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.

/pdf/cyclooxygenase-2-is-associated-with-the-macula-densa-of-rat-30r294o76t.pdf

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

In adult mammalian kidney, cyclooxygenase-2 (COX-2) expression is found in a restricted subpopulation of cells. The two sites of renal COX-2 localization detected in all species to date are the mac...

Physiological regulation of cyclooxygenase-2 in the kidney

Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl](L)) and transmit signals to the mesangial cellafferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch-clamp studies of macula densa cells, we identified an [NaCl](L)-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 microM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl](L), by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca(2+) concentration to extracellular application of ATP (EC(50) 0.8 microM). Importantly, we also found increases in cytosolic Ca(2+) concentration with elevations in [NaCl](L), when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y(2) receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

https://www.pnas.org/content/pnas/100/7/4322.full.pdf

Macula densa cell signaling involves ATP release through a maxi anion channel

The protease renin is the key enzyme of the renin-angiotensin-aldosterone cascade, which is relevant under both physiological and pathophysiological settings. The kidney is the only organ capable o...

https://www.physiology.org/doi/pdf/10.1152/physrev.00011.2009

Physiology of Kidney Renin

In the kidney nitric oxide (NO) has numerous important functions including the regulation of renal haemodynamics, maintenance of medullary perfusion, mediation of pressure-natriuresis, blunting of tubuloglomerular feedback, inhibition of tubular sodium reabsorption and modulation of renal sympathetic neural activity. The net effect of NO in the kidney is to promote natriuresis and diuresis. Significantly, deficient renal NO synthesis has been implicated in the pathogenesis of hypertension. All three isoforms of nitric oxide synthase (NOS), namely neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3) are reported to contribute to NO synthesis in the kidney. The regulation of NO synthesis in the kidney by NOSs is complex and incompletely understood. Historically, many studies of NOS regulation in the kidney have emphasized the role of variations in gene transcription and translation. It is increasingly appreciated, however, that the constitutive NOS isoforms (nNOS and eNOS) are also subject to rapid regulation by post-translational mechanisms such as Ca(2+) flux, serine/threonine phosphorylation and protein-protein interactions. Recent studies have emphasized the role of post-translational regulation of nNOS and eNOS in the regulation of NO synthesis in the kidney. In particular, a role for phosphorylation of nNOS and eNOS at both activating and inhibitory sites is emerging in the regulation of NO synthesis in the kidney. This review summarizes the roles of NO in renal physiology and discusses recent advances in the regulation of eNOS and nNOS in the kidney by post-translational mechanisms such as serine/threonine phosphorylation.

Nitric oxide in the kidney: functions and regulation of synthesis

The macula densa cells of the juxtaglomerular apparatus probably serve as the sensor cells for the signal which leads to the appropriate tubuloglomerular feedback response. The present study reports basolateral membrane voltage (PDbl) measurements in macula densa cells. We isolated and perfused in vitro thick ascending limb segments with the glomerulus, and therefore the macula densa cells, and the early distal tubule still attached. Macula densa cells were impaled with microelectrodes under visual control. PDbl was recorded in order to examine how these cells sense changes in luminal NaCl concentrations. The addition of furosemide, a specific inhibitor of the Na+2Cl-K+ cotransporter in the thick ascending limb, to the lumen of the perfused thick ascending limb hyperpolarized PDbl from -55 +/- 5 mV to -79 +/- 4 mV (n = 7). Reduction of NaCl in the lumen perfusate from 150 mmol/l to 30 mmol/l also hyperpolarized PDbl from -48 +/- 3 mV to -66 +/- 5 mV (n = 4). A Cl- concentration step in the bath from 150 mmol/l to 30 mmol/l resulted in a 24 +/- 4 mV (n = 4) depolarization of PDbl. This depolarization of PDbl was absent when furosemide was present during the Cl- concentration step. These data suggest that the macula densa cells sense changes in luminal NaCl concentration via coupled uptake of Na+ and Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)

Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport.

This review will address several recent findings regarding the interaction of so called loop diuretics of the furosemide type with the Na+2Cl−K+ cotransporter in the thick ascending limb of the loop of Henle (TAL): (i) The organ selectivity of these transport inhibitors is caused by their secretion by the proximal tubule leading to an increase in their luminal concentration. We have examined whether probenecid, a selective inhibitor of proximal organic anion secretion, reduces the diuretic effect of known loop diuretics. We show that all compounds tested: furosemide (FUR), piretanide (PIR), bumetanide (BUM), and azosemide (AZO) lose part of their diuretic effect when administered in the presence of probenecid. (ii) Previously, we have shown that all loop diuretics of the furosemide type require an acidic group for binding to the Na+2Cl−K+ cotransporter. This is a carboxylate group in the case of FUR, PIR, and BUM; a tetrazolic acid group in the case of AZO; and a sulfonylurea group in the case of torasemide (TOR). Now we show that TOR-derivates with an even less acidic sulfonylurea group (pKa > 8) are still very potent inhibitors of the Na+2C1−K+ cotransporter. Experiments at various pH values indicate that, even for these substances, it is only the anionic form which inhibits the Na+2C1−K+ cotransporter. (iii) Most of the loop diuretics, with the exception of TOR, are rather hydrophilic (FUR, PIR, BUM, AZO). Now we have examined whether one can design compounds which sustain their inhibitory effect on the Na+2C1−K+ cotransporter, although they are highly lipophilic. We found that cyclo-alkyl substitutions at the tolyl-and sulfonylurea-moieties of TOR led to highly lipophilic compounds with very high affinity for the Na+2C1−K+ cotransporter. (iv) Previous data suggested that loop diuretics bind to the Na+2Cl−K+ cotransporter at the extracellular side of the luminal membrane. Now we have designed impermeable macromolecular derivatives of PIR and we show that these macromolecular probes inhibit the Na+2Cl−K+ cotransporter as well as PIR. (v) Previous data indicated that loop diuretics interrupt the macula densa feedback mechanism, and circumstantial evidence suggested that the uptake of Cl− and/or Na+ by macula densa cells may occur via the Na+2Cl−K+ cotransporter. Now we show that macula densa cells sense the luminal NaCl concentration via FUR sensitive Na+2C1−K+ cotransport.

M. Salomonsson

Papers

Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport.

The Mode of Action of Diuretics