Contribution of Angiotensin II to Late Renal Injury after Acute Ischemia

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1 J Am Soc Nephrol 11: , 2000 Contribution of Angiotensin II to Late Renal Injury after Acute Ischemia MARIA ENRICA PAGTALUNAN,* JEAN L. OLSON, and TIMOTHY W. MEYER* *Department of Medicine, VA Palo Alto Health Care System and Stanford University, Palo Alto, California; and Department of Pathology, University of California, San Francisco, California. Abstract. Rats recovering from acute renal ischemia exhibit tubule loss and interstitial fibrosis followed by development of proteinuria and glomerular sclerosis. The current study assessed the contribution of angiotensin II (AngII) to these processes. The contribution of AngII to early tubule loss and interstitial fibrosis was assessed in rats studied for 35 d after right nephrectomy and transient occlusion of the left renal artery. One group of rats received no treatment, while a second group received losartan beginning at 2 d following ischemia. Studies at 35 d showed that losartan did not improve GFR ( ml/min treated, ml/min untreated), reduce the fraction of glomeruli that were no longer connected to normal tubule segments (42 9% treated, 42 13% untreated), or limit expansion of the interstitial volume fraction (25 3% treated, 25 4% untreated). The contribution of AngII to progressive glomerular injury following initial recovery from ischemia was assessed in similarly prepared rats studied for 140 d. One group of rats received no treatment, while a second group received enalapril beginning at 35 d following ischemia. Enalapril markedly reduced proteinuria (78 17 mg/d treated, mg/d untreated) and the prevalence of segmental glomerular sclerosis (14 9% treated, 45 10% untreated). Untreated rats developed sclerotic lesions in glomeruli not connected to normal tubules, as well as in glomeruli connected to normal tubules. Enalapril prevented injury in both classes of glomeruli. These results indicate that AngII does not contribute to interstitial fibrosis during recovery from ischemic injury. Reduction of AngII activity, can, however, prevent secondary glomerular injury in kidneys initially damaged by ischemia. Chronic renal injury follows acute renal ischemia in rats with a solitary kidney. Recent studies have shown that at least two processes contribute to chronic injury in this setting (1). First, incomplete recovery from ischemia is accompanied by reduction in the number of functioning nephrons and by the appearance of interstitial fibrosis. This initial tubulointerstitial injury is followed by the development of progressive proteinuria and glomerular sclerosis. The current study assessed the contribution of angiotensin II (AngII) to these processes. Studies suggesting that AngII contributes to interstitial fibrosis in a variety of conditions prompted us to study the effects of AngII blockade on tubulointerstitial injury in kidneys recovering from acute ischemia (2 5). We found that AngII blockade did not limit tubule atrophy or interstitial fibrosis in this setting. Studies showing that AngII contributes to remnant glomerular injury after surgical reduction in nephron number prompted us to perform a longer-term study of the effect of AngII blockade on progressive glomerular injury following recovery from acute ischemia (6,7). Previous studies have shown that the course of glomerular injury is somewhat different in these two settings, in that rats recovering from acute ischemia develop Received August 9, Accepted December 1, Correspondence to Dr. Timothy W. Meyer, Palo Alto VA Medical Center, Nephrology 111R, 3801 Miranda Avenue, Palo Alto, CA Phone: , extension 63314; Fax: ; twmeyer@leland.stanford.edu / Journal of the American Society of Nephrology Copyright 2000 by the American Society of Nephrology glomerular injury in the absence of significant hypertension (1). We found that reduction of AngII activity by converting enzyme inhibition was nevertheless very effective in preventing progressive glomerular injury after acute renal ischemia. Materials and Methods Studies at 35 Days Male Sprague Dawley rats weighing 290 to 350 g were anesthetized with thiopental (50 mg/kg intraperitoneally). The right kidney was removed and the left renal artery was occluded for 30 to 35 min. Rats subjected to ischemia were assigned to one of two groups on the basis of serum creatinine measurements at 24 and 48 h after operation. Assignment was made so that group average creatinine values were similar. One group (n 10) then received losartan (180 mg/l) in the drinking water, while the second group (n 8) received no treatment. An additional group (n 8) underwent sham operation and received no treatment. Systolic BP (tail-cuff method), serum creatinine (Beckman Creatinine 2 analyzer; Beckman Instruments, Fullerton, CA), and 24-h urine protein excretion (Coomassie blue) were then assessed periodically over 35 d, during which rats had free access to water and standard laboratory chow. Clearance and morphologic studies were performed at the end of this period. Morphologic studies were performed in only four of the eight sham-operated rats. Studies at 140 Days Male Sprague Dawley rats underwent right nephrectomy and ischemic injury to the left kidney as described above. They then received no treatment for 35 d before being divided into two groups on the basis of serum creatinine measurements. Assignment was made so that group average creatinine values measured on days 1, 5, 20, and 35 after operation were similar. One group (n 7) then began treatment

2 J Am Soc Nephrol 11: , 2000 Late Renal Injury after Acute Ischemia 1279 with enalapril (150 mg/l) in the drinking water, while the other group (n 8) received no treatment. Clearance and morphologic studies were performed at 105 d after beginning treatment (140 d after initial injury). Systolic BP, serum creatinine, and 24-h urine protein excretion were assessed periodically over the study period. Functional Studies Rats were anesthetized with Inactin (100 mg/kg intraperitoneally) and placed on a temperature-regulated table. A PE-50 tubing catheter was inserted in the right femoral artery and used for subsequent blood sampling. After tracheotomy, PE-50 catheters were inserted in the right and left jugular veins for infusion of rat plasma, saline, and radiolabeled inulin. Plasma was infused in an amount equal to 1% body weight over 40 to 45 min, followed by a reduction of the infusion rate to 0.4 ml/h for the duration of the study. Saline was infused at 2.4 ml/h throughout the study. After 60 min, tritiated methoxy-inulin was added to the saline to achieve an infusion rate of approximately 7 Ci/h following a loading dose of approximately 6 Ci. A PE-10 catheter was installed into the left ureter for collection of urine. Clearance measurements were carried out over two or three 30-min periods. In each period, an arterial blood sample was obtained for determination of plasma inulin concentration. Morphologic Studies After functional studies, kidneys were fixed by retrograde aortic perfusion with 2.5% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer. Kidneys were sliced perpendicular to the long axis at approximately 2-mm intervals. Slices from the midportion of the kidney were further fixed in 10% formalin for embedding in paraffin and in 1.25% glutaraldehyde in 0.1 M cacodylate for embedding in Epon. For each kidney, one or two smaller blocks of tissue extending from the capsule into the medulla were embedded in Epon. These blocks were sectioned serially at 3- m intervals so that glomeruli at all levels of the cortex were included in the sections. Every other serial section was mounted and stained with toluidine blue so that the tissue could be examined at 6- m intervals. One hundred to two hundred sections were examined in each rat in the ischemia groups, and 60 sections were examined in rat in the sham group. Every fourth mounted section was photographed, and a series of prints was prepared for use as maps of the serial sections. These maps served as a guide to identification of individual glomeruli, as described previously (8). Only glomeruli contained entirely within the serial sections were examined. The average number of glomeruli examined was 27 7 (SD) in rats subjected to ischemia and 20 5 in rats subjected to sham operation. Each glomerulus was first classified as being either connected to a normal proximal tubule, connected to an atrophic proximal tubule, or without any tubular connection as described by Marcussen (9). The proximal tubule segment connected to a glomerulus was considered atrophic when there was thinning of tubular cells accompanied by loss of brush border and narrowing of the tubular lumen. Glomeruli were then further examined at three levels evenly spaced along the diameter perpendicular to the plane of section to assess the prevalence of sclerotic injury to the tuft. Segmental sclerotic injury was defined by the presence of adhesions of the tuft to Bowman s capsule with collapse of capillary lumina in a portion of the tuft. The volume of each glomerulus was calculated from the area of its midsection, using the maximal planar area method (10). The fractional volume of cortical components was determined by point counting using a 6 6 point eyepiece reticule grid and a magnification of 400. In each case, a minimum of 500 points in three sections spaced 120- m apart was counted. Cortex was divided into tubular cells, tubular lumina, glomeruli, vessels larger than capillaries, and interstitium. For the purposes of point counting, tubular basement membranes and capillaries were included in the interstitium. Tubular cells included both proximal and distal cells, as well as cells whose original type was difficult to ascertain due to tubular injury. Volume fractions were calculated as the number of points falling on each of these components divided by the number of points evaluated. One trichrome and one periodic acid-schiff-stained section were prepared from a paraffin-embedded kidney slice in each animal. Trichrome sections were ranked to compare the extent of fibrosis, and periodic acid-schiff sections were ranked to compare the extent of cast formation in treated and untreated rats. Because slices from near the midportion of the kidney were used, morphologic results apply to this portion of the kidney. Separate sections were not prepared to assure that losartan and enalapril did not have different effects on renal disease toward the poles. Statistical Analyses ANOVA was used to assess the significance of differences between groups at 35 d, and unpaired t test was used to assess the significance of differences between groups at 140 d. The paired t test was used to assess the significance of differences within each group, and the Mann-iWhitney test was used to compare rank values assigned to treated and untreated rats. Values are expressed as the mean SEM throughout. Results Studies at 35 Days Serum creatinine values are depicted in Figure 1. By design, the groups of rats subjected to ischemia of a solitary kidney exhibited the same elevation of serum creatinine over the first 2 d. Serum creatinine values thereafter declined rapidly in both groups and were not affected by AngII receptor blockade beginning at the end of the second day. Elevation of serum creatinine was associated with impairment of growth, so that Figure 1. Serum creatinine values in rats studied for 35 d. The average serum creatinine value declined rapidly following ischemic injury to a solitary kidney both in rats that received losartan (F) and in rats that received no treatment (E). *P 0.05 ischemia groups versus shams (ƒ). The arrow in this and subsequent figures indicates the point at which treatment was initiated.

3 1280 Journal of the American Society of Nephrology J Am Soc Nephrol 11: , 2000 body weight was lower in animals subjected to ischemic injury than in sham-operated animals at the end of the experiment (AngII blockade g, untreated g, shamoperated 502 5g;P 0.05 both injured groups versus shams). BP values are depicted in Figure 2. Rats subjected to ischemia of a solitary kidney exhibited a slight elevation of systolic BP at 10 and 20 d, which was prevented by AngII blockade. No difference in systolic BP was apparent at 35 d. Measurements of kidney function and structure at 35 d are summarized in Table 1. Rats subjected to injury and not treated exhibited a modest increase in urine protein excretion that was limited by AngII blockade. GFR was reduced to a similar extent in both groups of rats recovering from ischemic injury to a solitary kidney. GFR was reduced more than kidney weight, so that values for GFR/kidney weight were significantly reduced in rats subjected to injury compared with shams. Morphologic studies revealed tubulointerstitial changes at 35 d following acute ischemic injury, as illustrated in Figure 3. Widening of the space between tubules resulted from prominence of intertubular capillaries as well as an increase in matrix. Collecting ducts were dilated. In addition to these diffuse changes, there was patchy tubule injury characterized by dilation with attenuation of epithelial cells in some segments, and atrophy with thickening of the basement membrane in others. Interstitial fibrosis and a mild infiltrate composed of mononuclear cells accompanied tubule injury. Large cystic structures were observed, with increasing frequency in kidneys showing the most severe injury. Examination of serial sections revealed that many glomeruli were no longer connected to normal proximal tubules. Segmental glomerular sclerosis, however, was rare (treated 3 2%, untreated 5 2%; P NS). As described previously, the size of glomeruli that were no longer connected to normal tubules tended to be reduced Figure 2. Systolic BP in rats studied for 35 d. Compared with sham rats (ƒ), untreated rats exhibited an increase in BP at 10 and 20 d after ischemic injury. BP was lower in losartan-treated rats (F) at these intervals. Differences in BP were no longer apparent at 35 d. *P 0.05 ischemia groups versus shams (ƒ); P 0.05 losartan versus no treatment. (1,9). Morphometric studies showed that AngII blockade did not reduce the severity of tubulointerstitial injury. The fraction of glomeruli no longer connected to normal tubule segments was similar in rats receiving no treatment and rats receiving the AngII receptor blocker. The two groups also exhibited a similar increase in the fractional volume of the interstitium. Ranking of trichrome-stained whole kidney sections confirmed that treatment did not limit the development of fibrosis following ischemic injury (mean rank values: 9.9 treated and 9.1 untreated; P NS). AngII blockade did, however, tend to reduce the size of glomeruli connected to atrophic tubular segments and significantly reduced the size of glomeruli without any connection to tubular segments. The reduction in volume caused by AngII blockade was not associated with a qualitative difference in glomerular structure as assessed by light microscopy. Regression analysis showed that the extent of injury at 35 d was correlated with the severity of initial ischemic injury, as summarized in Table 2. Rats with higher serum creatinine values on days 1 and 2 had developed larger numbers of glomeruli without normal tubule connections, higher interstitial volume fractions, and lower tubule cell volume fractions. AngII blockade did not affect the relation of initial serum creatinine with subsequent structural injury as assessed by these parameters. Regression analysis also showed that the GFR at 35 d was correlated with the extent of tubulointerstitial injury, as reflected by loss of tubule connections to glomeruli, interstitial volume expansion, and reduction in tubule cell volume fraction. Studies at 140 Days Serum creatinine values in the groups of rats maintained for 140 d are depicted in Figure 4. During the first 35 d after ischemic injury, these rats exhibited serum creatinine levels similar to those observed in the experiment described above. Institution of converting enzyme inhibition at day 35 did not have a notable effect on serum creatinine levels. Values in both the treated and untreated groups remained stable between 35 and 140 d. Body weight was also not effected by converting enzyme inhibition (final values: g treated, g untreated; P NS). BP values are depicted in Figure 5. Converting enzyme inhibition beginning at day 35 caused a reduction in BP, which was sustained throughout the experiment. Converting enzyme inhibition also limited the development of proteinuria, as depicted in Figure 6. In rats that received no treatment, recovery from acute ischemic injury to a solitary kidney was followed by a progressive increase in proteinuria to a mean of mg/d at 140 d. In contrast, converting enzyme inhibition limited proteinuria to a mean of mg/d at 140 d, which was no greater than the value observed immediately before beginning treatment at 35 d. Measurements of kidney function and structure at 140 d are summarized in Table 3. The GFR tended to be lower in rats that received no treatment than in rats receiving the converting enzyme inhibitor, but this difference was not statistically significant. Morphologic examination revealed patchy tubular injury associated with interstitial fibrosis and infiltration of

4 J Am Soc Nephrol 11: , 2000 Late Renal Injury after Acute Ischemia 1281 Table 1. Functional and structural findings at 35 d a Parameter No Treatment (n 8) AngII Receptor Blockade (n 10) Sham (n 4to8) Proteinuria (mg/d) b c 24 2 GFR (ml/min) b b Kidney weight (g) b b GFR/kidney weight (ml/min per g) b b Glomeruli with normal tubules (%) b b Fractional volume of the interstitium (%) 25 4 b 25 3 b 13 2 Fractional volume of tubular cells (%) 41 5 b Volume of glomeruli with normal tubules (10 6 m 3 ) b b Volume of glomeruli with atrophic tubules (10 6 m 3 ) d Volume of glomeruli without tubules (10 6 m 3 ) d c,d a Values for total GFR and kidney weight in sham rats were obtained by doubling measured values for the left kidney. Morphologic studies were performed in four of the eight sham-operated rats that underwent functional studies. b P 0.05 versus sham. c P 0.05 AngII blockade versus no treatment. d P 0.05 versus glomeruli with normal tubules in the same group. mononuclear cells (Figure 3). These tubulointerstitial changes were similar to those observed at 35 d, but the density of the mononuclear cell infiltrate appeared greater on average in rats studied at 140 d. In addition, rats studied at 140 d showed segmental sclerotic changes in glomeruli and protein casts in tubule lumina. These latter changes were most prominent in rats that received no treatment. Segmental sclerotic lesions were seen in 45 10% of glomeruli in these animals compared with 14 9% of glomeruli in treated animals. Examination of serial sections from untreated rats revealed that segmental sclerotic lesions developed in a similar portion of glomeruli with normal tubule connections and glomeruli without normal tubule connections. Converting enzyme beginning at 35 d after acute ischemia appeared to prevent the development of segmental sclerotic lesions regardless of whether glomeruli were connected to normal tubule segments. However, the number of glomeruli in each class was small and the error values were therefore large (17 10% lesions in glomeruli with normal connections, 9 7% lesions in glomeruli without normal connections). Converting enzyme inhibition also reduced the extent of cast formation (mean rank values: 10.3 untreated, 5.4 treated; P 0.05). Converting enzyme inhibition did not, however, have a major effect on other features of tubulointerstitial injury. The fraction of glomeruli that were not connected to normal tubules was similar in the treated and untreated rats and similar to that observed in rats studied at 35 d. The treated and untreated groups also exhibited similar values for interstitial and tubule cell volume fraction. Ranking of trichromestained whole kidney sections suggested that there was a trend toward less fibrosis in the treated group, but this did not reach conventional significance (mean rank values: 5.8 treated, 9.9 untreated; 0.05 P 0.10). The effects of converting enzyme inhibition on glomerular volume at 140 d were similar to those seen with AngII blockade at 35 d. Converting enzyme inhibition had no effect on the size of glomeruli with normal tubule connections, but reduced the size of glomeruli connected to atrophic tubule segments and of glomeruli without tubule connections. Again, the reduction in volume of these glomeruli was not associated with a qualitative difference in glomerular structure as assessed by light microscopy. Rats studied at 140 d did not exhibit an association between final GFR or structural parameters and the severity of initial ischemic injury, as reflected by the elevation of serum creatinine on day 1. Regression analysis did show that the GFR at 140 d was correlated with the extent of tubulointerstitial injury as reflected by loss of tubule connections to glomeruli, interstitial volume expansion, and reduction in tubule cell volume fraction (Table 4). Discussion Kidney function and structure do not return completely to normal after ischemic renal injury in rodents (11). Fox (12) initially described late structural changes following acute renal artery clamping in mice. Atrophy of a portion of the tubules in these animals was accompanied by interstitial fibrosis and round cell infiltration. Finn (13) later showed that these structural changes were accompanied by reduction in the number of functioning nephrons at 4 wk after acute ischemic injury in rats. Recently, the hypothesis that ischemic injury at the time of transplantation contributes to late graft loss has resulted in more prolonged studies of the sequelae of acute renal ischemia (14). Azuma et al. (15) showed that tubular injury was followed by late development of proteinuria and segmental glomerular sclerosis in rats observed for 1 yr after acute ischemia. We have confirmed these findings in a morphometric study of rats subjected to ischemic injury of solitary kidney (1). Rats studied at 5 wk exhibited tubule loss accompanied by interstitial fibrosis and inflammation. Rats studied at 20 wk showed tubulointerstitial injury and also segmental glomerular sclerosis. Glomerular sclerosis developed in the absence of hyper-

5 1282 Journal of the American Society of Nephrology J Am Soc Nephrol 11: , 2000 Figure 3. Renal cortex following recovery from acute ischemia. (A) Normal tissue from a sham-operated rat. (B) Tubulointerstitial injury at 35 d after ischemia is characterized by patchy interstitial fibrosis and focal tubular dilation as well as tubular atrophy. (C) Injury at 140 d after ischemia is again characterized by patchy interstitial fibrosis. There is prominent variation in tubular diameter associated with the appearance of protein casts in tubule lumina and focal and segmental glomerular sclerosis (toluidine blue; magnification, 25). Inset in Panel C shows infiltration of mononuclear cells accompanying tubular injury (toluidine blue; magnification, 150).

6 J Am Soc Nephrol 11: , 2000 Late Renal Injury after Acute Ischemia 1283 Table 2. Correlation of function and structure in rats studied for 35 d Variable r 2 P Value Structure at 35 days against mean serum creatinine on days 1 and 2 glomeruli with normal tubules (%) fractional volume of the interstitium (%) fractional volume of tubular cells (%) GFR at 35 d against structure at 35 d glomeruli with normal tubules (%) fractional volume of the interstitium (%) fractional volume of tubular cells (%) Figure 5. Systolic BP in rats studied for 140 d. After initiation of enalapril at 35 d, treated rats (F) exhibited a reduction in BP compared with rats receiving no treatment (E). P 0.05 enalapril versus no treatment. Figure 4. Serum creatinine values in rats studied for 140 d. Creatinine values were not altered in rats that received enalapril beginning at 35 d (F) compared with rats receiving no treatment (E). tension and was observed in atubular glomeruli as well as glomeruli attached to normal tubules. The present study examined the effect of agents that reduce AngII activity on the sequelae of acute renal ischemia. These agents have been found effective in a remarkable variety of clinical and experimental renal diseases. Their beneficial effect has been demonstrated most extensively in models of glomerular injury characterized by proteinuria (6,7,16 19). Recent studies have shown, however, that reduction of AngII activity can also limit the development of tubular injury and interstitial fibrosis (2,3). Direct effects of AngII blockade on tubulointerstitial structure are difficult to demonstrate in animals with primary glomerular injury, because any benefit observed can be ascribed to reduction of proteinuria or some other effect of AngII blockade on glomerular function. However, AngII blockade has been shown to protect against injury in some disease models in which there is little proteinuria. In particular, AngII blockade has been found to limit interstitial fibrosis during cyclosporine administration and after unilateral ureteral Figure 6. Twenty-four-hour urinary protein excretion in rats studied for 140 d. Rats receiving no treatment (E) exhibited steadily increasing proteinuria. Initiation of enalapril at 35 d prevented any further increase in proteinuria in treated rats (F). P 0.05 enalapril versus no treatment.; # P 0.05 versus 35 d same group. obstruction (3 5,20). In contrast, AngII blockade appears to have a lesser effect on interstitial fibrosis in rats with genetic polycystic kidney disease (Han:SPRD) and no effect on fibrosis in rats with chronic lithium toxicity (21 24). It is unclear at present why fibrosis is more dependent on AngII in some conditions than others. The present study first assessed the effect of AngII receptor blockade on kidney structure and function at 35 d after acute injury. Reduction in the tubule cell volume fraction at this interval was accompanied by the appearance of glomeruli without normal connections to tubule segments and by interstitial expansion. The GFR was correlated with the fraction of

7 1284 Journal of the American Society of Nephrology J Am Soc Nephrol 11: , 2000 Table 3. Functional and structural findings at 140 d a Parameter No Treatment (n 8) Converting Enzyme Inhibition (n 7) GFR (ml/min) Kidney weight (g) Glomeruli with sclerotic lesions (%) b Glomeruli with normal tubules (%) Fractional volume of the interstitium (%) Fractional volume of tubular cells (%) Volume of glomeruli with normal tubules (10 6 m 3 ) Volume of glomeruli with atrophic tubules (10 6 m 3 ) c b,c Volume of glomeruli without tubules (10 6 m 3 ) c b,c a No GFR value was obtained in one untreated rat. b P 0.05 versus no treatment. c P 0.05 versus glomeruli with normal tubules in the same group. Table 4. Correlation of function and structure in rats studied for 140 d Variable r 2 P Value GFR against structure glomeruli with normal tubules (%) fractional volume of the interstitium (%) fractional volume of tubular cells (%) glomeruli with normal tubular connections, suggesting that dropout of nephrons which suffered the most severe tubule injury accounted for loss of kidney function. The extent of injury at 35 d was correlated with the magnitude of the rise in serum creatinine immediately after acute ischemia and was not affected by AngII blockade. In particular, AngII blockade beginning at 2 d did not prevent reduction in the tubule cell volume, appearance of glomeruli without normal tubule connections, or interstitial fibrosis. The latter finding suggests that the contribution of AngII to interstitial fibrosis following acute, severe ischemic cell injury is relatively small. Not surprisingly, treatment that failed to prevent tubule loss had no effect on the GFR. It should be emphasized that the present study assessed the effect of AngII blockade on recovery from ischemic injury and not on the development of acute ischemic injury. Previous studies have shown that introduction of AngII blockade at the time of ischemia confers variable but generally incomplete protection against the development of renal dysfunction (25,26). We next examined the effect of converting enzyme inhibition on the course of injury between 35 and 140 d after acute ischemia. In addition to the features of injury present at 35 d, rats left untreated for 140 d developed increasing proteinuria and focal glomerular sclerosis. They did not, however, exhibit a late increase in serum creatinine or a clear worsening of tubulointerstitial injury. Results of previous studies suggest we would have observed more deterioration if the initial ischemic injury had been more severe or if the follow-up period had been longer. In a previous study using this model, we observed a late rise in serum creatinine and progressive loss of function in rats that had very high creatinine values 1 d after acute ischemia (1). Studies in other disease models have shown that heavy proteinuria leads eventually to tubule loss and interstitial fibrosis (27 29). These studies suggest that tubulointerstitial injury following recovery from acute ischemia can be divided into two components. First, there is tubule loss and interstitial expansion due to incomplete recovery from initial injury as was observed in animals studied at 35 d. Second, we presume that additional tubulointerstitial injury gradually develops in association with sustained proteinuria, but that this secondary tubulointerstitial injury was not severe enough to be detected in the present study. Converting enzyme inhibition largely prevented the development of proteinuria and glomerular injury after initial recovery from acute ischemia. The beneficial effect of converting enzyme inhibition was similar to that observed in rats with renal insufficiency induced by renal ablation. It should be emphasized, however, that the present model differs from renal ablation in at least two important ways. First, as documented previously, glomerulosclerosis develops while BP remains near normal (1). Second, sclerosis develops in small nonfiltering glomeruli as well as in remnant functioning glomeruli. The mechanisms that cause sclerosis under these conditions remain to be defined, but the presence of AngII is apparently required. The finding that AngII receptor blockade and converting enzyme inhibition reduce the size of atubular glomeruli suggests that AngII might contribute to the maintenance of perfusion in these structures. However, Tanner and Knopp (30) found that AngII blockade increased blood flow in glomeruli of nephrons in which filtration was stopped by tubule obstruction for 1 wk. Glomerular pressure was not measured in their study, and we are not aware of other studies of the effect of AngII on glomerular perfusion during long-term cessation of filtration in single nephrons.

8 J Am Soc Nephrol 11: , 2000 Late Renal Injury after Acute Ischemia 1285 In the present study, rats treated during the first 35 d received an AngII receptor blocker and rats treated from 35 to 140 d received a converting enzyme inhibitor. It seems likely that the results would have been the same if the drugs had been used in the opposite order. Previous studies have identified similar effects of AngII receptor blockade and converting enzyme inhibition in glomerular disease models including renal ablation, Heymann nephritis, anti-thy1 glomerulopathy, and radiation nephritis (7,17,19,31). Agents of both types have also been found to have similar effects in models of tubulointerstitial disease including cyclosporine nephrotoxicity and the Han:SPRD rat (5,21). Finally, AngII receptor blockade and converting enzyme inhibition have similar effects on glomerular function in humans with proteinuric renal disease (32). Together, these results suggest that the effects of both AngII receptor blockade and converting enzyme inhibition are mediated by reduction in AngII activity. The current study found that reduction in AngII activity did not prevent tubule loss or interstitial fibrosis during the early phase of recovery from acute ischemia. Thus, acute ischemia appears not to be among the forms of tubule injury that trigger interstitial fibrosis by increasing local AngII activity. However, AngII blockade did largely prevent late development of proteinuria and glomerular injury in solitary kidneys that had initially been damaged by ischemia. The ability of AngII to prevent late glomerular injury in this setting is potentially relevant to the treatment of patients who suffer ischemic injury to graft kidneys at the time of transplantation. 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