRC) to 20 to 40 times basal PRC in wild-type mice but did not significantly alter PRC in Gsalpha-deficient mice. Captopril also completely abrogated renin stimulation in wild-type mice after co-administration of propranolol, indomethacin, and L-NAME. Treatment with enalapril and a low-NaCl diet for 7 days led to a 35-fold increase in PRC among wild-type mice but no significant change in PRC among Gsalpha-deficient mice. Three different pharmacologic inhibitors of AC reduced the stimulatory effect of captopril by 70% to 80%. In conclusion, blockade of angiotensin II stimulates renin synthesis and release indirectly through the action of ligands that activate the cAMP/PKA pathway in a Gsalpha-dependent fashion, including catecholamines, prostaglandins, and nitric oxide.

Pyrogallol-induced As4.1 juxtaglomerular cell death is attenuated by MAPK inhibitors via preventing GSH depletion.

Pyrogallol (PG) induces apoptosis in several types of cells mediated by superoxide anion (O(2*-)). Here, we investigated the effects of PG and/or MAPK (MEK, JNK, and p38) inhibitors on the changes in cell growth, cell death, reactive oxygen species (ROS), and GSH levels in As4.1 juxtaglomerular (JG) cells. PG inhibited the growth of As4.1 cells. It also induced apoptosis and the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)) and increased the level of p53 protein. Intracellular O2(*-) level was increased in PG-treated As4.1 cells. PG also increased the number of GSH deleted cells in As4.1 cells. All the MAPK inhibitors significantly attenuated the growth inhibition and death mediated by PG. They decreased the levels of p53 protein and MMP (DeltaPsi(m)) loss in PG-treated As4.1 cells. They also reduced O2(*-) level and GSH-depleted cell number in these cells. In conclusion, MAPK inhibitors attenuated As4.1 cell growth inhibition and death mediated by PG treatment. The changes in O2(*-) and GSH levels by PG and/or MAPK inhibitors appeared to affect the growth and death of As4.1 cells.

Treatment with p38 inhibitor intensifies the death of MG132-treated As4.1 juxtaglomerular cells via the enhancement of GSH depletion.

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MG132 and/or MAPK inhibitors on As4.1 juxtaglomerular cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)) and activation of caspase-3 and -8. MG132 increased ROS levels, and GSH depleted cell numbers. The MEK inhibitor slightly reduced cell growth and caspase-3 activity in MG132-treated As4.1 cells and mildly increased MMP (DeltaPsi(m)) loss and O(2)(*-) level. However, it did not increase apoptosis and GSH depletion. The JNK inhibitor did not strongly influence cell growth, cell death, and GSH depletion by MG132, but increased caspase-3 activity, MMP (DeltaPsi(m)) loss, and O(2)(*-) level. Treatment with the p38 inhibitor magnified cell-growth inhibition and apoptosis by MG132. This agent also strongly increased caspase-8 activity, MMP (DeltaPsi(m)) loss, O(2)(*-) level, and GSH depletion. Conclusively, the p38 inhibitor strongly intensified cell death in MG132-treated As4.1 cells. The changes of GSH content by MG132 and/or MAPK inhibitors were closely related to the death of As4.1 cells.

Calcium-dependent phosphodiesterase 1C inhibits renin release from isolated juxtaglomerular cells.

Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by calcium. We previously showed JG cells contain a calcium sensing receptor (CaSR), which, when stimulated, decreases cAMP formation and inhibits renin release. We hypothesize CaSR activation decreases cAMP and renin release, in part, by stimulating a calcium calmodulin-activated phosphodiesterase 1 (PDE1). We incubated our primary culture of JG cells with two selective PDE1 inhibitors [8-methoxymethil-IBMX (8-MM-IBMX; 20 microM) and vinpocetine (40 microM)] and the calmodulin inhibitor W-7 (10 microM) and measured cAMP and renin release. Stimulation of the JG cell CaSR with the calcimimetic cinacalcet (1 microM) resulted in decreased cAMP from a basal of 1.13 +/- 0.14 to 0.69 +/- 0.08 pM/mg protein (P < 0.001) and in renin release from 0.89 +/- 0.16 to 0.38 +/- 0.08 microg ANG I/mlxh(-1)xmg protein(-1) (P < 0.001). However, the addition of 8-MM-IBMX with cinacalcet returned both cAMP (1.10 +/- 0.19 pM/mg protein) and renin (0.57 +/- 0.16 microg ANG I/mlxh(-1)xmg protein(-1)) to basal levels. Similar results were obtained with vinpocetine, and also with W-7. Combining 8-MM-IBMX and W-7 had no additive effect. To determine which PDE1 isoform is involved, we performed Western blot analysis for PDE1A, B, and C. Only Western blot analysis for PDE1C showed a characteristic band apparent at 80 kDa. Immunofluorescence showed cytoplasmic distribution of PDE1C and renin in the JG cells. In conclusion, PDE1C is expressed in isolated JG cells, and contributes to calcium's inhibitory modulation of renin release from JG cells.

The effect of angiotensin II on the number of macula densa cells through the AT1 receptor.

The present study was performed to obtain information about a possible correlation between the activity of the renin-angiotensin system and the stereological features of the macula densa (MD). In normal kidneys, the numbers of angiotensin II AT1 receptors were estimated in MD cells and in the neighboring tubular cells. The total volumes of MD, MD cells, neighboring tubular cells, and the total number of MD cells, were measured in normal and candesartan-treated rats (15 mg/kg/day over 21 days). In the normal kidneys, the relative number of AT1 receptors in MD cells [mean = 0.17 (CV = 0.23)] was significantly (p = 0.03) lower than that in normal tubular cells [0.25 (0.21)]. A significant difference (p < 0.01) was observed in the total volume between MD cells [515 microm(3) (0.14)] and normal tubular cells [984 microm(3) (0.19)]. Candesartan treatment significantly elevated (p < 0.01) the total volume of the MD, whereas the total number of MD cells was increased [from 14.2 (0.11) to 19.5 (0.11)]. The results demonstrated that the transdifferentiation from normal tubular cells to MD cells can be controlled by pharmacological means. The structural features of MD controlled by the renin-angiotensin system may be one of the important factors governing the sensitivity of tubuloglomerular feedback.

Oscillating cortical thick ascending limb cells at the juxtaglomerular apparatus.

While studying the intracellular calcium dynamics in cells of the macula densa, the observation was made that tubular epithelial cells located near the macula densa and associated with the renal arterioles exhibit spontaneous Ca2+ oscillations. In this study, the cortical thick ascending limb-distal tubule, with attached glomerulus, was isolated and perfused. At a low luminal sodium chloride concentration, Ca2+ oscillations at a frequency of 63 mHz were observed in tubular cells that were within 100 microm of the macula densa plaque using four-dimensional multiphoton microscopy and wide-field fluorescence microscopy with fura-2. The Ca2+ oscillations were absent in the macula densa cells. Spontaneous oscillations in basolateral membrane potential suggested that Ca2+ oscillations occurred, at least in part, through depolarization-induced increases in Ca2+ entry. The amplitude of these Ca2+ oscillations was significantly enhanced by the activation of the Ca2+-sensing receptor. Increasing the luminal sodium chloride concentration or luminal flow resulted in a significant increase in both the amplitude of Ca2+ oscillations and the intracellular Ca2+ concentration in perimacular cortical thick ascending limb cells. In addition, luminal furosemide attenuated the [NaCl]L-dependent changes in intracellular Ca2+ concentration, but hydrochlorothiazide had no effect. These findings demonstrate that tubular epithelial cells at the perimeter of the macula densa exhibit spontaneous oscillations in intracellular Ca2+ concentration, enhanced by tubular flow and luminal sodium chloride. These oscillatory patterns may play a role in juxtaglomerular signaling.

2,4-Dinitrophenol induces apoptosis in As4.1 juxtaglomerular cells through rapid depletion of GSH.

2,4-Dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in mitochondria. Here, we investigated the in vitro effect of DNP on apoptosis and the involvement of reactive oxygen species (ROS) in As4.1 juxtaglomerular cell death. Dose- and time-dependent induction of apoptosis was evidenced by flow cytometric detection of sub-G1 DNA content and annexin V binding assay. The intracellular H(2)O(2) and O(2)(-) levels were markedly increased in DNP-treated cells. However, the reduction of intracellular H(2)O(2) level by Tiron and catalase did not prevent apoptosis induced by DNP. Moreover, DNP rapidly reduced intracellular GSH content in As4.1 cells. Taken together, apoptosis in DNP-treated As4.1 cells is correlated with the rapid change of intracellular GSH levels rather than ROS levels.

Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction.

The kidney is a rich source of prostaglandins. These eicosanoids, formed by cyclooxygenase-dependent metabolism of arachidonic acid, are important physiologic mediators of renal glomerular hemodynamics and tubular sodium and water reabsorption. Two separate isoforms of cyclooxygenase (COX) have now been identified: constitutive COX-1, encoded by a 2.8-kb mRNA, and mitogen-activated COX-2, encoded by a 4.0-4.5-kb mRNA. COX-2 expression increases during development and inflammation, but, except for brain, constitutive expression is low. It has been generally accepted that physiologic renal production of prostaglandins is mediated by COX-1. However, in the absence of inflammation, low levels of COX-2 mRNA are also detectable in the kidney. To examine the role of COX-2 in the kidney and determine its intrarenal localization, we used a 1.3-kb cDNA probe specific for the 3' untranslated region of rat COX-2 and COX-2-specific antiserum. The COX-2-specific cDNA probe hybridized with a 4.4-kb transcript in total RNA from adult rat kidney. Immunoblots of microsomes isolated from kidney cortex and papilla indicated immunoreactive COX-2 in both locations. In situ hybridization and immunohistochemistry indicated that renal cortical COX-2 expression was localized to the macula densa of the juxtaglomerular apparatus and to adjacent epithelial cells of the cortical thick ascending limb of Henle. In addition, COX-2 immunoreactivity was detected in interstitial cells in the papilla. No COX-2 message or immunoreactive protein was detected in arterioles, glomeruli, or cortical or medullary collecting ducts. When animals were chronically sodium restricted, the level of COX-2 in the region of the macula densa increased threefold (from 0.86 +/- 0.08 to 2.52 +/- 0.43/mm2) and the total area of the COX-2 immunoreactive cells in cortex increased from 34 microns2/mm2 of cortex to 226 microns2/mm2 of cortex. The intrarenal distribution of COX-2 and its increased expression in response to sodium restriction suggest that in addition to its proposed role in inflammatory and growth responses, this enzyme may play an important role in the regulation of salt, volume, and blood pressure homeostasis.

Effect of adenosine1-receptor blockade on renin release from rabbit isolated perfused juxtaglomerular apparatus.

Adenosine has been proposed to act within the juxtaglomerular apparatus (JGA) as a mediator of the inhibition of renin secretion produced by a high NaCl concentration at the macula densa. To test this hypothesis, we studied the effects of the adenosine1 (A1)-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (CPX) on renin release from single isolated rabbit JGAs with macula densa perfused. The A1-receptor agonist, N6-cyclohexyladenosine (CHA), applied in the bathing solution at 10(-7) M, was found to inhibit renin secretion, an effect that was completely blocked by adding CPX (10(-5) M) to the bath. Applied to the lumen, 10(-5) M CPX produced a modest stimulation of renin secretion rates suppressed by a high NaCl concentration at the macula densa (P less than 0.05). The effect of changing luminal NaCl concentration on renin secretion rate was examined in the presence of CPX (10(-7) and 10(-5) M) in the bathing solution and in vehicle control experiments. The control response to increasing luminal NaCl concentration was a marked suppression of renin secretion, that was maintained as long as luminal NaCl concentration was high and was promptly reversible when concentration was lowered. CPX did not alter renin release when luminal NaCl was low, but diminished the reduction caused by high NaCl (P less than 0.01). It is concluded that A1-receptors are located within the JGA, and that A1-receptor activation inhibits renin release. A high NaCl concentration at the macula densa appears to influence A1-receptor activation, but a low NaCl concentration does not. The findings support participation of adenosine in macula densa control of renin secretion.

The changes of intracellular H2O2 are an important factor maintaining mitochondria membrane potential of antimycin A-treated As4.1 juxtaglomerular cells.

We investigated an involvement of ROS, such as H2O2 and O2- and GSH in the As4.1 cell death by antimycin A and examined whether ROS scavengers rescue antimycin A-induced As4.1 cell death and its mechanism. Levels of intracellular H2O2 and O2- were markedly increased in antimycin A-treated cells. Antimycin A reduced the intracellular GSH content. A ROS scavenger, Tiron down-regulated the production of intracellular H2O2. However, the reduction of intracellular H2O2 level did not change the apoptosis parameters, such as sub-G1 DNA content and annexin V binding. Interestingly, treatment of Tiron could partially prevent the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment of SOD and catalase also reduced the intracellular H2O2 and loss of mitochondrial transmembrane potential (DeltaPsi(m)) without reducing O2- level and apoptosis in antimycin A-treated As4.1 cells. All the ROS scavengers, SOD and catalase did not inhibit GSH depletion induced by antimycin A, resulting in failure of preventing the apoptosis. In addition, all the reagents including antimycin A did not induce any specific phase arrest of cell cycle in As4.1 cells. In summary, these results demonstrate that antimycin A generates potently ROS, H2O2 and O2- and induces the depletion of GSH content in As4.1 JG cells, and that Tiron, SOD and catalase inhibited partially the loss of mitochondrial transmembrane potential (DeltaPsi(m)) via the reduction of intracellular H2O2 level.

Pyrogallol, ROS generator inhibits As4.1 juxtaglomerular cells via cell cycle arrest of G2 phase and apoptosis.

Pyrogallol as a catechin compound has been employed as an O(2)(*-) generator and often used to investigate the role of ROS in the biological system. Here, we investigated the in vitro effect of pyrogallol on cell growth, cell cycle and apoptosis in As4.1 juxtaglomerular cells. Dose-dependent inhibition of cell growth was observed with IC(50) of about 60 microM for 48 h using MTT assay. Pyrogallol (100 microM) did not alter intracellular H(2)O(2) level and catalase activity, but increased the intracellular O(2)(-) level and decreased SOD activity in As4.1 cells. DNA flow cytometric analysis indicated that 50 and 100 microM pyrogallol significantly increased G2 phase cells as compared with those of pyrogallol-untreated cells. Also, pyrogallol induced apoptosis as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptosis process was accompanied with the loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation and PARP cleavage. Pan caspase inhibitor (Z-VAD) could significantly rescue As4.1 cells from pyrogallol-induced cell death. But, the inhibitors of caspase-3, caspase-8, and caspase-9 did not prevent apoptotic events in pyrogallol-treated As4.1 cells. Taken together, we have demonstrated that an ROS inducer, pyrogallol inhibits the growth of As4.1 JG cells via cell cycle arrest and apoptosis, and suggest that the compound exhibits an anti-proliferative efficacy on these cells.