Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.

Macula densa (MD) cells express COX-2 and COX-2-derived PGs appear to signal the release of renin from the renal juxtaglomerular apparatus, especially during volume depletion. However, the synthetic machinery and identity of the specific prostanoid released from intact MD cells remains uncertain. In the present studies, a novel biosensor tool was engineered to directly determine whether MD cells release PGE2 in response to low luminal NaCl concentration ([NaCl]L). HEK293 cells were transfected with the Ca2+-coupled E-prostanoid receptor EP1 (HEK/EP1) and loaded with fura-2. HEK/EP1 cells produced a significant elevation in intracellular [Ca2+] ([Ca2+]i) by 29.6 +/- 12.8 nM (n = 6) when positioned at the basolateral surface of isolated perfused MD cells and [NaCl]L was reduced from 150 mM to zero. HEK/EP1 [Ca2+]i responses were observed mainly in preparations from rabbits on a low-salt diet and were completely inhibited by either a selective COX-2 inhibitor or an EP1 antagonist, and also by 100 microM luminal furosemide. Also, 20-mM graduated reductions in [NaCl]L between 80 and 0 mM caused step-by-step increases in HEK/EP1 [Ca2+]i. Low-salt diet greatly increased the expression of both COX-2 and microsome-associated PGE synthase (mPGES) in the MD. These studies provide the first direct evidence that intact MD cells synthesize and release PGE2 during reduced luminal salt content and suggest that this response is important in the control of renin release and renal vascular resistance during salt deprivation.

Amyloid beta peptide 1-40 stimulates the Na+/Ca2+ exchange activity of SNCX.

The Na+/Ca2+ exchangers, RNCX and SNCX, were cloned from mesangial cells of salt sensitive and salt resistant Dahl/Rapp rats, respectively, and differ at amino acid 218 (RNCXi/SNCXf) and in the exons expressed at the alternative splice site (RNCXB, D/SNCXB, D, F). These isoforms are also expressed in myocytes, neurons, and astrocytes where they maintain cytosolic calcium homeostasis. We demonstrated that cells expressing SNCX were more susceptible to oxidative stress than cells expressing RNCX. Others demonstrated that amyloid beta peptide (Abeta) augments the adverse effects of oxidative stress on calcium homeostasis. Therefore, we sought to assess the effect of Abeta 1-40 on the abilities of OK-PTH cells stably expressing RNCX and SNCX and human glioma cells, SKMG1, to regulate cytosolic calcium homeostasis. Our studies showed that Abeta 1-40 (1 microM) did not affect RNCX activity, as assessed by changes in [Ca2+]i (Delta[Ca2+]i, 260+/-10 nM to 267+/-8 nM), while stimulating exchange activity 2.4 and 3 fold in cells expressing SNCX (100+/-8 to 244+/-12 nM) and in SKMG1 cells (90+/-11 nM to 270+/-18 nM), respectively. Our results also showed that Abeta 1-40, while not affecting the rate of Mn2+ influx in cells expressing RNCX, stimulated the rate of Mn2+ influx 2.8 and 2.9 fold in cells expressing SNCX and in SKMG1 cells. Thus, our studies demonstrate that Abeta-induced cytosolic calcium increase is mediated through certain isoforms of the Na+/Ca2+ exchanger and reveals a possible mechanism by which Abeta 1-40 can alter cytosolic calcium homeostasis.

Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells.

Recent genetic analysis has identified a pivotal role of primary cilia in the pathogenesis of polycystic kidney disease (PKD). However, little is known regarding how cilia loss/dysfunction contributes to cyst development. In epithelial cells, changes in apical fluid flow induce cilia-mediated Ca2+ entry via polycystin-2 (PC2), a cation channel. The Oak Ridge Polycystic Kidney (orpk) mouse contains a mutated Tg737 gene that disrupts expression of polaris, a protein required for ciliogenesis. These studies examine the effect of cilia malformation on Ca2+ entry in orpk cilia(-) collecting duct principal cells, and in orpk cells in which wild-type Tg737 was reintroduced, orpk cilia(+). [Ca2+]i was monitored in confluent cell monolayers using fluorescence microscopy. Intrinsic apical Ca2+ entry was measured by Mn2+ quenching and Ca2+ depletion/readdition under flow conditions below the threshold for stimulation. We found that unstimulated apical Ca2+ entry was markedly increased in cilia(-) cells and was sensitive to Gd3+, an inhibitor of PC2. Electrophysiological measurements demonstrate increased abundance of an apical channel, consistent with PC2, in cilia(-) cells. Immunofluorescence studies revealed that PC2, normally expressed on and at the base of cilia in orpk cilia(+) cells, was observed throughout the apical membrane in cilia(-) cells. Furthermore, cilia(-) cells displayed elevated subapical Ca2+ levels measured with the near-membrane Ca2+ indicator FFP-18. We propose that cilia exert a tonic regulatory influence on apical Ca2+ entry, and absence of cilia results in loss of spatial organization of PC2, causing unregulated Ca2+ entry and elevations in subapical [Ca2+], a factor which may contribute to cyst formation.

Characterization of basolateral chloride/bicarbonate exchange in macula densa cells.

Functional and immunohistological studies were performed to identify basolateral chloride/bicarbonate exchange in macula densa cells. Using the isolated, perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, macula densa intracellular pH (pH(i)) was measured with fluorescence microscopy and BCECF. For these experiments, basolateral chloride was reduced, resulting in reversible macula densa cell alkalinization. Anion exchange activity was assessed by measuring the maximal net base efflux on readdition of bath chloride. Anion exchange activity required the presence of bicarbonate, was independent of changes in membrane potential, did not require the presence of sodium, and was inhibited by high concentrations of DIDS. Inhibition of macula densa anion exchange activity by basolateral DIDS increased luminal NaCl concentration-induced elevations in pH(i). Immunohistochemical studies using antibodies against AE2 demonstrated expression of AE2 along the basolateral membrane of macula densa cells of rabbit kidney. These results suggest that macula densa cells functionally and immunologically express a chloride/bicarbonate exchanger at the basolateral membrane. This transporter likely participates in the regulation of pH(i) and might be involved in macula densa signaling.

Immunolocalization of a microsomal prostaglandin E synthase in rabbit kidney.

PGE2, the major cyclooxygenase (COX) metabolite of arachidonic acid, is an important paracrine regulator of numerous tubular and vascular functions in the kidney. To date, COX activity has been considered the key step in prostaglandin synthesis and is well characterized. However, much less is known about the recently cloned microsomal PGE2 synthase (mPGES), the terminal enzyme of PGE2 synthesis, which converts COX-derived PGH2 to the biologically important PGE2. Present studies provide the detailed localization of mPGES protein in the rabbit kidney using immunohistochemistry. In the cortex, strong mPGES labeling was found in the macula densa (MD) and principal cells of the connecting segment and cortical collecting tubule but not in intercalated cells. The medulla was abundant in mPGES-positive structures, with heavy labeling in the collecting duct system. In descending thin limbs and renal medullary interstitial cells, mPGES expression was less intense, and it was below the limits of detection in the vasa recta. Expression of MD mPGES, similarly to COX-2, was greatly increased in response to low-salt diet and angiotensin I-converting enzyme inhibition by captopril. These findings suggest autocrine regulation of renal salt and water transport by PGE2 in descending thin limb and collecting tubule and a paracrine effect of PGE2 on the glomerular and medullary vasculature. Similar to other organs, mPGES in the kidney is an inducible enzyme and may be similarly regulated and acts in concert with COX-2.

Real-time imaging of renin release in vitro.

Renin release from juxtaglomerular granular cells is considered the rate-limiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect, and work with significant internal errors. To directly visualize and study the dynamics of both the release and tissue activity of renin, we isolated and perfused afferent arterioles with attached glomeruli dissected from rabbit kidneys and used multiphoton fluorescence imaging. Acidotropic fluorophores, such as quinacrine and LysoTrackers, clearly and selectively labeled renin granules. Immunohistochemistry of mouse kidney with a specific renin antibody and quinacrine staining colocalized renin granules and quinacrine fluorescence. A low-salt diet for 1 wk caused an approximately fivefold increase in the number of both individual granules and renin-positive granular cells. Time-lapse imaging showed no signs of granule trafficking or any movement, only the dimming and disappearance of fluorescence from individual renin granules within 1 s in response to 100 microM isoproterenol. There appeared to be a quantal release of the granular contents; i.e., an all-or-none phenomenon. Using As4.1 cells, a granular cell line, we observed further classic signs of granule exocytosis, the emptying of granule content associated with a flash of quinacrine fluorescence. Using a fluorescence resonance energy transfer-based, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS)-conjugated renin substrate in the bath, an increase in EDANS fluorescence (renin activity) was observed around granular cells in response to isoproterenol. Fluorescence microscopy is an excellent tool for the further study of the mechanism, regulation, and dynamics of renin release.

Neuronal nitric oxide synthase: its role and regulation in macula densa cells.

Macula densa (MD) cells detect changes in distal tubular sodium chloride concentration ([NaCl](L)), at least in part, through an apical Na:2Cl:K co-transporter. This co-transporter may be a site for regulation of tubuloglomerular feedback (TGF), and recently angiotensin II (Ang II) was shown to regulate the MD Na:2Cl:K co-transporter. In addition, nitric oxide (NO) produced via neuronal NO synthase (nNOS) in MD cells attenuates MD-TGF signaling. This study investigated [NaCl](L)-dependent MD-NO production, the regulation of co-transporter activity by NO, and the possible interaction of NO with Ang II. MD cell Na(+) concentration ([Na(+)](i)) and NO production were measured using sodium-binding benzofuran isophthalate and 4-amino-5-methylamino-2',7'-difluorescein diacetate, respectively, using fluorescence microscopy. Na:2Cl:K co-transport activity was assessed as the initial rate of increase in [Na(+)](i) when [NaCl](L) was elevated from 25 to 150 mM. 10(-4) M 7-nitroindazole, a specific nNOS blocker, significantly increased by twofold the initial rate of rise in [Na(+)](i) when [NaCl](L) was increased from 25 to 150 mM, indicating co-transporter stimulation. There was no evidence for an interaction between the stimulatory effect of Ang II and the inhibitory effect of NO on co-transport activity, and, furthermore, Ang II failed to alter MD-NO production. NO production was sensitive to [NaCl](L) but increased only when [NaCl](L) was elevated from 60 to 150 mM. These studies indicate that MD-NO directly inhibits Na:2Cl:K co-transport and that NO and Ang II independently alter co-transporter activity. In addition, generation of MD-NO seems to occur only at markedly elevated [NaCl](L), suggesting that NO may serve as a buffer against high rates of MD cell transport and excessive TGF-mediated vasoconstriction.

Macula densa basolateral ATP release is regulated by luminal [NaCl] and dietary salt intake.

One component of the macula densa (MD) tubuloglomerular feedback (TGF) signaling pathway may involve basolateral release of ATP through a maxi-anion channel. Release of ATP has previously been studied during a maximal luminal NaCl concentration ([NaCl](L)) stimulus (20-150 mmol/l). Whether MD ATP release occurs during changes in [NaCl](L) within the physiological range (20-60 mmol/l) has not been examined. Also, because TGF is known to be enhanced by low dietary salt intake, we examined the pattern of MD ATP release from salt-restricted rabbits. Fluorescence microscopy, with fura 2-loaded cultured mouse mesangial cells as biosensors, was used to assess ATP release from the isolated, perfused thick ascending limb containing the MD segment. The mesangial biosensor cells, which contain purinergic receptors and elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) on ATP binding, were placed adjacent to the MD basolateral membrane. Elevations in [NaCl](L) between 0 and 80 mmol/l, in 20-mmol/l increments, caused stepwise increases in [Ca(2+)](i), with the highest increase at [NaCl](L) of approximately 60 mmol/l. Luminal furosemide at 10(-4) mol/l blocked ATP release, which suggests that the efflux of ATP required MD Na-2Cl-K cotransport. A low-salt diet for 1 wk increased the magnitude of [NaCl](L)-dependent elevations in biosensor [Ca(2+)](i) by twofold, whereas high-salt intake had no effect. In summary, ATP release occurs over the same range of [NaCl](L) (20-60 mmol/l) previously reported for TGF responses, and, similar to TGF, ATP release was enhanced by dietary salt restriction. Thus these two findings are consistent with the role of MD ATP release as a signaling component of the TGF pathway.

Angiotensin I conversion to angiotensin II stimulates cortical collecting duct sodium transport.

Angiotensin (Ang) II directly stimulates epithelial sodium channel activity in the rabbit cortical collecting duct. Because Ang I and converting enzyme analogues might be present in the distal nephron, this raises the possibility of intraluminal generation of Ang II. Conversion of Ang I to Ang II was monitored by Ang II-dependent changes in intracellular sodium concentration as a reflection of sodium transport across the apical membrane. This involved imaging-based fluorescence microscopy with sodium-binding benzofuran isophthalate in isolated, perfused, cortical collecting-duct segments from rabbit kidney. Principal and intercalated cells were differentiated by rhodamine-conjugated peanut lectin. Control principal cell intracellular sodium concentration, during perfusion with 25 mmol/L NaCl and zero sodium in the bath plus monensin (10(-5) mol/L) averaged 5.8+/-0.14 mmol/L (n=156). The increase in intracellular sodium concentration, when luminal NaCl was increased from 25 to 150 mmol/L, was elevated by 3.5-fold in the presence of intraluminal Ang I (10(-6) mol/L). Also, the effects of Ang I on sodium transport were not significantly different from the effects of Ang II (10(-9) mol/L). Ang I was used in micromolar concentrations to ensure that there was sufficient substrate available for conversion to Ang II. Inhibition of the angiotensin-converting enzyme with captopril reduced the stimulatory effect of Ang I. These results suggest that intraluminal conversion of Ang I to Ang II can occur in the cortical collecting duct, resulting in enhanced apical sodium entry.

Adaptations to diving hypoxia in the heart, kidneys and splanchnic organs of harbor seals (Phoca vitulina).

Pinnipeds (seals and sea lions) have an elevated mitochondrial volume density [VV(mt)] and elevated citrate synthase (CS) and beta-hydroxyacyl-CoA dehydrogenase (HOAD) activities in their swimming muscles to maintain an aerobic, fat-based metabolism during diving. The goal of this study was to determine whether the heart, kidneys and splanchnic organs have an elevated VV(mt) and CS and HOAD activities as parallel adaptations for sustaining aerobic metabolism and normal function during hypoxia in harbor seals (Phoca vitulina). Samples of heart, liver, kidney, stomach and small intestine were taken from 10 freshly killed harbor seals and fixed in glutaraldehyde for transmission electron microscopy or frozen in liquid nitrogen for enzymatic analysis. Samples from dogs and rats were used for comparison. Within the harbor seal, the liver and stomach had the highest VV(mt). The liver also had the highest CS activity. The kidneys and heart had the highest HOAD activities, and the liver and heart had the highest lactate dehydrogenase (LDH) activities. Mitochondrial volume densities scaled to tissue-specific resting metabolic rate [VV(mt)/RMR] in the heart, liver, kidneys, stomach and small intestine of harbor seals were elevated (range 1.2-6.6x) when compared with those in the dog and/or rat. In addition, HOAD activity scaled to tissue-specific RMR in the heart and liver of harbor seals was elevated compared with that in the dog and rat (3.2x and 6.2x in the heart and 8.5x and 5.5x in the liver, respectively). These data suggest that organs such as the liver, kidneys and stomach possess a heightened ability for aerobic, fat-based metabolism during hypoxia associated with routine diving. However, a heightened LDH activity in the heart and liver indicates an adaptation for the anaerobic production of ATP on dives that exceed the animal's aerobic dive limit. Hence, the heart, liver, kidneys and gastrointestinal organs of harbor seals exhibit adaptations that promote an aerobic, fat-based metabolism under hypoxic conditions but can provide ATP anaerobically if required.

The diving paradox: new insights into the role of the dive response in air-breathing vertebrates.

When aquatic reptiles, birds and mammals submerge, they typically exhibit a dive response in which breathing ceases, heart rate slows, and blood flow to peripheral tissues is reduced. The profound dive response that occurs during forced submergence sequesters blood oxygen for the brain and heart while allowing peripheral tissues to become anaerobic, thus protecting the animal from immediate asphyxiation. However, the decrease in peripheral blood flow is in direct conflict with the exercise response necessary for supporting muscle metabolism during submerged swimming. In free diving animals, a dive response still occurs, but it is less intense than during forced submergence, and whole-body metabolism remains aerobic. If blood oxygen is not sequestered for brain and heart metabolism during normal diving, then what is the purpose of the dive response? Here, we show that its primary role may be to regulate the degree of hypoxia in skeletal muscle so that blood and muscle oxygen stores can be efficiently used. Paradoxically, the muscles of diving vertebrates must become hypoxic to maximize aerobic dive duration. At the same time, morphological and enzymatic adaptations enhance intracellular oxygen diffusion at low partial pressures of oxygen. Optimizing the use of blood and muscle oxygen stores allows aquatic, air-breathing vertebrates to exercise for prolonged periods while holding their breath.