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From the Department of Medicine,*
Austin and
Repatriation Medical Centre, and Department of
Physiology,
University of
Melbourne, Australia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The existence of an intrarenal RAS is suggested by the high concentration of angiotensin II in the glomerular filtrate and proximal tubular lumen relative to plasma.3 Angiotensinogen and angiotensin converting enzyme are both present in the kidney and while the juxtaglomerular apparatus is the main source of systemic renin it may also contribute in a paracrine fashion to the intrarenal RAS .4 While the glomerulus is often the primary site of injury in renal disease, it is the extent of tubulointerstitial rather than glomerular injury which correlates most closely with and predicts future loss of renal function in patients with primary glomerular disease.5,6 Indeed, recent studies indicate that production of various cytokines by the proximal tubular epithelium may be a critical factor in the development of tubulointerstitial fibrosis7 as indicated by the formation of the prosclerotic cytokine, transforming growth factor-ß (TGF-ß), predominantly in this region.8 However, under physiological conditions renin can only be detected in the proximal tubule with the use of ultrasensitive, reverse transcriptase polymerase chain reaction (RT-PCR) methods.9
Apart from its actions on glomerular hemodynamics and electrolyte transport, angiotensin II (AII), the effector molecule of the RAS, may also function as a growth factor leading to the stimulation of extracellular matrix synthesis.10 While the precise mechanisms underlying this effect have not been fully delineated, several studies suggest that the ability of AII to stimulate extracellular matrix production may be mediated by increased expression of TGF-ß.11
Previous studies examining the intrarenal RAS in models of renal disease such as the remnant kidney have yielded conflicting results that may reflect differences in methodology.12,13 The present study sought to examine in detail the cell specific changes in the renin-angiotensin system following renal mass reduction, and furthermore to investigate the relationship between the RAS and TGF-ß expression and to determine the effects of ACE inhibition on the intrarenal RAS in this model of kidney disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirty male Sprague-Dawley rats weighing 200–250 g were randomized to three groups of 10 animals each. Anesthesia was achieved by the intraperitoneal administration of pentobarbital (6 mg/100 g body weight, Boehringer Ingelheim, Artarmon, NSW, Australia). The control group underwent sham surgery consisting of laparotomy and manipulation of both kidneys before wound closure. The other 20 rats all underwent subtotal nephrectomy (STNx) performed by right subcapsular nephrectomy and infarction of approximately two-thirds of the left kidney by selective ligation of two of three to four extrarenal branches of the left renal artery.14 Animals were then randomly assigned to two groups: STNx alone or STNx with the ACE inhibitor perindopril 8 mg/l drinking water (Servier, Neuilly, France). Rats were housed in a temperature (22°C) controlled room with ad libitum access to commercial standard rat chow (Norco Co-Operative Ltd., Lismore N.S.W., Australia) and water during the entire study. Rats from each group were sacrificed at 12 weeks after surgery. At sacrifice the remnant (left) kidney of STNx and the left kidney of sham animals were excised and the renal capsule removed. The kidney was then immersion fixed in 10% neutral buffered formalin and embedded in paraffin for later in situ hybridization and immunohistochemical studies. Experiments were approved by the Animal Welfare and Ethics Committee of the Austin and Repatriation Medical Center.
Renal Function
Body weight was measured weekly. Plasma urea and creatinine were measured by autoanalyzer (Beckman Instruments, Palo Alto, CA) at the beginning and end of the study. Glomerular filtration rate was measured at the end of the study by single shot Tc99m-DTPA clearance.15 Systolic blood pressure was measured in conscious rats using an occlusive tail-cuff plethysmograph attached to a pneumatic pulse transducer (Narco Bio-System Inc., Houston, Texas).16 Before sacrifice, rats were housed in metabolic cages for 24 hours for subsequent measurement of urinary protein excretion using the Coomassie Brilliant Blue method.17 Plasma renin activity was measured by radioimmunoassay.15
Tubulointerstitial Morphology
Paraffin-embedded sections were stained with hematoxylin and eosin, periodic acid-Schiff (PAS) and Masson's Trichrome. Tubulointerstitial injury was defined as tubular dilatation and/or atrophy, interstitial fibrosis, and inflammatory cell infiltrates. Morphological analyses were performed as previously described18 with the extent of injury graded on a scale of 0 to 4 by an observer blinded to the animal treatment group. With this method injury is graded as 0 for normal; 1 for involvement of <10% of the cortex; 2 for involvement of 10 to 25% of the cortex; 3 for involvement of 25 to 75% of the cortex, 4 for extensive damage involving >75% of the cortex.
Glomerular Morphology
Glomerular injury was assessed in PAS-stained sections by
examining 50 glomeruli in PAS-stained sections. Each glomerulus was
graded as normal (0); mildly sclerotic (1+, lesion occupying <25% of
glomerular tuft); moderately sclerotic (2+, lesion occupying 25 to 50%
of glomerular tuft); severely sclerotic (3+, lesion occupying more than
50% of glomerular tuft); or globally sclerotic (4+, lesion occupying
100% of glomerular tuft). A semiquantitative score was thus derived
for each animal using the formula:
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In Situ Hybridization
The 1.4-kb cDNA probe for rat renin (gift of Dr. D.J. Campbell,
Melbourne, Australia) was cloned into pGEM 4 (Stratagene, La Jolla, CA)
and linearized with BamHI to produce an antisense riboprobe
with T7 RNA polymerase. The 945-bp cDNA probe for TGF-ß1 (gift of Dr.
Qian, Bethesda, MD) and the 600-bp cDNA probe for 
1 (IV) collagen
(gift of Dr. R. Timpl, Martinsried, Germany) were also cloned into
pBluescript KS+ (Stratagene) and linearized with XbaI and
HindIII to produce an antisense riboprobes with T7 RNA
polymerase. In situ hybridization was performed as
previously described.20
In brief, sections 4 µm thick
cut from formalin-fixed paraffin-embedded kidney tissue were placed
onto slides precoated with 3-aminopropyltriethoxysilane and baked
overnight at 37°C. Tissue sections were dewaxed and rehydrated in
graded ethanol and milliQ water, equilibrated in P buffer (50 mmol/L
Tris-HCl, pH 7.5, 5 mmol/L EDTA) and incubated in 125 µg/ml Pronase E
in P buffer for 10 minutes at 37°C. Sections were then washed in 0.1
mol/L sodium phosphate buffer (pH 7.2), briefly refixed in 4%
paraformaldehyde for 10 minutes, rinsed in milliQ water, dehydrated in
70% ethanol, and air dried. Hybridization buffer containing 2 x
104
cpm/µl riboprobe in 300 mmol/L NaCl, 10 mmol/L
Tris-HCl (pH 7.5), 10 mmol/L Na2HPO4, 5 mmol/L
EDTA (pH 8.0), 1x Denhardt's solution, 50% formamide, 17 mg/ml yeast
RNA, 10% w/v dextran sulfate was heated to 85°C for 5 minutes.
Twenty-five µl of this solution was then added to each section.
Hybridization was performed overnight at 60°C in 50% formamide
humidified chambers. Sections hybridized with sense probe for renin,
TGF-ß1 and 1 (IV) collagen were used as controls for nonspecific
binding. After hybridization, slides were washed in 2x SSC containing
50% formamide prewarmed to 50°C to remove coverslips. Sections were
then washed in the above solution for 1 hour at 55°C, rinsed three
more times in RNase buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA,
pH 8.0, 0.5 mol/L NaCl) and then incubated with RNase A (150 µg/ml)
for 1 hour at 37°C. Sections were later washed in 2x SSC for 45
minutes at 55°C, dehydrated in graded ethanol, air dried, and exposed
to Kodak X-Omat autoradiographic film for 1–3 days. Slides were then
dipped in Ilford K5 nuclear emulsion (Ilford, Mobberley, Cheshire, UK),
stored in a light-free box with desiccant at room temperature for 2 to
3 weeks, immersed in Kodak D19 developer, fixed in Ilford Hypam, and
stained with hematoxylin and eosin or periodic acid-Schiff (PAS).
Quantitative Autoradiography
Densitometry of autoradiographic images obtained by in
situ hybridization was performed by computer-assisted image
analysis as previously described14,21,22
in accordance
with guidelines for computer assisted densitometric quantitative
autoradiography for in situ hybridization with
33P.23
In brief, using the MCID system (Image
Research, Ontario, Canada), in situ autoradiographic images
were placed on a uniformly illuminating fluorescent light box (Northern
Light Precision Luminator Model C60, Image Research, Ontario, Canada)
and captured using a video camera (Sony Video Camera Module CCD, Japan)
connected to an IBM AT computer with a 512 x 512 pixel array
imaging board with 256 gray levels. Following appropriate calibration
by constructing a curve of optical density versus
radioactivity density,24,25
quantitation of digitized
autoradiographic images was performed with the MCID software and
expressed as relative optical density (ROD). Control kidneys were
arbitrarily assigned a value of 1. TGF-ß1 and 1 (IV) collagen mRNA
were quantitated only in the superficial cortex, the most distant area
from the infarct-related scar in STNx animals. In addition to
quantitation of gene expression, localization and relative abundance of
transcript were visualized using pseudocolorized computer images.
Immunohistochemistry
Immunohistochemistry for renin and angiotensin II was performed as previously described.26 In brief, sections 4 µm thick were placed onto slides, deparaffinized, and rehydrated. To block endogenous peroxidase sections were pretreated with 1% H2O2/methanol. Sections were next incubated in protein blocking agent (Lipshaw-Immunon, Pittsburgh, PA) for 20 minutes at room temperature followed by incubation with either renin or angiotensin II antiserum. Polyclonal renin anti-serum27 was raised by immunizing rabbits with renin purified from mouse submandibular gland. Angiotensin II antibody (gift of Dr. D. Caranzaro, Cornell University Medical Center, New York)28 was raised by immunizing rabbits with a conjugate of synthetic angiotensin II combined with rabbit serum albumin.29 Sections were incubated with specific anti-serum for 18 hours at 4°C, washed in PBS and then incubated with universal biotinylated immunoglobulin (DAKO, Carpinteria, CA) and avidin-biotin complex (Vector, Burlingame, CA). Peroxidase conjugates were subsequently localized using diaminobenzidine tetrahydrochloride (DAB) as a chromogen and counterstained with Mayer's hematoxylin. Negative controls included omitting the primary antibody or replacing it with normal rabbit IgG at an equivalent protein concentration.
To quantify the presence of renin in the juxtaglomerular apparatus, 50 JGAs were counted in kidney sections from each rat and assessed by a blinded observer. The number of JGAs with positive immunostaining for renin was then expressed as a percentage of the total.13 The extent of tubular staining was assessed according to the percentage of the gridfield showed positive staining using a scoring system where 0 = absent staining, I = 1 to 5%, II = 5 to 25%, III = 25 to 50%, IV = 50 to 75%, and V = >75% as previously described.18
Statistics
Results are expressed as mean ± SEM unless stated otherwise. Data were analyzed by ANOVA with comparisons between groups using Fisher's least significant difference method.30 Semiquantitative data obtained from immunohistochemical studies were expressed as median (range) and between group comparisons were analyzed using the Kruskal-Wallis test. Analyses were performed using the Statview SE+ Graphics package (Abacus Concepts, Berkeley, CA) on an Apple Macintosh Performa 6360 computer. A p value less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functional data are shown in Table 1
. Rats that had undergone STNx became
hypertensive. This was ameliorated by perindopril treatment. Similarly
STNx rats developed chronic renal impairment characterized by elevated
serum creatinine and heavy proteinuria, both of which were
reduced by treatment with perindopril. Receipt of ACE inhibitor was
confirmed by elevation of plasma renin activity in comparison with
untreated STNx rats.
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Kidneys from STNx rats demonstrated glomerulosclerosis. The
proportion of normal tubules was also substantially reduced in STNx
animals as a result of tubular dilatation, tubular atrophy, and
interstitial pathology (Table 2
, Figure 1
). Treatment with perindopril
significantly reduced the extent of both glomerular and
tubulointerstitial injury in STNx rats.
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Subtotal nephrectomy and its treatment with ACE inhibition was
associated with altered distribution of renin gene expression in the
kidney (Figure 2)
. In sham kidneys
abundant expression of renin mRNA was noted in the juxtaglomerular
apparatus and not in the tubular epithelium (Figure 3A)
. In contrast, in STNx kidneys, in
areas distant from the site of infarction, de novo renin
expression was noted in renal tubular epithelial cells with minimal or
absent expression in the JGA (Figure 3, B and C)
. This aberrant
expression of renin by the tubular epithelium was particularly apparent
in areas of marked structural injury in comparison with areas in
which only minor damage was noted (Figure 3, B and D)
. In
perindopril-treated STNx rats, areas distant from the infarct scar
demonstrated a pattern of renin gene transcription similar to that of
control animals (Figures 2 and 3E)
. Sections incubated with renin sense
probe showed no hybridization (Figure 3F)
.
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The pattern of renin immunostaining corresponded to that
of its mRNA in sham, STNx and perindopril-treated STNx rats in areas
distant from the site of infarction (Figure 4)
and in the peri-infarct region (Figure 5)
. In the non infarct-adjacent regions,
the proportion of JGAs containing immunostainable renin was
significantly reduced in STNx rats compared with sham and
perindopril-treated STNx rats (Table 2)
. However, in the peri-infarct
area JGA and vascular renin was present and similar in both untreated
and perindopril-treated STNx rat kidneys (Figure 5)
.
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. In STNx kidneys, distant from the
site of infarction, AII was noted in renal tubular epithelial cells and
not in the JGA (Figure 6B)
. In perindopril-treated STNx rats AII
staining was not detected in the kidney being absent from both
scar-adjacent region and in areas distant from the site of infarction
(Figure 6C)
. Sections incubated with normal serum or normal rabbit IgG
showed no specific labeling (Figure 6D)
.
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1 (IV) Collagen
Transforming growth factor-ß1 mRNA was expressed at low levels
in glomeruli and tubulointerstitium of sham animals (Figure 7, A and B)
. In contrast, diffuse
overexpression of TGF-ß was noted in subtotal nephrectomy rat kidneys
(Figures 2, 7C, and 7D)
in comparison with sham and perindopril-treated
STNx rats. Abundant TGF-ß transcript was noted in both in the
epithelium of dilated and atrophic tubules (Figure 7C)
and in glomeruli
(Figure 7D)
. In areas adjacent to the infarct diffuse overexpression of
TGF-ß1 was noted in all glomerular, tubular and vascular structures.
In perindopril-treated STNx rats TGF-ß1 expression was reduced
compared with untreated STNx animals in non-infarct adjacent tissue
(Figure 7, E and F)
, paralleling the amelioration of histological
injury (Table 2)
. However, TGF-ß1 transcript was still overexpressed
in the region adjacent to the infarct scar (Figure 2)
. Sections
incubated with TGF-ß1 sense probe showed no specific hybridization
(Figure 7G)
. The pattern of gene expression for 1 (IV) collagen was
similar that of TGF-ß1 in sham, STNx, and perindopril-treated STNx
rats (Figure 2)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The intrarenal renin-angiotensin system has been the subject of intense investigation following the appreciation of the paracrine effects of angiotensin II and the dissociation between its plasma and intrarenal concentrations.34,35 The substrate, angiotensinogen is primarily formed in the liver leading to circulating levels that are in excess of 1000-fold greater than angiotensins I and II.36 In addition, angiotensinogen is also synthesized by epithelial cells of the proximal tubule37,38 as is ACE39 so that under most circumstances renin activity is the main determinant of angiotensin II formation from its abundant substrate.40
Studies using in situ hybridization have not found renin mRNA outside the JGA and afferent arteriole in the normal kidney.31,32 This was interpreted as indicating that the immunohistochemical localization of renin in subapical vessels of the proximal tubule41 reflects the endocytotic activity of these cells rather than endogenous synthesis of renin. More recently, with the use of ultra-sensitive techniques such as RT-PCR (reverse transcription polymerase chain reaction) low levels of renin mRNA have been detected in the microdissected tubules of normal rats.9 In addition, using quantitative competitive RT-PCR, modulation of renin expression in response to uninephrectomy,9 salt depletion42 and ACE inhibition43 have also been noted. However, in other studies using RT-PCR, proximal tubule renin mRNA has not been found either under basal conditions, or after ACE inhibition and given the ultrasensitivity of RT-PCR and the abundance of renin in the JGA it is possible that contamination with JGA transcripts may account for these divergent findings.44
In the present study, renal mass reduction was associated with dramatic changes in the microtopography of renin expression with a substantial reduction in JGA renin and its de novo appearance in the tubular epithelium. The presence of renin mRNA in the tubular epithelium indicates that the renin protein detected immunohistochemically within these cells was synthesized intracellularly rather than derived by pinocytosis. Indeed, the paucity of juxtaglomerular renin synthesis in this model suggests that little, if any, of the renin detected immunohistochemically would have been derived from the glomerular filtrate. Furthermore, the co-localization of angiotensin II to the tubular epithelial cells suggests that all of the necessary components of the renin-angiotensin system are present within these cells in the setting of renal injury following subtotal nephrectomy. However, despite its presence in the tubular epithelium and throughout the peri-infarct region, JGA renin expression was substantially reduced thus accounting for the relative reduction in total kidney and non-scar adjacent renin mRNA noted previously.45
Angiotensin II has several non-hemodynamic actions which may contribute to the progression of renal disease. These include induction of oxidant stress by stimulating sodium transport,46 increased macromolecular trafficking47 and the increased secretion of various peptide growth factors, most notably TGF-ß.48 In vitro, angiotensin II induces TGF-ß expression in a variety of cell types that may contribute to the pathogenesis of progressive renal injury including proximal tubular cells48 and renal interstitial fibroblasts49 as well as mesangial50 and vascular smooth muscle cells.51 Furthermore, the stimulation of matrix protein synthesis which accompanied AII-induced induction of TGF-ß transcription can be blocked by either neutralizing TGF-ß antibody or by AII-receptor antagonism.50 Similarly, in vivo, infusion of angiotensin II leads to increased TGF-ß and matrix protein expression52 further suggesting a link between activation of the renin-angiotensin system, TGF-ß expression and extracellular matrix (ECM) synthesis.
There is substantial evidence to support a pathogenetic role for TGF-ß in orchestrating the accumulation of ECM, leading to fibrosis and ultimately, renal dysfunction in several models of experimental renal injury and in human kidney disease (reviewed in Ref. 52 ). Its prosclerotic effects include stimulation of ECM production, inhibition of its degradation, up-regulation of cell-matrix adhesion molecules and chemoattraction of macrophages and fibroblasts.53 In the present study, renal mass reduction was associated with greatly increased expression of TGF-ß1 mRNA throughout the nephron.
In many primary glomerular diseases the extent of interstitial damage correlates better with the degree of renal dysfunction than do indices of glomerular injury54 and the mechanisms by which such glomerular changes lead to tubulointerstitial injury have been the focus of considerable recent attention.55-57 The model of renal mass reduction, as used in the present study, is believed to lead to progressive renal injury as a consequence of altered intraglomerular hemodynamics.58 The finding that in this form of primarily glomerular injury, there is activation of the renin-angiotensin system and TGF-ß1 expression within the tubular epithelium, as demonstrated in the present study, provides a pathophysiological link between glomerular injury and the development of tubulointerstitial fibrosis.
The findings of the present study do not suggest that intrarenal-derived AII is the only stimulus for TGF-ß expression following renal mass reduction. Indeed, in this study neither renin nor AII were detected in glomeruli of subtotal nephrectomized rats despite abundant TGF-ß1 mRNA at this site suggesting that other factors such as physical hemodynamic forces59 and other vasoactive hormones such as the endothelins60 may also be involved in up-regulating TGF-ß expression. Furthermore, although tubular expression of renin and AII were noted following subtotal nephrectomy this does not indicate that AII is the only stimulus for TGF-ß expression and fibrosis at this site. For instance, as in the glomerulus other factors such as protein trafficking may also be involved in tubulointerstitial TGF-ß expression and fibrosis.7
The expression of renin by tubular epithelial cells, described in the present study may reflect a phenotypic change that occurs as a nonspecific response to injury. Indeed, this phenomenon of cell transdifferentiation in which there is de novo expression of proteins such as osteopontin61 and SPARC21 has been well described in the setting of renal disease.
Interruption of the renin-angiotensin system by either ACE inhibition or angiotensin II-receptor antagonism reduces renal injury in several models of experimental and human kidney disease.52 In the absence of activation of the systemic RAS, these pharmacological intervention studies provide further evidence for the role of the local, intrarenal RAS in the progression of kidney disease. In the present study, administration of the ACE inhibitor, perindopril was associated with disappearance of aberrant tubular expression of renin and angiotensin II along with the restoration of high level expression to the JGA. In addition, ACE inhibition was associated with reduced expression of the AII-inducible mediator of renal fibrosis, TGF-ß along with the amelioration of the structural and functional manifestations of renal injury.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from the Baxter Health Corporation Extramural Grant Program.
Accepted for publication April 11, 1999.
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