Identification of renal natriuretic peptide receptor subpopulations by use of the non-peptide antagonist, HS-142-1.

1. The renal actions of natriuretic peptides are dictated by the distribution of guanylyl cyclase-linked (NPRA and NPRB) and non-guanylyl cyclase-linked (NPRC) receptors. Natriuretic peptide receptors have previously been distinguished on the basis of their differential affinity for peptide fragments and analogues; however, most of the available ligands are not fully selective. We have used the specific guanylyl cyclase-linked receptor antagonist, HS-142-1, to investigate the differential distribution of natriuretic peptide receptor subtypes in the human, bovine and rat kidney. 2. Specific, high affinity 3-([125I]-iodotyrosyl)-rat-ANP-(1-28)([125I]-rANP1-28) binding sites were identified in all three species, localized to glomeruli, inner medulla, intrarenal arteries and regions in the outer medulla corresponding to vasa recta bundles. Binding sites were also identified in the smooth muscle lining of the hilar region in the bovine and rat kidney. 3. In the rat, [125I]-rANP1-28 binding was inhibited by unlabelled peptide sequences with a rank order of potency (rANP1-28 > pCNP1-22 > C-ANP4-23). The glomeruli exhibited a heterogeneous population of binding sites, C-ANP4-23 and pCNP1-22 producing a significantly better fit to a two component inhibition curve compared to the single component curve for rANP1-28. 4. Competitive inhibition experiments with the receptor selective ligands, C-ANP4-23 and HS-142-1, suggested that, like the rat, human and bovine glomeruli possessed a heterogeneous population of binding sites, whilst those in the inner medulla and intrarenal arteries of all three species represented a homogeneous population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of ET(A) and ET(B) in the renal tissue of rats with compensated and decompensated heart failure

Endothelin-1 (ET-1) exerts its biological actions through two receptor subtypes: endothelin-A (ETA) receptor and endothelin-B (ETB) receptor. We demonstrated previously that ET-1 induces systemic and renal cortical vasoconstriction via ETA whereas ETB mediates medullary vasodilation. Congestive heart failure (CHF) is characterized by increased vascular resistance and impaired renal hemodynamic and excretory function. While the pathophysiological effects of ET-1 in CHF are well established, the status of ETA and ETB in the kidney is poorly characterized. The present study examined the immunostaining and localization of ETA and ETB in the renal cortex and medulla of rats with experimental CHF induced by aorto-caval fistula. Rats with CHF were further subdivided, based on their daily urinary sodium excretion, into rats with compensated (urinary sodium excretion > 1200 microEq/day) and decompensated CHF (urinary sodium excretion < 200 microEq/day). ETA is predominantly localized to the cortex mainly in the peritubular capillaries, and is upregulated in rats with compensated and decompensated CHF compared with sham controls. In contrast, ETB is preferentially expressed in the outer and inner medulla, mainly in the vasa recta, the thick ascending limb of Henle's loop and the collecting duct. While compensated CHF is associated with upregulation of ETB in the collecting duct and vasa recta, decompensated CHF is accompanied with enhanced ETB abundance in the vasa recta and remarkable downregulation of this receptor subtype in the collecting duct. The findings suggest that upregulation of ETA may lead to a decrease in cortical blood flow while upregulation of ETB in the vasa recta probably contributes to the preservation of medullary blood flow. Furthermore, downregulation of ETB in the collecting duct, only in rats with decompensated CHF, could contribute to sodium retention in that subgroup.     

INTRARENAL BLOOD FLOW: MICROVASCULAR ANATOMY AND THE REGULATION OF MEDULLARY PERFUSION

1. The microcirculation of the kidney is arranged in a manner that facilitates separation of blood flow to the cortex, outer medulla and inner medulla. 2. Resistance vessels in the renal vascular circuit include arcuate and interlobular arteries, glomerular afferent and efferent arterioles and descending vasa recta. 3. Vasoactive hormones that regulate smooth muscle cells of the renal circulation can originate outside the kidney (e.g. vasopressin), can be generated from nearby regions within the kidney (e.g. kinins, endothelins, adenosine) or they can be synthesized by adjacent endothelial cells (e.g. nitric oxide, prostacyclin, endothelins). 4. Vasoactive hormones released into the renal inner medullary microcirculation may be trapped by countercurrent exchange to act upon descending vasa recta within outer medullary vascular bundles. 5. Countercurrent blood flow within the renal medulla creates a hypoxic environment. Relative control of inner versus outer medullary blood flow may play a role to abrogate the hypoxia that arises from O₂ consumption by the thick ascending limb of Henle. 6. Cortical blood flow is autoregulated. In contrast, the extent of autoregulation of medullary blood flow appears to be influenced by the volume status of the animal. Lack of medullary autoregulation during volume expansion may be part of fundamental processes that regulate salt and water excretion.     

Comparison of endothelin-A and endothelin-B receptor distribution visualized by radioligand binding versus immunocytochemical localization using subtype selective antisera

Molecular studies have predicted the existence in human tissue of splice variants and modifications to the amino acid sequence of endothelin receptors that may modulate function. Endothelin-A (ETA) receptors were visualized by ligand binding and autoradiography to the renal vasculature throughout the cortex and medulla, including the large arcuate arteries, adjacent veins and arterioles. Lower binding densities were visualized to the vasa recta and glomeruli. A similar pattern of staining was revealed by ETA selective antisera, with the higher resolution demonstrating ETA receptors confined to smooth muscle cells. Staining was also detected to the vasa recta and glomeruli. Ligand binding revealed a more heterogeneous endothelin-B (ETB) receptor distribution with high densities concentrated in the medulla. Three different site-directed ETB antisera demonstrated a similar pattern of staining to the endothelium lining all renal vessels but not to the smooth muscle. Staining was also detected to glomerular endothelial cells as well as epithelial cells lining the renal tubule, particularly the collecting ducts, consistent with high binding densities observed in the medulla by autoradiography. There was no evidence for a differential distribution in either ETA or ETB receptors visualized by the two techniques that might have indicated modified receptors or further subtypes in the human kidney.     

Molecular mechanisms and therapeutic strategies of chronic renal injury: physiological role of angiotensin II-induced oxidative stress in renal medulla

Renal medullary circulation has now been found to play a fundamental role in regulating long-term blood pressure control and fluid balance. Elevation of superoxide or reduction of nitric oxide (NO) in renal medulla decreases medullary blood flow and Na excretion, resulting in sustained hypertension. Angiotensin II (Ang II)-induced interaction of superoxide and NO was determined in thin tissue strips isolated from the renal outer medullary region of Sprague-Dawley rats using fluorescent microscopy techniques. Ang II can induce diffusion of NO, but not superoxide, from the medullary thick ascending limb (mTAL) to the surrounded vasa recta. However, when NO is reduced by the NO scavenger carboxy-PTIO, Ang II can induce superoxide diffusion from mTAL to vasa recta pericytes. Therefore, the physiological action of oxidative stress in renal medullary region is demonstrated as balance of superoxide and NO diffusion ("tubulo-vascular cross-talk"). These results explain how chronically hypoxic medulla can maintain blood flow. In other studies using chronically instrumented rats, we found that nearly 70% of Ang II-induced medullary renal injury was dependent on pressure determined by servo-control of renal perfusion pressure, whereas 30% of the injury was non-hemodynamic. We conclude that oxidative stress within the renal medulla can induce hypertension and also make the kidney functionally more vulnerable to the effects of Ang II.     

IMPACT OF CYCLO-OXYGENASE BLOCKADE ON JUXTAMEDULLARY MICROVASCULAR RESPONSES TO ANGIOTENSIN II IN RAT KIDNEY

1. Experiments were designed to evaluate the hypothesis that cyclo-oxygenase products modulate the influence of angiotensin II (AII) on the renal juxtamedullary microvasculature of enalaprilat-treated rats. 2. The in vitro blood-perfused juxtamedullary nephron technique was utilized to provide access to afferent arterioles, efferent arterioles and descending vasa recta located in the outer stripe of the outer medulla. 3. Baseline afferent arteriolar diameter was 20.8 ± 1.9 μm in kidneys subjected to cyclo-oxygenase blockade (1μmol/L piroxicam), a value significantly lower than that observed in untreated kidneys (26.1 ± 1.0 μm). Baseline diameters of efferent arterioles and outer medullary descending vasa recta did not differ between untreated and piroxicam-treated groups. 4. Topical application of 1 nmol/L AII reduced blood flow through outer medullary descending vasa recta by 22 ± 6% in untreated kidneys and by 24 ± 7% in piroxicam-treated kidneys. 5. In untreated kidneys, AII (0.01–100nmol/L) produced concentration-dependent afferent and efferent arteriolar constrictor responses of similar magnitudes. Neither afferent nor efferent arteriolar AII responsiveness was significantly altered in piroxicam-treated kidneys, although afferent responses exceeded efferent responses at AII concentrations ≥ 10 nmol/L. 6. We conclude that endogenous cyclo-oxygenase products exert a vasodilator influence on juxtamedullary afferent arterioles under baseline conditions. Although cyclo-oxygenase inhibition had little effect on juxtamedullary microvascular responses to AII, the response to high AII concentrations may be modulated by cyclo-oxygenase products in a manner which delicately alters the relative influence of the peptide on pre- vs postglomerular resistances.     

Visualization of adenosine A1 receptors in the human and the guinea-pig kidney

Adenosine A1 receptors were localized in sections of human and guinea-pig kidney with quantitative receptor autoradiography and [3H]cyclohexyladenosine [( 3H]CHA) used as ligand. The binding sites had the characteristics of an A1 receptor. In the human kidney a high density of receptor sites was measured over the glomeruli. In the guinea-pig kidney the receptor sites were localized in the inner and outer medulla although a low density of binding was also seen over the glomeruli. The functional significance of the findings is discussed.     

Rat atrial natriuretic polypeptide increases net water, sodium and chloride absorption across rat small intestine in vivo

Localization of binding sites for synthetic rat atrial natriuretic polypeptide (alpha-rANP) and the effect of the peptide on net water and electrolyte movement in vivo perfused small intestine of the rat were studied. Autoradiographic study demonstrated that specific binding sites were localized on a space between the base of epithelia and lamina propria of the small intestine. Alpha-rANP (0.25 micrograms/min) infused into the superior mesenteric artery of rats increased net absorption of water (46% increase in comparison with controls), Na (84% increase) and Cl (70% increase) across the small intestinal tract perfused with Ringer's solution. These increases were also observed when glucose-free Ringer's solution was employed. Thus it would be concluded that the Na-glucose cotransport system is not involved in the action of alpha-rANP. These observations suggest that alpha-rANP controls circulating water-electrolyte balance through regulating not only renal function but also intestinal water, Na and Cl absorption.     

Presence of angiotensin II AT2 receptor binding sites in the adventitia of human kidney vasculature

1. Angiotensin II (AngII) receptor subtypes in adult human kidney were pharmacologically characterized by in vitro autoradiography using the AngII receptor subtype-selective antagonists, losartan and PD 123319, and the sensitivity to the reducing agent, dithiothreitol. 2. High densities of AngII AT(1) receptor binding occur in the glomeruli and the inner stripe of the outer medulla, while a moderate AT(1) receptor binding is localized in the proximal convoluted tubules. 3. AT(2) receptor binding is observed predominantly in the intrarenal large blood vessels, including the arcuate, inter- and intra-lobular arteries, and in the renal capsule. 4. In the major renal artery, AT(1) receptor binding is abundant in the media and adventitia, while AT(2) receptor binding is observed mainly in the adventitia. 5. At the light microscopic level using emulsion autoradiography, AT(1) receptors are localized in the glomeruli and juxtaglomerular apparatus, as expected. However, in larger renal blood vessels, including the arcuate arteries, inter- and intra-lobular arteries, intense AT(2) receptor labelling occurs primarily in the adventitia, while the endothelium and vascular smooth muscle layers contain only low levels of AngII receptor binding. 6. These results indicate that the adult human kidney displays two pharmacologically distinct AngII receptor subtypes, with AT(1) predominating in the glomeruli, juxtaglomerular apparatus, proximal tubules and the inner stripe of the outer medulla, while AT(2) predominates in the adventitia of the arcuate and interlobular arteries and the renal capsule. The functional significance of AT(2) receptor binding sites in the adventitia of adult human kidney vessels remains to be elucidated.     

The autoradiographic distribution of substance P binding sites in guinea-pig vas deferens is altered by capsaicin pretreatment

The distribution of binding sites for [125I]Bolton-Hunter substance P (BHSP) was investigated in vasa deferentia from normal, capsaicin-pretreated and vehicle-pretreated guinea-pigs, using qualitative and quantitative autoradiography. Dense binding of BHSP was seen over the outer longitudinal muscle with less over the inner longitudinal muscle. Very low specific binding occurred to the circular muscle and was absent in the mucosa. Characterization in slide-mounted sections showed that binding was saturable and of high affinity, with equilibrium dissociation constant (KD) 91 +/- 15 pM. BHSP was displaced by substance P greater than neurokinin A greater than neurokinin B, suggesting binding to NK-1 receptors. Capsaicin pretreatment had no effect on the lengthwise distribution of binding sites but significantly altered their relative distribution between the different smooth muscle layers. There was a very marked increase in BHSP binding over the inner longitudinal muscle and the inner part of the circular muscle layer, whereas binding was virtually abolished over the outer longitudinal muscle, compared with vehicle control. Capsaicin-sensitive binding sites over the outer longitudinal muscle may be located presynaptically on capsaicin-susceptible primary afferent sensory neurons. In contrast, the increase in number of binding sites associated with the inner longitudinal muscle may be due to receptor upregulation resulting from loss of sensory innervation, and suggests that these binding sites are postsynaptic.     

Similar distribution of [125I]sarafotoxin-6b and [125I]endothelin-1, -2, -3 binding sites in the human kidney

The distribution of binding sites for 125I-labelled endothelins (ET-1, ET-2, ET-3) and sarafotoxin-6b (SRTX-6b) was visualized in human kidneys using autoradiography. The glomeruli, the internal medulla and the renal blood vessels presented the highest levels of labelling, while the outer medulla exhibited intermediate and the cortex lower densities of sites. The relative enrichment of binding sites in different renal areas was similar for each peptide, suggesting the existence of a homogeneous population of endothelin/sarafotoxin receptors.     

Localization of Binding Sites for Epidermal Growth Factor (EGF) in Rat Kidney: Evidence for the Existence of Low Affinity EGF Binding Sites on the Brush Border Membrane

We investigated the renal distribution of ¹²⁵I-EGF in the filtering perfused rat kidney using an acid washing technique. Trichloroacetic acid-precipitable ¹²⁵I-EGF radioactivity was eluted from both the renal vein and the urinary cannulae, the former regarded as representing the antiluminal, and the latter the luminal, cell surface bound ¹²⁵I-radioactivity. The addition of excess unlabeled EGF (20 nM) to the perfusate completely inhibited the binding of ¹²⁵I-EGF to the antiluminal membrane but did not inhibit that of ¹²⁵I-EGF to the luminal membrane. On the other hand, the order of relative density of ¹²⁵I-EGF binding sites in the in vivo kidney determined by auto-radiography was cortex > inner medulla > outer medulla. After the i.v. administration of excess unlabeled EGF together with ¹²⁵I-EGF, the renal uptake of ¹²⁵I-EGF was inhibited completely in the inner medulla, but only by 50% in the cortex and outer medulla, suggesting the presence of nonsaturable luminal uptake of EGF in the cortex and outer medulla. After i.v. administration of ¹²⁵I-EGF, a change in position of silver grains from the luminal cell surface membrane to the intracellular space was observed in the proximal convoluted tubules. In conclusion, in addition to the previously identified uptake mechanism of circulating EGF through high-affinity binding sites on the antiluminal cell surface membrane, the reabsorption mechanism of filtered EGF through low-affinity binding sites on the luminal cell surface membrane was demonstrated. In vivo autoradiography showed the gradual internalization of EGF from the luminal cell surface membrane to the intracellular space of the proximal convoluted tubule.     

Stimulation of guanylate cyclase activity by irreversible binding of atrial natriuretic peptide to its receptor

We examined receptor binding profiles of atrial natriuretic peptide (ANP) in rat tissue using 125I-labeled alpha-rat ANP [( 125I]alpha-rANP). Specific [125I]alpha-rANP binding to its receptor was reversible following addition of unlabeled free alpha-rANP, but it became increasingly irreversible with time during incubation. Irreversible binding of alpha-rANP was observed both at 0 degrees and 25 degrees in homogenates of adrenal capsules and lungs, crude membranes of renal glomeruli, partially purified membranes of lung, solubilized membrane preparations from renal glomeruli, and intact renal glomeruli. Irreversible binding increased in a time- and temperature-dependent manner. HPLC analysis demonstrated that the irreversibly bound radioactivity, which was extracted by 1 N CH3COOH from both intact renal glomeruli and from partially purified membranes, was associated with intact [125I]alpha-rANP. Irreversibly bound alpha-rANP increased cGMP concentrations by activating guanylate cyclase activity. These findings suggest that the appearance of irreversible binding of alpha-rANP to its receptor is independent of its internalization, and may be involved in message transduction and subsequent biological responses.     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: PRESENCE OF ANGIOTENSIN II AT2 RECEPTOR BINDING SITES IN THE ADVENTITIA OF HUMAN KIDNEY VASCULATURE

• 1 Angiotensin II (AngII) receptor subtypes in adult human kidney were pharmacologically characterized by in vitro autoradiography using the AngII receptor subtype-selective antagonists, losartan and PD 123319, and the sensitivity to the reducing agent, dithiothreitol.
• 2 High densities of AngII AT₁ receptor binding occur in the glomeruli and the inner stripe of the outer medulla, while a moderate AT₁ receptor binding is localized in the proximal convoluted tubules.
• 3 AT₂ receptor binding is observed predominantly in the intrarenal large blood vessels, including the arcuate, inter- and intra-lobular arteries, and in the renal capsule.
• 4 In the major renal artery, AT₁ receptor binding is abundant in the media and adventitia, while AT₂ receptor binding is observed mainly in the adventitia.
• 5 At the light microscopic level using emulsion autoradiography, AT₁ receptors are localized in the glomeruli and juxtaglomerular apparatus, as expected. However, in larger renal blood vessels, including the arcuate arteries, inter- and intra-lobular arteries, intense AT₂ receptor labelling occurs primarily in the adventitia, while the endothelium and vascular smooth muscle layers contain only low levels of AngII receptor binding.
• 6 These results indicate that the adult human kidney displays two pharmacologically distinct AngII receptor subtypes, with AT¹ predominating in the glomeruli, juxtaglomerular apparatus, proximal tubules and the inner stripe of the outer medulla, while AT₂ predominates in the adventitia of the arcuate and interlobular arteries and the renal capsule. The functional significance of AT₂ receptor binding sites in the adventitia of adult human kidney vessels remains to be elucidated.

     

Garlic (Allium sativum) down-regulates the expression of angiotensin II AT1 receptor in adrenal and renal tissues of streptozotocin-induced diabetic rats

The up-regulation of angiotensin II AT₁ receptors has been implicated as a major mediator in the development of hypertension and progressive nephropathy in experimental diabetes. In spite of the documented potential of garlic treatments in ameliorating diabetic complications, the possible involvement of the angiotensin II AT₁ receptor, as a central target for the anti-diabetic potential of garlic, has not been explored. Three groups of rats were studied after 8 weeks following diabetes induction: normal, streptozotocin-induced diabetic (control diabetic), and garlic-treated diabetic rats. A polyclonal antibody of proven specificity to the AT₁ receptor, as verified by western blotting, indicated in immunohistochemical assays that AT₁ receptor labeling was significantly increased in adrenal and renal tissues of control diabetic rats compared to the normal group. The increased AT₁ receptor labeling involved all cortical zones and medullary chromaffin cells of the adrenal gland. Except for glomerulii, increased AT₁ receptor labeling was also evident in proximal convoluted tubules in the renal cortex, and all tubular segments and interstitial cells outlining the vasa recta bundles in the inner stripe of the outer renal medulla. Compared with control diabetic rats, the labeling of the AT₁ receptor in the garlic-treated diabetic group was significantly reduced throughout adrenal and renal regions to levels comparable to those observed in normal rats. The capacity of garlic to modulate diabetes-induced AT₁ receptor up-regulation may be implicated in reversing the detrimental consequences of excessive Ang II signaling, manifested by the development of hypertension and nephropathy.     

Differential expression of renal adenosine A(1) receptors induced by acute renal failure

The distribution of renal adenosine A(1) receptors was investigated in rats with glycerol- or mercuric chloride (HgCl(2))-induced acute renal failure. Receptors were localised by autoradiography using [(3)H]8-cyclopentyl-1,3-dipropylxanthine ([(3)H]DPCPX), a selective A(1) adenosine receptor antagonist. In saline-injected control animals, significant labelling with [(3)H]DPCPX was detected in glomeruli, the inner stripe of outer medulla, and the inner medulla. Sixteen hours following induction of glycerol-induced acute renal failure (ARF), a 34% increase in labelling in glomeruli was noted compared to saline-injected controls, and by 48 hr, glomerular labelling had increased by 200%. In addition, 48 hr following glycerol injection, significant labelling was now detected in the cortical labyrinth and medullary rays whilst, in the inner medulla, labelling had decreased by 34%. By contrast to glycerol-induced ARF, the only significant change noted 48 hr following induction of HgCl(2)-induced ARF was a 39% decrease in labelling in the inner medulla. It is concluded that glycerol-induced ARF results in differential expression of renal adenosine A(1) receptors with increased expression in the cortex and reduced expression in the inner medulla. Increased density of A(1) receptors in glomeruli may account, at least in part, for the increased renal vasoconstrictor response to adenosine and depressed glomerular filtration rate noted previously in this type of acute renal failure.     

Localization of Epidermal Growth Factor (EGF) Binding Sites on Antiluminal Plasma Membrane of Rat Kidney: Autoradiographic Study Using Nonfiltering Perfused Rat Kidney

We previously demonstrated that the specific binding of EGF to the antiluminal plasma membrane was a prerequisite step for the renal uptake of EGF. In the present study, the localization of ¹²⁵I-EGF binding sites on the antiluminal plasma membrane was investigated by tissue sampling and X-ray autoradiography in the nonfiltering kidney. The binding of ¹²⁵I-EGF was recognized over the whole kidney and was highest in the inner medulla followed by the cortex and outer medulla. The binding of ¹²⁵I-EGF in the nonfiltering kidney was completely inhibited in the presence of 20 nM unlabeled EGF, suggesting specific binding of ¹²⁵I-EGF to its receptor. Further, we used a histologic tissue staining method to confirm the location of the ¹²⁵I-EGF binding sites. Binding of ¹²⁵I-EGF was demonstrated on the proximal straight tubules (PST), cortical collecting ducts (CCD), inner medullary collecting ducts (IMCD), and thin limb of Henle in the inner medulla (IMTLH). We found that the binding of ¹²⁵I-EGF was high in the IMTLH. In addition, we determined the grain density both on the cell surface membrane and in the intracellular space of the proximal straight tubules, where the grain density on the antiluminal plasma membrane was approximately 50% that in the intracellular space at 20 min after the start of ¹²⁵I-EGF perfusion, suggesting the internalization of ¹²⁵I-EGF from the antiluminal plasma membrane to the intracellular compartment. In conclusion, the binding sites of ¹²⁵ I-EGF, which were accessible from the antiluminal side, were broadly distributed over the whole kidney and were most dense around the IMTLH.     

Yohimbine-induced alterations in alpha(2)-adrenoceptors in kidney regions of the spontaneously hypertensive rats: an autoradiographic analysis

We have tested the hypothesis that a pharmacologically determined alteration in renal alpha(2)-adrenoceptor (alpha(2)-AR) density might be a pathophysiologically important factor of genetic hypertension in the spontaneously hypertensive rats (SHRs). First, we compared he regional distribution and biochemical parameters of alpha(2)-ARs in SHRs and Wistar-Kyoto (WKY) rats, using the full agonist [(3)H]UK 14304. Secondly, we evaluated the effect of selective blockade and stimulation of alpha(2)-ARs on the development of hypertension and on renal alpha(2)-AR density and regional distribution in SHRs. [(3)H]UK 14304 binding was distributed predominantly over the outer medulla, less abundantly over the inner medulla and was almost absent from the renal cortex. Renal alpha(2)-ARs were found to be increased in SHRs at the ages tested compared with their respective controls and the increase was completely localized to the outer medulla. In these rats, blood pressures immediately before sacrifice were significantly higher in the hypertensive group compared with normotensive controls. The daily administration of SK&F 86466 or clonidine significantly decreased the blood pressure but the autoradiographic studies showed that the prolonged administration of yohimbine to rats for two weeks resulted in a large increase in the density of alpha(2)-ARs in some areas of the rat kidney but not in others. Taken together these data do not support the hypothesis that alteration in renal alpha(2)-ARs (as measured by autoradiography) is crucial for the maintenance of hypertension in the SHR model.     

Metabolic oxidation of acetaminophen (Paracetamol) mediated by cytochrome P-450 mixed-function oxidase and prostaglandin endoperoxide synthetase in rabbit kidney

Long-term abuse of acetaminophen, in combination with other antipyretic analgesics, is thought to be responsible for papillary necrosis in analgesic nephropathy. Cytochrome P-450-mediated oxidative metabolism requiring NADPH and O₂ has been believed, to date, to be the sole pathway for the toxic metabolic activation of acetaminophen. Using microsomes from different regions of rabbit kidney, protein covalent binding of acetaminophen by NADPH-dependent metabolism was highest in cortex, less in outer medulla, and minimal in inner medulla. In vivo studies have shown that the highest binding of acetaminophen occurred in renal inner medulla compared to liver and renal cortex. However, cytochrome P-450 could not be detected in rabbit renal inner medulla. Hence, another metabolic pathway for the activation of acetaminophen was presumed to be operative in renal inner medulla. This alternate pathway was recognized as cooxidation of acetaminophen mediated by prostaglandin endoperoxide synthetase, requiring arachidonic acid as well as O₂, and was found to be active predominantly in renal inner medulla. Glutathione, ascorbic acid, and ethoxyquin inhibited protein covalent binding of acetaminophen arising from both pathways. Indomethacin and aspirin inhibited only the arachidonic acid-dependent cooxidation of acetaminophen. Butylated hydroxyanisole inhibited both NADPH- and arachidonic acid-dependent metabolism, the latter more effectively. Arachidonic acid-dependent metabolism of acetaminophen is probably mediated by the hydroperoxidase activity of prostaglandin endoperoxide synthetase. This alternate pathway could be a significant contributing factor for the genesis of papillary necrosis, as manifested in analgesic nephropathy.