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1Division of Nephrology, Departments of Medicine and Physiology, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 2Institute of Pathophysiology, Hungarian Academy of Sciences and Semmelweis University Nephrology Research Group, Budapest H-1089, Hungary
Submitted 22 September 2003 ; accepted in final form 19 January 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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 [Ca2+]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. tubuloglomerular feedback; fluorescent microscopy; salt diet; purinergic receptors
It is generally accepted that there is a direct relationship between luminal NaCl concentration ([NaCl]L) and TGF responses at 15–60 mmol/l, with maximal feedback responses at [NaCl]L of 
60 mmol/l (3, 27). This relationship between [NaCl]L and TGF responses is due, at least in part, to the transport characteristics of the apically located Na-2Cl-K cotransporter, which has been shown in MD cells to saturate at an [NaCl]L of 60 mmol/l (22). Similarly, furosemide, a loop diuretic that blocks the Na-2Cl-K cotransporter, is very effective in inhibiting TGF responses (36). Although TGF responses are primarily a function of [NaCl]L at the MD, TGF responsiveness can be altered as the result of a number of physiological conditions and pharmacological manipulations. In this regard, it is well known that there is a resetting of TGF during short-term alterations in salt and water balance, with an inverse relationship between effective circulating volume and TGF responsiveness (4, 16, 21, 29, 33). Also, long-term dietary salt restriction has been shown to increase TGF sensitivity (6).
In terms of MD cell signaling, we recently reported that MD cells possess a basolateral membrane maxi-anion channel that is ATP permeable (2). We also used a recently developed biosensor assay that can detect cellular release of ATP to directly demonstrate the release of ATP across the MD basolateral membrane. In these initial studies, we found a large increase in the transport of ATP across the MD basolateral membrane when [NaCl]L was increased from 25 to 150 mmol/l. However, we did not determine whether ATP was released in response to alterations in [NaCl]L within the same range as TGF responses and did not examine the effects of apical furosemide on ATP release. Thus one purpose of the present studies was to define the operating range for [NaCl]L-induced MD ATP release as well as its sensitivity to furosemide. In addition, other work was performed to establish the pattern of MD ATP release from rabbits that were maintained on different levels of dietary salt intake. Specifically, we wanted to determine whether there was enhanced ATP release from the MD of rabbits maintained on a low-NaCl diet.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Salt diet.
Separate groups of New Zealand White rabbits (0.5–1.0 kg; Myrtle's Rabbitry, Thompson Station, TN; n = 36 animals total) were fed standard (8630 Harlan Teklad, Madison, WI; 0.3% NaCl), low-salt (TD 90188; 0.01% NaCl) rabbit chow and were given tap water ad libitum for 1 wk. High-salt rabbits were given high-salt (TD 98164; 7.7% NaCl) rabbit chow and 0.45% (wt/vol) NaCl solution as drinking water.
Cell preparation.
Cultured mouse mesangial cells (SV40 MES 13, American, Type Culture Collection, Manassas, VA) were cultured in 1:1 DMEM-Ham's F-12 medium supplemented with 5% FBS (Mediatech, Herndon, VA) and loaded in PBS containing 10–5 mol/l fura 2-AM and 15% (wt/vol) pluronic acid in DMSO for 1 h at 37°C to facilitate dye loading. Subsequently, cells were incubated at 37°C for 30 min in PBS.
Tubule perfusion. Individual cortical thick ascending limbs containing the MD segment with attached glomeruli were dissected from rabbit kidneys and perfused in vitro using methods similar to those described previously (2, 24). For these biosensor studies, it was necessary to carefully dissect the MD plaque away from the glomerulus so that the basolateral surface of MD cells was accessible from the bath. The dissection solution was an isosmotic, low-NaCl Ringer solution consisting of (in mmol/l) 25 NaCl, 120 N-methyl-d-glucamine (NMDG) cyclamate, 5 KCl, 1 MgSO4, 1.6 Na2HPO4, 0.4 NaH2PO4, 1.5 CaCl2, 5 d-glucose, and 10 HEPES. Dissection was performed at 4°C. After transfer to the chamber that was mounted on the microscope, the tubule was cannulated and perfused with the same Ringer solution, except NaCl was isosmotically substituted with NMDG cyclamate, KCl with potassium gluconate, and CaCl2 with calcium gluconate to achieve an [NaCl]L of 0 mmol/l. For experiments in Figs. 2–4, a separate holding pipette was used to gently position fura 2-loaded mesangial cells at the basolateral surface of the MD segment. Changes in [NaCl]L between 0 and 80 mmol/l were achieved by isosmotic substitution of NMDG cyclamate with NaCl. In most experiments, only one or two different [NaCl]L were tested. The bathing solution was 150 mmol/l NaCl Ringer solution, and temperature was maintained at 37°C.
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Statistical analyses. Values are means ± SE. Statistical significance was tested using ANOVA. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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10–5 mol/l ATP, [Ca2+]i responses were composed of an initial peak and a sustained plateau; at lower concentrations, only the slower, sustained response was observed (data not shown). In contrast, addition of adenosine at 10–7–10–3 mol/l did not alter mesangial cell [Ca2+]i. Also, administration of an adenosine A1 receptor agonist, N6-cyclopentyl-adenosine (10–7–10–4 mol/l), failed to alter mesangial cell [Ca2+]i. Finally, preincubation with 8-cyclopentyl-1,3-dipropylxanthine (10–8 mol/l), an adenosine A1 receptor antagonist, did not reduce the increase in [Ca2+]i obtained with 10–5 mol/l ATP.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In our previous work, we used a pheochromocytoma cell line (PC12 cells) as the biosensor cell to monitor ATP release from the MD (2). In addition to this cell line, we also demonstrated that cultured mesangial cells, when placed at the basolateral membrane of the MD plaque, also responded with ATP-dependent increases in [Ca2+]i when [NaCl]L was increased from 25 to 150 mmol/l. In the present studies, we wanted to study the effects of [NaCl]L at 0–60 mmol/l, which is the range in which TGF is most sensitive. Thus we needed to be able to detect lower levels of ATP release; i.e., we needed to use a cell line that was very sensitive to ATP. In preliminary studies, we compared dose-response relationships between exogenous ATP and [Ca2+]i for PC12, HEK293, and mesangial cells (data not shown). Mesangial cells gave the most homogeneous response and were the most sensitive to ATP. We were able to detect increases in [Ca2+]i at <10–6 mol/l ATP. The fact that this increase in [Ca2+]i was due to activation of purinergic receptors is supported by inhibition of increases in [Ca2+]i from exogenous addition of ATP, as well as under experimental conditions of increased [NaCl]L at the MD, by suramin, a P2 receptor blocker. Also other studies have found that mesangial cells express isoforms of P2Y and P2X receptors (8, 9, 26, 28, 34).
Mesangial cells produce mRNA for adenosine receptor subtypes, but the pattern of isoform protein expression may vary depending on species. For instance, cultured mesangial cells from the mouse have been reported to express only the adenosine A3 receptor (37), whereas mesangial cells from the rat may express other isoforms of the adenosine receptor, including the A1 receptor (14, 18–20, 30, 35). The concept that ATP acted directly on P2 receptors, and not through its conversion to adenosine, was also supported by the finding that specific blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine (10–8 mol/l) did not diminish the responses to extracellular ATP. In addition, the possibility of direct adenosine release from MD cells and A1 receptor-mediated [Ca2+]i signaling can be ruled out, because mesangial cells were not responsive to adenosine or the selective A1 agonist N6-cyclopentyl-adenosine.
Because mesangial cells normally lie beneath the MD plaque, one might question why it was necessary to use cultured mesangial cells, instead of the native mesangial cells that are present in the cortical thick ascending limb glomerular preparation. The reasons for this are as follows. 1) The space beneath the MD plaque contains a high ratio of interstitial space and matrix material to mesangial cell bodies. When an attempt is made to load this area with fura 2 or other intracellular fluorescent probes, the fluorescent image is very dim; thus it is difficult to obtain measurements of mesangial [Ca2+]i, at least using conventional imaging. 2) Fluorescent probes, such as fura 2-AM, are cell permeable but do not penetrate very far into tissues or into layers of cells. Thus loading of MD cells from the lumen with fluorescent probes is very efficient, and one can obtain high intracellular concentrations of a fluorescent probe over a short period of time in MD cells, but not in the underlying mesangial cell field. Also, addition of fura 2-AM to the bath results in the loading of peripheral cells with little penetration of the dye into the interior of the glomerulus.
In the present studies, [NaCl]L-dependent ATP release was sensitive to [NaCl]L over the range 0–60 mmol/l (Fig. 2). This is the same dynamic range of [NaCl]L that induces TGF responses (27) and produces changes in MD basolateral membrane potential (1) and intracellular Na+ concentration (22). Thus the identical pattern of MD ATP release and TGF sensitivity suggests that ATP release might play a role in TGF signaling.
It has generally been thought that the Na-2Cl-K cotransporter is the primary apical step in TGF signaling. The results shown in Fig. 4 support this conclusion. However, there is still a residual [NaCl]L-dependent release of ATP that is not apparently sensitive to furosemide. There are two possible reasons for this finding. 1) We failed to use sufficient furosemide to totally block cotransporter activity. 2) ATP release may not depend entirely on the cotransporter, but there may be another transport pathway(s) that influences ATP release and is insensitive to this loop diuretic. One possibility is the Na+ antiporter, NHE2 (23), which has been shown to mediate 20% of the apical entry of Na+ into the MD cells (22).
Studies were performed in MD plaques from rabbits maintained on a normal-salt intake, as well as on low- and high-salt diets. Dietary salt manipulations have been shown to alter TGF responses; thus it was of interest to determine whether ATP release was likewise affected by NaCl intake. We found that ATP release was significantly increased from MD plaques from salt-restricted rabbits. As shown in Fig. 2, this occurred over the entire range of [NaCl]L from 20 to 80 mmol/l. Thus these results are consistent with previous micropuncture studies that found enhanced TGF responses with dietary NaCl restriction (6). However, we did not find attenuated ATP release in MD plaques from rabbits that were maintained on a high-salt diet. The reason for this is not entirely clear but may be due to the difficulties in maintaining the physiological state that is associated with a high-salt diet, i.e., low levels of angiotensin II and other vasoconstrictive hormones and suppression of sympathetic neural activity.
TGF is thought to be a cascade of events involving changes in [NaCl]L, increased MD NaCl transport, activation of intracellular signaling pathways, and release of a mediator or mediators from the MD basolateral surface. Upregulation of ATP release by MD cells under low-salt conditions suggests that the enhanced TGF responsiveness under this condition is due to some change in MD cell function. In other words, enhanced TGF signaling, as a result of salt restriction, may not be due to an increased sensitivity of afferent arteriole smooth muscle cells or enhanced transmission through the mesangial cell field.
These studies clearly demonstrate that MD cells release ATP across the basolateral membrane in a manner that is consistent with TGF responses. These results support the notion that ATP plays a critical role in TGF signaling. However, TGF responses have been shown to be absent in the adenosine A1 receptor knockout mice, suggesting that adenosine may also be an important component in TGF signaling (5, 31). The concept that adenosine is formed by 5'-nucleotidases from ATP (32) that is released from the MD cells is an attractive idea, although our work does not directly provide evidence for this hypothesis. If adenosine is formed in this manner, it would most likely act directly on A1 receptors that are expressed on afferent arteriolar smooth muscle cells, because mesangial cells, at least cultured mouse mesangial cells, do not express adenosine A1 receptors. The recent findings of Ren et al. (25) that demonstrate the necessity of an intact and functional mesangium for TGF would seem to suggest that ATP, when released across the basolateral membrane of MD cells, could directly activate mesangial cell P2 receptors, causing Ca2+ signaling throughout the mesangial cell field. However, whether mesangial cell Ca2+ waves propagate to the afferent arteriolar smooth muscle cells and, thereby, participate in TGF remains unknown. Clearly, further studies are needed to define the site(s) and the roles of ATP and adenosine in the TGF signaling pathway.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) did not alter mesangial cell [Ca2+]i. Studies were also performed to test the effects of the adenosine A1 receptor agonist N6-cyclopentyl-adenosine (10–7–10–4 mol/l, n = 20; 
) and also the effects of the 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist (10–8 mol/l, n = 20, inset), on the response of [Ca2+]i to 10–5 mol/l ATP.