 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
REGULAR ARTICLES |
*
Department of Medicine, VA Palo Alto Health Care System and Stanford
University, Palo Alto, California
Department of Pathology, University of California, San Francisco,
California
Correspondence to Dr. Timothy W. Meyer, Palo Alto VA Medical Center, Nephrology 111R, 3801 Miranda Avenue, Palo Alto, CA 94303. Phone : 650-493-5000, extension 63314 ; Fax : 650-849-0213 ; E-mail : twmeyer{at}leland.stanford.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsStatistical AnalysesResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsStatistical AnalysesResultsDiscussionReferences |
|---|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsStatistical AnalysesResultsDiscussionReferences |
|---|
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 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 µCi/h following a loading dose of approximately 6 µCi. 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-µm 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-µm 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 x 6 point eyepiece reticule grid and a magnification of
x400. In each case, a minimum of 500 points in three sections spaced
120-µm 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsStatistical AnalysesResultsDiscussionReferences |
|---|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsStatistical AnalysesResultsDiscussionReferences |
|---|
|
|
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 (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 : 465 ± 11 g treated, 490
± 10 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 229 ± 52
mg/d at 140 d. In contrast, converting enzyme inhibition limited proteinuria
to a mean of 78 ± 17 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 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsStatistical AnalysesResultsDiscussionReferences |
|---|
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, 16,17,18,19). 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 obstruction (3,4,5, 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 (21,22,23,24). 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 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 (27,28,29). 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.
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.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsStatistical AnalysesResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  R. L. Chevalier and M. S. Forbes Generation and Evolution of Atubular Glomeruli in the Progression of Renal Disorders J. Am. Soc. Nephrol., February 1, 2008; 19(2): 197 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Spurgeon-Pechman, D. L. Donohoe, D. L. Mattson, H. Lund, L. James, and D. P. Basile Recovery from acute renal failure predisposes hypertension and secondary renal disease in response to elevated sodium Am J Physiol Renal Physiol, July 1, 2007; 293(1): F269 - F278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Forbes, B. A. Thornhill, M. H. Park, and R. L. Chevalier Lack of Endothelial Nitric-Oxide Synthase Leads to Progressive Focal Renal Injury Am. J. Pathol., January 1, 2007; 170(1): 87 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Devarajan Update on Mechanisms of Ischemic Acute Kidney Injury J. Am. Soc. Nephrol., June 1, 2006; 17(6): 1503 - 1520. [Full Text] [PDF]
|
|
|
|
 |
|
 U. Danilczyk and J. M. Penninger Angiotensin-Converting Enzyme II in the Heart and the Kidney Circ. Res., March 3, 2006; 98(4): 463 - 471. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Basile, D. L. Donohoe, S. A. Phillips, and J. C. Frisbee Enhanced skeletal muscle arteriolar reactivity to ANG II after recovery from ischemic acute renal failure Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1770 - R1776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Basile, K. Fredrich, M. Alausa, C. P. Vio, M. Liang, M. R. Rieder, A. S. Greene, and A. W. Cowley Jr. Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure Am J Physiol Renal Physiol, May 1, 2005; 288(5): F953 - F963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Garin, C. Badid, B. McGregor, M. Vincent, S. Guerret, K. Zibara, A. Hurlstone, M. Laville, and J. L. McGregor Ischemia Induces Early Expression of a New Transcription Factor (6A3-5) in Kidney Vascular Smooth Muscle Cells: Studies in Rat and Human Renal Pathology Am. J. Pathol., December 1, 2003; 163(6): 2485 - 2494. [Abstract] [Full Text]
|
|
|
|
|
|
Y.-C. Tian and A. O. Phillips TGF-{beta}1-Mediated Inhibition of HK-2 Cell Migration J. Am. Soc. Nephrol., March 1, 2003; 14(3): 631 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Basile, D. L. Donohoe, K. Roethe, and D. L. Mattson Chronic renal hypoxia after acute ischemic injury: effects of L-arginine on hypoxia and secondary damage Am J Physiol Renal Physiol, February 1, 2003; 284(2): F338 - F348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Gerlofs-Nijland, K. J. M. Assmann, H. B. P. M. Dijkman, J. W.C. Dieker, J. P. H. F. van Son, S. Mentzel, J. P. van Kats, A. H. J. Danser, O. Smithies, P. J. T. A. Groenen, et al. Albuminuria in Mice after Injection of Antibodies against Aminopeptidase A: Role of Angiotensin II J. Am. Soc. Nephrol., December 1, 2001; 12(12): 2711 - 2720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Benigni, E. Gagliardini, A. Remuzzi, D. Corna, and G. Remuzzi Angiotensin-Converting Enzyme nhibition Prevents Glomerular-Tubule Disconnection and Atrophy in Passive Heymann Nephritis, an Effect Not Observed with a Calcium Antagonist Am. J. Pathol., November 1, 2001; 159(5): 1743 - 1750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Basile, D. Donohoe, K. Roethe, and J. L. Osborn Renal ischemic injury results in permanent damage to peritubular capillaries and influences long-term function Am J Physiol Renal Physiol, November 1, 2001; 281(5): F887 - F899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. JAVAID, J. L. OLSON, and T. W. MEYER Glomerular Injury and Tubular Loss in Adriamycin Nephrosis J. Am. Soc. Nephrol., July 1, 2001; 12(7): 1391 - 1400. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
). *P < 0.05 ischemia groups
versus shams (
). The arrow in this and subsequent figures
indicates the point at which treatment was initiated.
