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(Hypertension. 1997;30:455.)
© 1997 American Heart Association, Inc.
Articles |
Increased Density of Renal Amylin Binding Sites in Experimental Hypertension
From the Department of Medicine, University of Melbourne (P.J.W., Z.C., R.C.I. van G., M.V., M.E.C.), and the Wound Foundation of Australia (I.A.D.), Austin and Repatriation Medical Centre, Repatriation Campus, Victoria, Australia; and the Institute for Clinical and Experimental Medicine, Prague, Czech Republic (R.K.).
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Abstract |
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Abstract High-affinity binding sites for the pancreatic ß-cell hormone amylin have been reported in the kidney, and it has been postulated that these sites may be involved in the genesis of hypertension. In the present study, we have used in vivo injection of 125I-amylin and in vitro autoradiographic techniques to assess renal amylin binding in both a genetic and a surgically induced model of hypertension. In the spontaneously hypertensive rat (SHR) at 6 weeks of age, before the rise in systolic blood pressure, there was a 36% increase in density of amylin binding compared with their normotensive counterpart, the Wistar-Kyoto rat (WKY). In SHR, there was a further increase in the density of amylin binding (to 53% greater) as the systolic blood pressure rose between 6 and 12 weeks of age. Histological examination of kidneys from SHR at 12 weeks of age revealed staining for a brush border glycoprotein, normally restricted to the proximal tubules, extending from the urinary pole into half of the epithelial lining of the glomerular capsule. In contrast to WKY, these cells also bound 125I-amylin with high density in SHR. In a rat model of renal ablation and hypertension, systolic blood pressure correlated with the density of 125I-amylin binding in the renal cortex (r=.54, P=.003, n=28). The changes in amylin binding reported here suggest a possible role for this peptide and/or activation of its receptor in the genesis as well as the maintenance of hypertension.
Key Words: receptors • kidney tubules, proximal • ablation, renal • rats, inbred SHR
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Introduction |
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Amylin is a 37–amino acid polypeptide1 2 that is cosecreted with insulin3 4 from pancreatic ß-cells. We have recently proposed that amylin is an important hormone, with novel functions in renal physiology,5 6 7 although it is also thought to be an endocrine regulator of carbohydrate8 9 10 and bone metabolism.11 12 The involvement of amylin with functions in the kidney is based on five key findings. First, we have identified high-affinity amylin binding sites in renal cortex that can be distinguished from those of the homologues calcitonin and calcitonin gene-related peptide on the basis of regional distribution in kidney tissue sections and pharmacological characterization of the binding site with peptide antagonists.5 The inhibition of 125I-amylin binding by the hydrolysis-resistant nucleotide GTP
S suggests the receptor is a member of
that superfamily coupled to G proteins.5 Second, using in
vivo injection of 125I-amylin, we have demonstrated that
amylin binding sites are located on proximal tubules rather than distal
tubules, collecting ducts, interstitium, or glomeruli.6
Third, amylin, when administered to human volunteers13 14
or rats,5 14 stimulated plasma renin activity fivefold and
twofold, respectively. Fourth, in micropuncture experiments, amylin was
a potent stimulant of sodium/water reabsorption, mediated by the
Na+/H+ exchanger of the proximal
tubules.6 Finally, amylin acts as a mitogen, stimulating
hyperplasia of epithelial cells isolated from proximal tubules and
cultured in vitro.6 These potent effects of amylin
suggested it and its receptor(s) may have important
physiological links with the
renin-angiotensin system and may have a role in
sodium/water homeostasis and tubular growth. These discoveries and the
reported proposal15 16 that amylin may be involved in
hypertension associated with type II diabetes suggested to us that
amylin and its renal receptor may be involved in the development of
hypertension generally. For these reasons we decided to investigate the
putative role of amylin and its receptors by using two rat models of
hypertension: a genetic model, the SHR, and a surgical counterpart, the
5/6 Nx model of renal ablation. |
Methods |
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The handling of animals and surgical procedures were in accordance with the guidelines set down by the National Health and Medical Research Council in Australia. In this study, male Sprague-Dawley rats, WKY, and SHR aged 6 and 12 weeks were used. Animals were anesthetized with 60 mg/kg (IP) pentabarbitone.
The operation of renal ablation, using male Sprague-Dawley rats, aged 10 weeks, weighing 280 to 300 g, involved uninephrectomy followed by subtotal ablation of the remaining kidney by ligation of all but one of the segmental renal arterial branches.17 Initially this procedure resulted in approximately two-thirds ablation of this kidney. However, 2 weeks postoperation, extensive hyperplasia of the healthy, nonischemic portion led to regrowth, such that this portion now constituted greater than two thirds the volume of the remaining kidney.18 In addition, at this time postoperation, the animals displayed a range of SBP between 125 mm Hg (<160 mm Hg is considered normotensive; mean=137±3 mm Hg, n=11) and 210 mm Hg (>160 mm Hg is considered hypertensive; mean= 178±4 mm Hg, n=17), while the mean SBP of the shams remained unchanged at 122±3 mm Hg (n=12). The reason for the range of SBPs found in the 5/6 Nx animals remains unexplained; however, it has proved to be a useful tool for this research, particularly as these animals are of the same genetic stock. SBP was measured weekly by tail-cuff plethysmography in prewarmed, unanesthetized, slightly restrained animals19 and the mean SBP calculated. The SBP of WKY and SHR was measured just before they were killed.
Rat amylin was from Bachem. 125I-Rat amylin was iodinated (Amersham) at the N-terminal lysine (specific activity >2000 Ci/mmol) using the Bolton and Hunter reagent. The tracer was purified by reverse-phase high-performance liquid chromatography.
In Vitro Autoradiography
Binding studies, using dry-film autoradiography,
were performed as described previously.5 To determine the
density of binding, five slides, each with two frozen sections per
kidney (animal), were incubated with 60 pmol/L
125I-amylin (for total binding), and for nonspecific
binding, together with 1 µmol/L unlabeled peptide. The
density of tracer binding (dpm/mm2) was determined in the
cortical areas, as developed on the X-ray film, and analyzed by
using a computer-aided image analysis system (MCID, Imaging
Research).
Cellular Localization of Binding Sites
The methods involved in vivo infusion of 125I-amylin
into the descending aorta of anesthetized rats, followed by
perfusion under pressure with 2.5% glutaraldehyde,
postfixation and blocking in paraffin before thin sectioning (4
µm), emulsion autoradiography, and light microscopy,
as previously described.6 For emulsion
autoradiography,20 the first section of
each series was dipped in Ilford Nuclear Research Emulsion K5 (Ilford
Limited). The identification of the proximal tubules was performed on
the second serial section by using a lectin stain (Phaseolus Vulgaris
PHA-E, Sigma Chemical Company) at a concentration of 1 µg/mL,
a horseradish-peroxidase color development system, and light
microscopy.21 After the lectin stain and before the
staining with hematoxylin, the emulsion was applied.
Statistical Methods
Data were analyzed by ANOVA, using Statview SE
(Brainpower) on a Macintosh Computer. Correlations were derived by
using the Statview SE and Graphics program (Abacus Concepts Inc).
Comparisons of group means were performed by Fisher’s least
significant difference method. Data were shown as mean±SE, and a value
of P<.05 was viewed as statistically significant.
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Results |
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In the SHR and WKY inbred strains, the density of 125I-amylin binding sites was measured by in vitro autoradiography. It was shown that at 6 weeks of age, before the onset of hypertension in SHR (Fig 1A), the density of amylin binding in the renal cortex was 36% greater than in WKY (102±7 dpm/mm2 versus 75±9 dpm/mm2; Fig 1B) despite similar SBPs between the two groups. This density increased further over the next 6 weeks as the SBP of SHR rose into the hypertensive range (Fig 1) to a value 53% greater in SHR (123±8 dpm/mm2) than in WKY (80±10 dpm/mm2) at 12 weeks of age. In WKY, the density of amylin binding remained unchanged over this period.
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The cellular localization of 125I-amylin binding sites in SHR and WKY at 12 weeks of age was determined by in vivo injection of 125I-amylin, as shown in Fig 2. The tissue sections were also stained with a lectin/peroxidase immunohistological stain that detects a glycoprotein associated with brush border epithelial cells. Microscopic examination revealed that silver grains were associated with proximal tubules in WKY. By contrast, in SHR, high density of 125I-amylin binding sites was associated with brush border epithelial cells that extended into approximately 50% of the capsular lining from the urinary pole of Bowman’s capsule (Fig 2). The lectin staining in cells lining the urinary pole of the glomeruli was not observed in WKY (data not shown).
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In the 5/6 Nx model, representative images of the total and nonspecific binding for 125I-amylin by in vitro autoradiography5 to 20-µm longitudinal sections of kidneys from the sham, normotensive, and hypertensive groups are shown in Fig 3. When the specific (total minus nonspecific) binding densities for all animals were measured, the normotensive group displayed a lower density of binding for 125I-amylin (19.3±4.5 dpm/mm2, n=11) than the hypertensive group (54.9±5.6 dpm/mm2, n=17) and sham group (60.3±4.1 dpm/mm2, n=12). It is worth noting that in the ischemic tissue (the dark renal poles shown in Fig 3), the density of the nonspecific binding of 125I-amylin was greatly increased and suggested that it represented a low-affinity binding site.
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Of particular interest was the significant positive relationship between the density of amylin binding and SBP in the 5/6 Nx rats. When the binding densities of 125I-amylin were plotted against the mean SBP (Fig 4), a significant positive relationship could be demonstrated (r=.54, P=.003) within the population of 5/6 Nx rats (n=28).
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Infusion of 125I-amylin into representative kidneys of the hypertensive, normotensive, and sham groups in vivo demonstrated that binding sites were associated with the proximal tubules rather than distal convoluted tubules, collecting ducts, interstitium, or glomeruli.6 Furthermore, when the location of the 125I-amylin binding sites was assessed in the representatives of the hypertensive group, a similar proximal tubular location was observed (Fig 5), albeit at higher density.
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Discussion |
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We have demonstrated that in two models of hypertension, one genetic (SHR) and the other created surgically by subtotal nephrectomy (5/6 Nx), there is significant correlation between an increase in the density of amylin binding sites in the renal cortex and the rise in SBP. These findings provide further evidence for a possible role for amylin and/or its receptor in the development of hypertension.
When the SHR group was compared with the WKY group, increased density of amylin binding sites in the cortex, as measured by autoradiography in vitro, was evident before and increased further after the onset of systemic hypertension. Thus, increased amylin binding in the renal cortex was apparent by 6 weeks of age, before the rise in SBP begins in SHR. There was a further rise in the density of amylin binding coincident with the rise in SBP between the ages of 6 and 12 weeks.
The mechanisms by which activation of the amylin system in either model leads to hypertension require elucidation; however, the following observations may provide a basis for further research. The amylin binding site represents a putative receptor of the G protein– coupled class.5 The formation of the active ternary complex in the absence of the natural ligand22 23 and activation of second-messenger systems, which modulate cellular functions such as the Na+/H+ exchanger,6 has been noted in different biological systems. In vitro autoradiography demonstrated an increased density of amylin binding throughout the renal cortex, which may result from sensitization of the amylin receptor. This sensitization might arise from decreased exposure to the natural ligand or spontaneous activation of the receptor, as has been documented for other receptors of the G protein–coupled class.22 23
The normal route through which amylin activates its receptor appears to involve penetration of the endothelial lining of the capillaries, since amylin stimulates sodium/water reabsorption from the basolateral, peritubular side rather than the luminal side of the epithelial cells.6 In SHR, there is evidence of afferent vasoconstriction24 25 and endothelial dysfunction.26 27 28 Thus, amylin and other hormones active on the peritubular side of proximal epithelia may not be reaching their receptor targets in significant amounts. The activation and desensitization of the amylin receptors might therefore result from limited access of the ligand due to the hemodynamic abnormalities observed in SHR.
Using in vivo injection of 125I-amylin, emulsion autoradiography, and microscopy, a striking feature of SHR was evident that was not present in WKY (Fig 2) or Sprague-Dawley rats.6 That is, brush border epithelial cells (lectin positive, Fig 2) extended from the proximal tubules into half of the urinary pole of the glomerular capsule. Furthermore, these brush border epithelia bound high densities of 125I-amylin. In both normotensive strains, WKY and Sprague-Dawley (Fig 5 and Reference 66 ), 125I-amylin was found associated exclusively with proximal tubules. There are no apparent lectin-positive cells lining the glomeruli of either normotensive strain (data not shown).
Clearly, it is important in the future to define more precisely the time of onset of anatomical changes as described in the glomerulus of SHR (Fig 2). The atypical appearance of brush border epithelial cells lining the urinary pole of Bowman’s capsule, which appear to express high levels of amylin binding sites, may have arisen from an abnormal induction of these epithelial cells during the development of the kidney or perhaps later during renal growth before 12 weeks of age. The functional significance of this abnormal anatomy is yet to be established, as is its contribution to the renal pathology of SHR.
In the case of the 5/6 Nx model, we found a positive and significant correlation between SBP and the density of 125I-amylin binding when the 125I-amylin binding was assessed in tissue slices incubated with tracer in vitro (Fig 4). When 125I-amylin was injected into the descending aorta in vivo, the density of silver grains in the emulsions was greater in representatives of the hypertensive group than those of the normotensive group (Fig 5), thus corroborating the result found by in vitro autoradiography. In both groups, the silver grains were associated only with proximal tubules, as we have previously reported for Sprague-Dawley rats.6
The reason for the range of blood pressures from 125 to 215 mm Hg in the 5/6 Nx animals 2 weeks after subtotal renal ablation is unclear. We and others18 have measured hyperplasia and found no significant difference between the groups of normotensives or hypertensives (data not shown). The numbers of proximal tubules evident in the kidneys of the normotensive and hypertensive groups appeared similar (data not shown). Therefore, the difference in the SBPs among the 5/6 Nx rats did not appear to be a function of hyperplasia or the proportion of tubules with brush borders. However, tubular dilation is an early event29 in the kidneys of the hypertensive but not the normotensive group (Fig 3), suggesting some alteration in tubular functions in the former. In this period postoperation, glomerular sclerosis and interstitial fibrosis are negligible (Fig 3 and Reference 2929 ). In the hypertensive group and in sham animals, amylin injected in vivo or introduced by incubation in vitro binds with equal density, whereas by both techniques, densities are much reduced in the normotensive group. In the latter case, this may be explained by desensitization of the amylin receptors brought about either by overexposure to the ligand or some other mechanism that inhibits the desensitization/sensitization cycle of the receptor. This explanation seems plausible in the light of our initial finding that an iodinated antagonist of the amylin receptor bound equally well in both hypertensive and normotensive groups (data not shown). Such binding only requires the desensitized state of G protein–coupled receptors.30 31 32 The important point to emphasize is that the inactivity or desensitization of the amylin receptor correlates with normotension in the 5/6 Nx model, whereas in SHR, the amylin receptor appears sensitized, which coincides with hypertension in both models.
In these assays, we have measured the density of sensitized amylin binding sites, but we are unable at this stage to report the overall activity of the amylin/amylin receptor/second-messenger system. We would hypothesize that although the density of the amylin binding sites is similar in the hypertensives and shams, the activity in the former is greatly elevated. It is possible that the putative G protein receptor is uncoupled as mentioned above,22 23 resulting in overactivity of the Na+/H+ exchanger in the case of the hypertensive rats. It has been demonstrated by familial analysis of Liddle’s syndrome that mutations of the Na+/H+ exchanger result in constitutive activation and hypertension.33 Thus, in principle, constitutive activation of the Na+/H+ exchanger as a result of activation of the amylin system would also promote hypertension. The amylin/amylin receptor system requires further research and definition to explore these possibilities.
Our findings suggest a causal relationship between activity of the amylin receptor and hypertension in two animal models. We have described here that increasing density of amylin receptors (presumably via a mechanism of sensitization) in SHR precedes the onset of hypertension and therefore may be considered to play a role in its development. We have also demonstrated elsewhere6 that amylin is a potent stimulant of sodium/water reabsorption from proximal tubules, and this factor was shown to be dependent on the activity of the Na+/H+ exchanger present on the luminal side of the tubular brush border epithelial cells. It has been hypothesized, and there is growing evidence,33 that a characteristic of essential hypertension is overactivity of the Na+/H+ antiporter. This has also been measured in hypertensive patients in which platelet Na+/H+ exchange activity was markedly elevated.34 A large proportion of these individuals had altered properties leading to salt retention.34 Furthermore, we have shown that in male volunteers, infused amylin stimulates plasma renin concentration fivefold8 and leads to significant increase in aldosterone levels, which results in salt retention.35 In hypertensive subjects, amylin levels have been reported to be significantly elevated.36 Possible mechanisms whereby amylin may be involved in the genesis of hypertension include activation of the renin-angiotensin system, either directly or indirectly, and promotion of sodium retention via stimulation of proximal tubular ion transport.6 Our results suggest that the activation of the amylin system (hyperamylinemia or activation of the renal receptor) is an important component in the development of hypertension. These considerations may be particularly relevant in states of hyperamylinemia and insulin resistance, such as obesity and glucose intolerance.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Australian Kidney Foundation, the Sir Edward Dunlop Foundation, the Austin Hospital Medical Research Foundation, the Rebecca Cooper Foundation, the Kidney Foundation of Holland for support of RCIvG and MV, and Amylin Pharmaceuticals Inc for provision of the peptides. We would also like to thank Julian Oldmeadow for technical assistance.
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Footnotes |
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Reprint requests to Dr P.J. Wookey, Department of Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Repatriation Campus, Heidelberg West, Victoria 3081, Australia.
Received December 9, 1996; first decision December 31, 1996; accepted February 13, 1997.
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References |
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