(Hypertension. 2000;36:569.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From the Division of Nephrology, VA Palo Alto Health Care System and Stanford University, Palo Alto, Calif (M.S., T.W.M.), and Department of Pathology, University of California, San Francisco (J.L.O.).
Correspondence to Timothy W. Meyer, MD, Nephrology 111R, VA Palo Alto HCS, 3801 Miranda Ave, Palo Alto, CA 94304. E-mail twmeyer{at}leland.stanford.edu
| Abstract |
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Key Words: hypertension, experimental mineralocorticoids angiotensin glomerular filtration rate interstitium
| Introduction |
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| Methods |
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At 8 weeks, rats were anesthetized with Inactin, 100 mg/kg IP, and subjected to studies of kidney function and structure. Glomerular filtration rate was measured over two 30-minute clearance periods in animals maintained euvolemic by infusion of saline and rat plasma. After the clearance measurements, the effectiveness of Ang II receptor blockade and converting enzyme inhibition was confirmed by assessing the pressor responses to 50-ng bolus infusions of angiotensin I (Ang I) and Ang II, and blood samples were collected for determination of plasma sodium and potassium concentrations. Kidneys were then fixed by retrograde aortic perfusion and weighed. Transverse kidney slices were embedded in paraffin, and sections were stained with hematoxylin and eosin and with the periodic acidSchiff technique. The prevalences of segmental glomerular lesions and glomerular microaneurysms were determined by examining all glomerular profiles in a single section from each animal (average, 150±5 profiles). Segmental lesions were most often characterized by areas of the tuft showing collapse of the glomerular capillaries, accompanied by hyalinosis and focal adhesion of the tuft to the Bowmans capsule. In addition, some glomeruli showed prominent distention of capillary loops by proteinaceous material. Microaneurysms were defined by the presence of mesangiolysis with extreme dilatation of glomerular capillary loops containing fibrin, erythrocytes, and other cellular elements.
Average values for the glomerular tuft volume and the fractional volume of cortical interstitium in each animal were determined as previously described.6 The extent of vascular injury was assessed by examining profiles of the arteries and arterioles in a single kidney section from each animal. Each profile was first categorized according to whether its shortest external diameter measured <50, 50 to 100, or >100 µm. Profiles exhibiting fibrinoid necrosis characterized by infiltration of the wall with amorphous, eosinophilic material or thrombotic microangiopathy characterized by fibrin-platelet thrombi and red cell fragments were further categorized as showing overt wall injury. The prevalence of wall injury was expressed as the percentage of vessel profiles showing these changes. Vascular wall thickness was then measured by a modification of the procedure described by Whitworth et al.7 Wall thickness was measured only in vascular profiles that did not show overt injury. For each profile, the total and luminal areas were measured with a computer-assisted morphometric unit. The ratio of the wall area to luminal area was then calculated as the ratio of the total area minus the luminal area to the luminal area. Vessels that appeared to have been cut very obliquely, as evidenced by a ratio of the longest to shortest external diameter of >5:1, were excluded from this analysis.
ANOVA and Fishers probability of least significant difference were used to assess the significance of differences between the groups. Significance was defined as P<0.05, and results are expressed as mean±SE throughout. The animal protocol was approved by the institutional review board.
| Results |
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Kidney function studies at 8 weeks are summarized in Table 1. Mineralocorticoid-salt hypertension was associated with impaired growth, so that body weight was less in groups 2, 3, and 4 than in group 1. Values for hematocrit and plasma protein concentration were similar in all 4 groups. The groups receiving aldosterone exhibited a slight increase in plasma sodium concentration without a significant reduction in plasma potassium concentration. Values for mean arterial pressure under anesthesia paralleled values for systolic blood pressure obtained in awake animals. The mean arterial pressure of 110±3 mm Hg in group 1 was similar to the value we have observed in normal rats. Chronic aldosterone infusion increased mean arterial pressure to 148±3 mm Hg in group 2. Neither Ang II receptor blockade nor converting enzyme inhibition altered mean arterial pressure, which averaged 145±2 mm Hg in group 3 and 143±2 mm Hg in group 4. The glomerular filtration rate in normotensive group 1 rats averaged 2.36±0.13 mL/min. The average glomerular filtration rate was reduced in each of the hypertensive groups but was not affected by drug treatment, averaging 1.95±0.11 mL/min in group 2, 1.86±0.12 mL/min in group 3, and 1.76±0.12 mL/min in group 4.
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Structural findings at 8 weeks are summarized in Table 2 and depicted in Figures 2 through 5. Significant structural abnormalities were not observed in group 1. Mineralocorticoid-salt hypertension, however, was associated with prominent structural changes in groups 2, 3, and 4. Kidney weight was increased in each of these groups. Thickening of the vascular wall was observed at all levels of the arterial tree. Morphometric studies showed an increase in the wall to lumen area ratio that was most prominent in small arteries and arterioles. The diffuse increase in wall thickness was accompanied by focal wall injury. Wall injury was characterized by the appearance of fibrinoid necrosis and thrombotic microangiopathy (Figure 2). Both features of injury were more common in smaller vessels. Neither diffuse wall thickening nor focal wall injury was affected by Ang II receptor blockade in group 3 or converting enzyme inhibition in group 4.
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Vascular injury was accompanied by glomerular injury in each of the hypertensive groups. Average glomerular volume was increased from 1.70±0.09x106 µm3 in group 1 rats subjected only to uninephrectomy to 2.23±0.13x106 µm3 in group 2 rats with mineralocorticoid-salt hypertension. Glomerular volume was not affected by Ang II receptor blockade or converting enzyme inhibition. The majority of glomeruli appeared normal, but a significant number exhibited segmental lesions in each of the hypertensive groups. Injured glomeruli most often showed segmental sclerosis characterized by collapse of capillary loops and adhesion of the tuft to Bowmans capsule (Figure 3). Segmental distention of capillary loops by proteinaceous material associated with decreased glomerular cellularity was also observed (Figure 4). These lesions sometimes involved the majority of the tuft. Visceral epithelial cells showed severe injury accompanied by denudation of the glomerular basement membrane. Some glomerular capillary loops showed fibrin thrombi and fragmented red cells characteristic of thrombotic microangiopathy. Occasional glomeruli contained microaneurysms characterized by mesangiolysis and extreme dilatation of glomerular capillary loops containing fibrin, erythrocytes, and other cellular elements (Figure 5). Microaneurysms often filled Bowmans space and appeared in many cases to have ruptured. Other glomeruli showed an appearance suggestive of ischemia characterized by wrinkling of capillary loops and collapse of capillary lumina. Glomerular injury, like vascular injury, was unaffected by Ang II receptor blockade and converting enzyme inhibition.
Glomerular and vascular injury in hypertensive rats was accompanied by patchy tubulointerstitial injury characterized by tubule epithelial cell damage, a chronic inflammatory infiltrate, and interstitial edema and fibrosis. Scattered tubule casts were also observed. The development of tubulointerstitial injury was reflected by an increase in the interstitial volume fraction from 15±1% in normotensive group 1 rats to 20±1% in group 2 rats with mineralocorticoid-salt hypertension. This change, like the other features of injury examined, was not affected by Ang II receptor blockade or converting enzyme inhibition.
The effectiveness of Ang II receptor blockade and converting enzyme inhibition was confirmed by assessing the pressor responses to intravenous bolus infusions of Ang I and Ang II in a subset of animals (n=5 to 9 in each group). The pressor response to 50 ng of Ang I was 49±5 mm Hg in group 1, 45±3 mm Hg in group 2, 4±2 mm Hg in group 3 (P<0.05 versus group 1 and 2), and 5±2 mm Hg in group 4 (P<0.05 versus group 1 and 2). The pressor response to 50 ng of Ang II was 52±3 mm Hg in group 1, 41±3 mm Hg in group 2, 4±1 in group 3 (P<0.05 versus groups 1, 2 and 4), and 56±5 mm Hg in group 4. Thus, both enalapril and losartan reduced the pressor response to Ang I, while losartan reduced the pressor response to Ang II.
| Discussion |
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The first finding of this study was that agents that reduce Ang II activity did not limit vascular injury in rats with mineralocorticoid-salt hypertension. As expected, hypertension was accompanied by an increase in the wall to lumen area ratio throughout the renal arterial tree. Two lines of evidence have suggested that Ang II can contribute to this structural change. First, Ang II has been shown to cause vascular smooth muscle cells to grow and produce matrix components.12 Second, mechanical strain has been shown to potentiate the local effects of Ang II produced within the vascular wall.13 14 These findings suggest that hypertension can promote vessel wall thickening by increasing local Ang II activity. We found, however, that losartan and enalapril did not reduce wall to lumen ratios in rats with mineralocorticoid-salt hypertension. It thus appears that a strain-induced increase in local Ang II activity does not contribute significantly to vessel wall thickening in this setting. In addition to generalized arterial wall thickening, hypertension was accompanied by the appearance of fibrinoid necrosis and thrombotic microangiopathy. The pathogenesis of these forms of vascular injury, which are commonly observed in severe hypertension, is not fully understood.1 Ang II has been considered a potential contributor to fibrinoid necrosis, however, because it increases endothelial permeability to plasma proteins.1 15 Ang II has likewise been identified as a potential contributor to thrombotic injury because it increases endothelial expression of plasminogen activator inhibitor.16 We found, however, that losartan and enalapril did not limit the extent of fibrinoid necrosis or thrombotic microangiopathy in rats with mineralocorticoid-salt hypertension.
The second finding of this study was that agents that reduce Ang II activity did not prevent glomerular injury in mineralocorticoid-salt hypertension. The development of glomerular injury was manifested by increasing proteinuria, which was not affected by treatment with losartan or enalapril. Morphological examination revealed glomerular segmental sclerosis similar to that seen in other models of hypertension.17 Studies analogous to those performed in arteries and smooth muscle cells have suggested that local Ang II action can contribute to this type of injury. Like smooth muscle cells, mesangial cells not only contract but grow and produce matrix components when exposed to Ang II.3 18 Studies in rats subjected to renal ablation have identified increased renin and angiotensinogen production in remnant glomeruli.19 20 We found, however, that losartan and enalapril did not reduce the extent of glomerular sclerosis in mineralocorticoid-salt hypertension. Losartan and enalapril also did not reduce microaneurysm formation, which has been identified as a possible precursor to glomerular sclerosis in mineralocorticoid-salt hypertension.8 The finding that losartan and enalapril did not affect these forms of glomerular injury suggests that local Ang II production was not important in their pathogenesis. In accord with previous reports, rats with mineralocorticoid-salt hypertension had larger kidneys and larger glomeruli than rats subjected to uninephrectomy alone.8 21 These interesting hypertrophic changes have not been associated with an increase in glomerular filtration rate, and their cause remains obscure. The present study found that glomerular size and kidney size were not effected by losartan or enalapril. Evidence from other disease models suggests that the increase in glomerular volume may have contributed to the development of glomerular sclerosis.22
Finally, the present study found that agents that reduce Ang II activity did not prevent tubulointerstitial injury in mineralocorticoid-salt hypertension. The pathogenesis of tubulointerstitial injury in this and other models of experimental hypertension remains uncertain. It has been suggested, however, that transmission of increased arterial pressure to interstitial capillaries may cause capillary injury resulting in tubular ischemia.23 There is also strong evidence that absorption of filtered proteins can cause tubule injury.24 Tubule injury, whether caused by ischemia or excessive protein absorption, is thought in turn to precipitate interstitial fibrosis. Recent studies have shown that local production of Ang II can contribute to interstitial fibrosis in damaged kidneys. Profibrotic actions of Ang II identified in these studies include stimulation of transforming growth factor-ß expression, mononuclear cell infiltration, and matrix production.4 5 25 The contribution of local Ang II to interstitial fibrosis is difficult to identify in disease models in which Ang II blockade prevents hypertension and/or reduces proteinuria. The strongest evidence that Ang II promotes interstitial fibrosis has thus come from studies showing a beneficial effect of Ang II blockade in models that develop fibrosis in the absence of hypertension and proteinuria, including rats with cyclosporine nephrotoxicity and ureteral obstruction.6 26 The present study, in contrast, examined the effect of Ang II blockade in rats that had severe hypertension and heavy proteinuria. The results showed that local Ang II production was not a major contributor to interstitial injury in this setting.
The present study was prompted in part by a discrepancy in previously reported results. Dworkin et al9 found that converting enzyme inhibition did not reduce blood pressure or proteinuria in rats with mineralocorticoid-salt hypertension. They concluded that failure to reduce proteinuria represented failure to prevent injury and did not examine renal tissue. In contrast, Kim et al10 found that both converting enzyme inhibition and Ang II receptor blockade limited proteinuria in mineralocorticoid-salt hypertension. Lower protein excretion was accompanied by reduced renal expression of matrix constituents. In a follow-up study, Wada et al11 again found that converting enzyme inhibition and Ang II receptor blockade limited proteinuria. Semiquantitative scoring of 12 structural parameters revealed significant, albeit incomplete, protection against injury. Converting enzyme inhibition significantly reduced the score for 1 structural parameter, while Ang II receptor blockade significantly reduced the score for 5.
The dose of enalapril used by Dworkin et al9 and by us exceeded that used by Kim et al10 and Wada et al.11 It should be also be noted that Dworkin et al9 observed no protection against injury when enalapril treatment was begun immediately after initiation of mineralocorticoid treatment. It is unclear why Kim et al10 and Wada et al11 saw some protection against injury, while we and Dworkin et al9 saw none. One possible explanation is that the rats studied by Kim et al10 and Wada et al11 had entered an accelerated phase of hypertension in which renin released in response to initial vascular injury was contributing to further renal damage. The observation of a late rise in plasma renin activity by Wada et al11 is consistent with this possibility. In this regard, it is important to emphasize that the present study should not be taken to suggest that local Ang II production cannot cause renal injury. There is strong evidence that the presence of Ang II speeds the operation of several injurious processes in vitro and in vivo. Blood pressure reduction with Ang II blockade has been shown to prevent injury more effectively than blood pressure reduction without Ang II blockade in several disease models. The hypothesis that increased intrarenal Ang II activity promotes injury when blood pressure is reduced without Ang II blockade provides an attractive explanation for these findings. The present study does show, however, that Ang II is not an essential participant in renal vascular or glomerular injury associated with hypertension and proteinuria. Likewise, the present study shows that interstitial injury can develop independent of Ang II activity in this setting.
| Acknowledgments |
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Received February 17, 2000; first decision March 14, 2000; accepted April 19, 2000.
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