A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat gl - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 26 下一页

返回列表

查看: 468525\|回复: 252	go A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat gl [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 12:59 |只看该作者 |倒序浏览 |打印

Department of Medicine, Veterans Affairs Hospital, Hines, Illinois, and Loyola University Stritch School of Medicine, Maywood
7 I- J' E, R3 s5 `
Hektoen Institute for Medical Research, Chicago, Illinois+ h/ T, k3 a; n  }5 k) p. v9 T

ABSTRACT6 a/ v! x. H& o& x6 E  ~

Recent evidence suggests that the intrarenal renin-angiotensin system (RAS) may play an important role in the development of glomerular changes associated with diabetic nephropathy. In this study, the glomerular RAS was examined in male Sprague-Dawley rats made diabetic with streptozotocin (STZ), and the findings compared with those obtained in control nondiabetic rats. In diabetic rat glomerular extracts, angiotensinogen and angiotensin II (ANG II) levels were increased significantly by 2.2- and 1.9-fold, respectively, compared with nondiabetic controls. No significant differences in ANG I and angiotensin-converting enzyme (ACE) levels were observed between these groups. The HPLC analysis of the glomerular extracts demonstrated that exogenous ANG I was converted into various ANG peptides including ANG II, ANG(1–9), and ANG(1–7). A significant increase in formation of ANG II from exogenous ANG I was observed in STZ rats compared with control rats. Preincubation of glomerular extracts with captopril resulted in a 20–30% decrease in ANG II conversion from exogenous ANG I in diabetic and control rats. The possible role of ANG(1–9) in formation of ANG II was examined by HPLC. Exogenous ANG(1–9) in glomerular extracts was converted into ANG II, this conversion being significantly higher in STZ rats than in control rats. These findings provide new information that ANG(1–9) is produced in rat glomerular extracts, can be converted to ANG II, and that this conversion is also stimulated in diabetic rat glomeruli. Thus this study demonstrates that in diabetic rats, glomerular ANG II levels are increased due to an increase in angiotensinogen and an increase in the formation of ANG II./ A( |) }2 h# s' [
: z: W. r( Q* ], ?
diabetes; angiotensin peptides; angiotensin(1–9)# }/ K  q5 }7 U) w: T) o
$ [0 S3 c) M0 n% _) k( p' _( i
THE INTRARENAL RENIN-ANGIOTENSIN system (RAS) has been postulated to play an important role in the pathogenesis of diabetic nephropathy (14). It is now well established that ANG II is locally produced in the kidney and that it is involved in the development of glomerulosclerosis. ANG II levels in various intrarenal compartments are found to be several-fold higher than those found systemically (18), suggesting that the intrarenal RAS may function independently of the systemic RAS. The beneficial effects of angiotensin-converting enzyme (ACE) inhibitors and ANG II receptor blockers in delaying the progression of glomerulosclerosis in animals (13, 16) and in humans (15) have been interpreted as evidence that the intrarenal RAS is activated in diabetes, despite the fact that the systemic RAS is generally suppressed. Previous studies in the streptozotocin (STZ)-diabetic rat model have demonstrated a significant increase in angiotensinogen and renin mRNA expression in the kidney (1). Also, in diabetic rats, glomerular ANG II receptors were downregulated (3, 12, 24), suggesting an increase in glomerular ANG II levels.6 O" \$ M: P! U+ W9 S
2 x5 }. E( p5 {+ U- C/ R
Previous studies with glomerular mesangial cells from our laboratory showed that addition of high glucose to the culture media resulted in increased ANG II production in these cells (19). In addition, we recently demonstrated that high glucose stimulates mesangial ANG II production by increasing its substrates angiotensinogen and ANG I (20). In the same study, it was also noted that other ANG peptides such as ANG(1–9) could be converted into ANG II and that this conversion was also stimulated under high-glucose conditions in mesangial cells (20). Because these findings indicated the existence of additional non-ACE mechanisms for ANG II production in mesangial cells, we tested the in vivo relevance of these mechanisms in diabetic rats. Thus the present study was designed to determine the mechanisms involved in diabetes-induced ANG II production in the rat glomeruli.
4 N& n+ {# o/ i" q% z
MATERIALS AND METHODS

Diabetic Rat Model' E# |1 N' M& s3 w6 @3 p+ V

Male Sprague-Dawley rats (200–300 g) were made diabetic by a single injection of STZ (65 mg/kg body wt; Sigma, St. Louis, MO) administered into the tail vein. Control animals received an equal volume of vehicle. The diabetic state of the animal was confirmed by the demonstration of a nonfasting blood glucose level >250 mg/dl 24 h after STZ injection. Blood glucose measurements were carried out twice a week throughout the 4-wk study period. Food and water intake were ad libitum throughout the study. No insulin was administered to STZ-diabetic rats., ~" T# I+ k- ^
# y- o  T1 {1 R8 a8 ~0 s
Preparation of Glomerular Extracts" ^+ l* A) ?( {' Y

Four weeks after STZ (or vehicle) injection, rats were anesthetized with pentobarbitol sodium (60 mg/kg ip). Kidneys were rapidly removed, weighed, and minced on ice. Because the state of kidney tissue (fresh vs. frozen) and the extraction time can significantly affect endogenous levels of ANG I and ANG II (9), fresh kidneys were used for the preparation of glomerular extracts with rapid removal and processing of the kidney tissue. Glomeruli were isolated by sequential sieving in PBS at 4°C. Glomerular suspensions were composed of >95% glomeruli as determined by light microscopy. The suspensions were then centrifuged, and pellets were resuspended in PBS and sonicated (Microscan ultrasonic cell distrupter, Heat Systems, Farmingdale, NY) to rupture cell membranes, followed by centrifugation at 13,000 g for 20 min at 4°C. The supernatant was collected and used for various measurements.

Because glomeruli contain many peptidase enzymes that can metabolize ANG peptides during the extraction procedure, glomerular extracts from normal and STZ-diabetic rats were also prepared in PBS containing peptidase enzyme inhibitors cocktail (Sigma). The supernatants were used for measurements of endogenous levels of ANG I and ANG II.

Measurement of Angiotensinogen$ b; N; D, k/ A% e6 o

Antibodies to angiotensinogen were prepared using renin substrate tetradecapeptide [angiotensinogen(1–14)] (Sigma) as the immunizing antigen and the specificity of the antibody to rat kidney angiotensinogen was confirmed by Western blot analysis as described previously (20). Angiotensinogen levels in glomerular extracts were measured by ELISA (20). In brief, a 96-well plate was coated overnight at 4°C with 4 μg/ml of angiotensinogen(1–14), the immunizing antigen. Incubation was carried out with standards or samples mixed with anti-angiotensinogen antibody (1:10,000) for 2 h at room temperature followed by incubation with a secondary antibody (anti-rabbit IgG-peroxidase, 1:1,000) for 1 h. The reaction was developed using TMB (3,3',5,5'-tetramethylbenzidine dihydrochloride) and H2O2 as substrate and read at 450 nm after addition of 2 N HCl in an ELISA reader (Molecular Devices, Sunnyvale, CA). A standard curve was run with each assay using angiotensinogen(1–14) as the standard and the levels of angiotensinogen in samples were calculated from the standard curve.
! c" q8 e! N% L- G  D* r
This antibody does not cross react to ANG I or ANG II (20). However, because it could cross react to des-ANG I angiotensinogen (due to the presence of 11–14 amino acids), assay values represent total angiotensinogen which includes intact angiotensinogen and des-ANG I angiotensinogen. Values using ELISA may therefore be higher than those obtained using the classical renin assay, which measures only intact angiotensinogen.
9 P: b1 m$ N: L& I1 f; i: e. u9 z% H
Measurement of ANG I and ANG II Levels in Glomerular Extracts3 D2 G  e; G! x' V) |8 j) B% y

HPLC. First, standard ANG peptides (Bachem Biosciences, King of Prussia, PA) were injected into the HPLC column and analyzed on a C18 μBond reverse-phase column using an ultraviolet detector set at 214 nm (Isco, Lincoln, NE). The flow rate was maintained at 1 ml/min. Mobile phase A consisted of water with 0.00005% trifluoroacetic acid and mobile phase B consisted of 100% acetonitrile containing 0.00005% trifluoroacetic acid. The gradient program was set as 0–5 min 90% A:10% B, 5–32 min 60% A: 40% B, 32–40 min 90% A: 10% B to elute the products. After each sample analysis, a wash cycle was run to prevent contamination from any residual peptide left from the previous run. A sample chromatogram showing different ANG peptide peaks and their elution times is shown in Fig. 1.

Separation of endogenous ANG I and ANG II in glomerular extracts was carried out using HPLC followed by ELISA. First, a glomerular extract sample from a control rat was injected into the HPLC column and fractions (1–30) were collected at 1-min intervals. Fractions were air dried, reconstituted in assay buffer (supplied with ANG peptide ELISA kit), and assayed for ANG II (fractions 1–23) and ANG I (fractions 19–30) immunoreactivity by ELISA (Peninsula Laboratories, Belmont, CA). As shown in Fig. 2, the peak immunoreactivity to ANG II was detected in the 20-min fraction that coincided with the elution time of standard ANG II, and the peak immunoreactivity to ANG I was detected in the 22-min fraction that coincided with the elution time of standard ANG I. In subsequent experiments, glomerular extracts of normal and STZ-diabetic rats were subjected to HPLC separation and only 20- and 22-min fractions were used for ANG II and ANG I measurements by ELISA, respectively." f6 T9 H% H6 e* I# R

ANG I and ANG II ELISA. ANG I and ANG II levels were measured by a competitive ELISA using ELISA kits purchased from Peninsula Laboratories. Briefly, ELISA plates precoated with goat anti-rabbit IgG were used for the assays. Samples were mixed with rabbit antibodies to ANG I or ANG II and biotinylated ANG I or ANG II peptides, respectively, and incubated in the wells for 2 h at room temperature. After incubation and washings, streptavidin-conjugated horseradish peroxidase (SA-HRP) was added and allowed to bind to the immobilized primary antibody/biotinylated peptide complex in the wells. The final reaction was developed with TMB and H2O2 substrate and after 20 min terminated with 2 N HCl. The absorbance was read at 450 nm using ELISA reader (Molecular Devices). The color intensity that develops depends on the quantity of biotinylated peptide bound to the immobilized antibody so a higher amount of peptide present in the sample will allow less binding of biotinylated peptide with the limited amount of antibody resulting in production of less color by the substrate. ANG I or ANG II standard curves were run with each assay and levels of these peptides in the samples were calculated.
- _) w" @# m( h4 O* Y3 P  c5 r
Measurement of ACE Levels in Glomerular Extracts
1 `, Z1 t" i; A6 b
ACE protein levels in glomerular extracts from STZ and control rats were measured by ELISA using commercially available ELISA kits (Chemicon International, Temecula, CA). In addition, ACE protein levels were also determined by Western blot analysis. Glomerular extracts were separated by 8% SDS-PAGE using a Bio-Rad Mini-Protein Cell (Bio-Rad Laboratories, Hercules, CA) and transferred to a polyvinylidene difluoride membrane (Bio-Rad) overnight at 4°C. Membranes were incubated in blocking buffer (PBS containing 0.1% Tween 20 and 5% nonfat milk protein) for 2 h, washed, and then incubated with 1:100 of primary antibody to ACE (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. Further incubation was carried out with a HRP-conjugated secondary antibody (1:000) for 1 h. The specific proteins on the membranes were detected by chemiluminescence using the ECL detection system (Amersham Biosciences, Piscataway, NJ).
% S1 G- ]0 I. {2 V" V4 c: k" u( Z$ c9 b
Analysis of ANG II Formation from Exogenous ANG I or ANG(1–9) Using HPLC& w0 B) P8 y7 o

Formation of ANG II from exogenous ANG I or ANG(1–9) was studied using HPLC as described previously (20). First, to identify ANG peptide peaks generated by glomerular extracts, the elution times for various ANG peptides and their breakdown products were established by running their standards. ANG I, ANG II, ANG(2–8) [ANG III], ANG(3–8) [ANG IV], ANG(1–9), and ANG(1–7) were purchased from Bachem Biosciences; breakdown products of ANG I [ANG(2–10), ANG(3–10), ANG(4–10), ANG(5–10)] and ANG(1–9) [ANG(2–9), ANG(3–9), and ANG(4–9)] were custom synthesized (Dr. B Wakim, Medical College of Wisconsin, Milwaukee, WI). These standard peptides were run individually and their elution times were recorded and presented in Table 1. After each sample analysis, a wash cycle was run to prevent contamination from any residual peptide left from the previous run. Most of the ANG peptides were separable by >1.0 min though elution times of ANG(2–8) and ANG(3–8) were found to be overlapping with elution times of ANG(5–10) and ANG(4–10), respectively (Table 1). Glomerular extracts were incubated with 10–4 M of exogenous ANG I or ANG(1–9) for 1 h at 37°C. A 12-μg sample of glomerular extract from a STZ or control rat was injected into the HPLC column and analyzed using the same conditions described above. Absorbance at 214 nm for each peptide was calculated from the peptide peak height and used to generate the representative chromatograms shown in RESULTS. Quantification of peptide peaks was carried out by measuring peak absorbance of each peptide and normalizing it by peak absorbance of the exogenous substrate, ANG I or ANG(1–9). The results are presented as the ratio of ANG peptides to exogenous ANG I or ANG(1–9). In some experiments, glomerular extracts were prepared in PBS containing EDTA to inhibit peptidases, and conversion of exogenous ANG I or ANG(1–9) to ANG II was analyzed by HPLC.
' W) {7 J, s+ f+ Y' V! K; O! K; {
View this table:

Also, in separate experiments, conversion of exogenous ANG II to ANG II metabolites in STZ and control rat glomerular extracts was studied by HPLC. Glomerular extracts from STZ and control rats were prepared in PBS without EDTA, incubated with 10–4 M of exogenous ANG II for 1 h at 37°C, and analyzed using HPLC. Quantification of peptide peaks was carried out as described above, and results were expressed as the ratio of ANG peptides to ANG II.

Statistics
& s8 n2 n* m; Q
Values are expressed as means ± SE, and n denotes the number of animals for each experiment. Results were compared using Student's t-test. In experiments where more than one determinant was present, one-way ANOVA was applied. A P value of $ @' W: t, ]+ ?* B, I5 H

RESULTS

Effect of Diabetes on Blood Glucose, Body Weight, and Kidney Weight0 {: A+ ?8 P+ R% I/ J. c
) W% l1 P+ p- z1 J1 n3 _
A significant increase in blood glucose levels of diabetic rats was observed as early as 24 h after STZ injection that was maintained throughout the 4-wk study period. At the time of euthanasia, blood glucose levels were significantly higher in STZ-diabetic rats compared with control animals (STZ: 480 ± 27 vs. 114 ± 6 mg/dl in control rats; P $ Y8 k1 S  H& k( P0 B, f( t; r

Effect of Diabetes on Glomerular Angiotensinogen, ANG I, and ANG II+ K! U  P/ K1 A8 k# \, J

In STZ rats, immunoreactive total angiotensinogen levels in glomerular extracts were increased significantly by 2.2-fold compared with control rats (Table 2). Also, glomerular ANG II levels in STZ rats were significantly increased by 1.9-fold compared with control rats (Table 2). In contrast, no significant differences were observed in ANG I levels between STZ and control rats (Table 2).; a' S8 l5 ^4 \4 u7 |6 n8 M8 q

View this table:, @" N! H1 Q. f* S: U8 r, W% l& q
! L; N" m. H% S& v1 j1 I
Similar results were obtained when glomerular extracts were prepared in PBS containing protease enzyme inhibitors. ANG II levels in STZ rats were 63 ± 8 vs. 39 ± 2 fmol/mg protein in control rats (n = 4; P - g# \3 h8 Y" d" K& B

Effect of Diabetes on Glomerular ACE
2 P) l6 Q* H8 E
Western blot analysis of glomerular extracts showed a distinct band at 75 kDa corresponding to the molecular mass for cellular ACE in both STZ and control rats. However, no significant differences in ACE protein were observed between the two groups (data not shown). Similarly, immunoreactive ACE levels in glomerular extracts did not differ between STZ and control rats (STZ: 28.6 ± 2 vs. control: 26 ± 4 ng/mg protein).

Formation of ANG II from Exogenous ANG I in Glomerular Extracts" l. f: c; x8 ~* ~4 ]/ O7 m
2 X9 G2 ?& v8 s) e- y& S  f8 r7 j2 x
First, to test whether exogenous ANG I was stable in incubation buffer (PBS), ANG I (10–4 M) was incubated in PBS buffer at 37°C for a period of 1–4 h. At the end of each hour, a 25-μl sample was injected into a HPLC column and analyzed. The ANG I peak was identified and peak absorbance at 214 nm was calculated from the ANG I peak height. ANG I peak absorbances were 1.6 units at 1, 2, 3, and 4 h. No other ANG peptide peaks were detected at any time point. These observations indicated that ANG I was stable in PBS buffer with no appearances of metabolites for up to 4 h of incubation.

Next, a 12-μg protein sample of glomerular extract from a STZ-diabetic or control rat was incubated in the presence of exogenous ANG I (10–4 M) for 1 h at 37°C and then injected into the HPLC column. As shown in Fig. 3, a representative chromatogram from a control and STZ rat, exogenous ANG I was converted to ANG II and other ANG peptides such as ANG(1–9) and ANG(1–7). The identity of another peak recorded at 19 min could not be established with certainty because both ANG(2–8) and ANG(5–10) peptides eluted at this time. This peak is shown as an unidentified peak in Fig. 3. The ANG II/ANG I ratio increased significantly in diabetic glomerular extracts indicating increased formation of ANG II from exogenous ANG I in diabetic rats compared with controls (Fig. 4A). There were trends toward an increase in the ANG(1–9)/ANG I ratio in diabetic rats suggesting increased formation of ANG(1–9) from exogenous ANG I and a decrease in ANG(1–7)/ANG I ratio, suggesting a decrease in formation of ANG(1–7) from exogenous ANG I in diabetic rats (Fig. 4A).& u$ ?0 [0 m; K/ d' _/ _( Q
7 `4 o' R2 X8 n- ?' Q, ^
Effect of ACE Inhibition on Conversion of Exogenous ANG I to ANG Peptides in Glomerular Extracts

Preincubation of glomerular extracts with captopril (10–4 M) for 30 min reduced ANG II generation from exogenous ANG I in both STZ and control rats (Fig. 4B). In the presence of captopril, the ratio of ANG II to ANG I remained higher in STZ glomerular extracts compared with control extracts (P = 0.06, n = 5; Fig. 4B). No effect of captopril treatment on ANG(1–7)/ANG I or ANG(1–9)/ANG I ratios was observed in either control or STZ diabetic glomerular extracts (data not shown).0 m4 x/ z% E( \9 n# |6 g" H! F
+ q/ v0 p  [+ m6 w8 b$ O
Role of ANG(1–9) in ANG II Formation in Glomerular Extracts

The possibility of ANG II formation from the intermediary angiotensin peptide ANG(1–9) was examined by HPLC. Glomerular extract samples from control and STZ-diabetic rats were incubated with exogenous ANG(1–9) (10–4 M) for 1 h at 37°C and analyzed by HPLC. Figure 5, a representative chromatogram from a control and STZ rat, shows conversion of exogenous ANG(1–9) to ANG(1–7) and ANG II. In STZ glomerular extracts, the ANG(1–7)/ANG(1–9) ratio was significantly decreased compared with control extracts (Table 3). In contrast, the ANG II/ANG(1–9) ratio was significantly increased in STZ rat glomerular extracts compared with control glomerular extracts (Table 3).& \7 H, F% Q' x6 q/ A
% O( a0 ^, ?- O2 G& {' f
View this table:
( E; g5 F& J: |5 n* ~. G4 G, L
Increased accumulation of ANG II in the presence of exogenous ANG I or ANG(1–9) in STZ rat glomerular extracts could be influenced by decreased breakdown of ANG II. Therefore, STZ and control glomerular extracts were prepared in PBS containing EDTA (to inhibit ANG II breakdown) and conversion of exogenous ANG I or ANG(1–9) to ANG II was studied by HPLC. In both STZ and control glomerular extracts, no ANG II peak was detected (data not shown), suggesting that EDTA inhibited ANG II-generating enzymes in addition to ANG II-degrading enzymes.

Effect of Enzyme Inhibitors on Conversion of ANG(1–9) to ANG II in Glomerular Extracts3 a7 `" H- I' u$ a

Although not much data are available on the putative carboxypeptidase enzyme involved in the conversion of ANG(1–9) to ANG II, the role of ACE and chymase in ANG II formation from exogenous ANG(1–9) was tested. Glomerular extracts from control or STZ rats were preincubated with captopril (10–4 M) for 30 min at 37°C followed by incubation with exogenous ANG(1–9) for 1 h and analysis by HPLC. Captopril treatment produced a significant decrease in the formation of ANG(1–7) and ANG II from exogenous ANG(1–9) in both STZ and control rats (Table 3). However, the ANG(1–7)/ANG(1–9) ratio remained lower and the ratio of ANG II/ANG(1–9) remained higher in STZ glomerular extracts in the presence of captopril (Table 3). In contrast, preincubation of glomerular extracts with chymostatin (chymase inhibitor) up to 60 min before incubation with exogenous ANG(1–9) had no effect on the conversion of ANG(1–9) to ANG II and ANG(1–7) in either STZ or control rats (data not shown).

Conversion of Exogenous ANG II in Glomerular Extracts
- ?0 e# w* h6 D. w7 I+ l) A
In separate experiments, when STZ and control glomerular extracts were incubated with exogenous ANG II, generation of ANG(1–6) (identified based on its elution time) and two other unidentified peaks were observed (Fig. 6). No significant differences were observed in the ANG(1–6)/ANG II ratio between the two groups (STZ: 0.078 ± 0.01 vs. control: 0.065 ± 0.01; P = NS, n = 3).
/ b1 b( [8 w% a) {* x
DISCUSSION: \' D* q4 G; H% l* j

This study demonstrated that endogenous ANG II levels in STZ-diabetic rat glomeruli were significantly increased compared with control nondiabetic rats. The increase in glomerular ANG II was accompanied by a significant increase in total angiotensinogen levels in diabetic rat glomeruli compared with controls, although ANG I and ACE levels did not differ between the diabetic and control rats. These findings led us to examine ANG II formation from exogenous ANG I in glomerular extracts using HPLC assays. In both STZ and control glomerular extracts, exogenous ANG I was converted into various ANG peptides such as ANG(1–9), ANG(1–7), and ANG II, and the conversion of ANG I to ANG II was significantly higher in STZ rats vs. controls. Because glomerular ACE levels and protein did not differ between the diabetic and control rats, increased ANG II conversion in diabetic rats did not appear to be due to stimulation of ACE. Furthermore, captopril treatment resulted in only 20–30% inhibition of exogenous ANG I conversion to ANG II in STZ and control glomerular extracts, suggesting that there are additional mechanisms or pathways for the conversion of ANG I to ANG II that employ enzymes other than ACE. Because in the present study a large amount of ANG(1–9) was generated from ANG I, its possible role in ANG II formation was explored using HPLC. Exogenous ANG(1–9) incubated with glomerular extracts was converted to ANG II in both STZ-diabetic and control rats, and this conversion was significantly higher in STZ glomerular extracts compared with control extracts. Increased accumulation of ANG II in the presence of exogenous ANG I or ANG (1–9) in STZ glomerular extracts was unlikely to be due to increased stability of ANG II in STZ glomerular extracts because breakdown of exogenous ANG II to its metabolic products was similar in STZ and control glomerular extracts.

The conversion of ANG I to ANG(1–9) may occur through a specific carboxypeptidase that has been recently identified from a human heart failure ventricle cDNA library (7) and from a human lymphoma cDNA library (21). This enzyme has been termed ACE2 or ACEH and although having a similar catalytic domain to ACE, it is present in only heart, kidney, and testis. Recent studies using ACE2 knockout mice demonstrated that ACE2 plays an important role in regulation of ANG II levels in kidney and heart (6). As a carboxypeptidase, ACE2 is also known to remove a COOH-terminal amino acid from various other angiotensin peptides (6, 22). Although it is not known whether this enzyme is present in the glomeruli, our recent studies (20) and the present study suggest that this enzyme is present in rat cultured mesangial cells as well as in glomeruli. Moreover, conversion of ANG(1–9) to ANG II points to the existence of yet another unidentified carboxypeptidase in the rat glomeruli that can convert ANG(1–9) to ANG II (Fig. 7). Because not much is known about the specificity of the enzyme involved in this conversion, the possible role of ACE or chymase in conversion of ANG(1–9) to ANG II was tested in the present study. Preincubation of glomerular extracts with chymostatin (chymase inhibitor) did not block formation of ANG II in the presence of exogenous ANG(1–9) in either STZ or control rats. These results indicate that chymases are probably not involved in the conversion of ANG(1–9) to ANG II, at least in the glomeruli. Also, chymostatin did not affect conversion of ANG(1–9) to ANG(1–7) in either STZ or control glomerular extracts. On the other hand, captopril (ACE inhibitor) produced a significant inhibition of ANG(1–7) generation from exogenous ANG(1–9) in both STZ and control glomerular extracts, indicating that ACE is involved in the conversion of ANG(1–9) to ANG(1–7). In addition, captopril inhibited conversion of exogenous ANG(1–9) to ANG II by >50% in both STZ and control glomerular extracts. This result was surprising, as ANG(1–9) is converted to ANG II by removal of a single amino acid possibly via carboxypeptidase activity; therefore, inhibition of this enzyme by captopril (a dipeptidase inhibitor) cannot be explained at this time. It appears that captopril may have nonspecific effects on enzymes other than ACE in rat glomeruli or that this enzyme may be another kind of ACE-related carboxypeptidase that, unlike ACE2, is susceptible to ACE inhibitors. Although captopril reduced the ANG II/ANG(1–9) ratio in both STZ and control glomerular extracts, the ANG II/ANG(1–9) ratio remained higher in STZ glomerular extracts in the presence of captopril. These results emphasize that the conversion of exogenous ANG(1–9) to ANG II, at least in part, employs an enzyme other than ACE and its activity is stimulated in diabetic rat glomeruli.+ W1 t) X/ s; s- L9 M
0 @7 H2 J- X- I( @
Our findings that immunoreactive angiotensinogen and ANG II levels are increased in 4-wk STZ-diabetic glomerular extracts suggest early changes in the RAS in diabetic rat glomeruli. Previously, angiotensinogen mRNA levels were noted in one study to be increased in 8-wk STZ-diabetic rat kidney (1), decreased in 4-wk STZ-diabetic kidney (5); and yet another study reported no change in angiotensinogen mRNA levels in the kidney of 2-wk STZ-diabetic rats (12). More recently, Wehbi et al. (23) reported no change in angiotensinogen mRNA in glomeruli obtained from 2-wk STZ-diabetic rats. It is known that protein levels can change without changes in the mRNA levels by virtue of increased stability and/or increased translational efficiency of the mRNA. This could explain our findings of increased total angiotensinogen levels in diabetic glomeruli vs. no change in angiotensinogen mRNA as reported by Wehbi et al. Of note, in the present study, the anti-angiotensinogen antibody used for ELISA may also react with des-ANG I angiotensinogen in addition to intact angiotensinogen as stated in MATERIALS AND METHODS. Thus levels measured represent total angiotensinogen, which includes intact angiotensinogen and des-ANG I angiotensinogen. Also, values for total angiotensinogen obtained in this study by ELISA may be higher than those obtained using the classic renin assay, which measures only intact angiotensinogen.

Despite an increase in angiotensinogen levels, no significant increase in ANG I levels was observed in STZ-diabetic glomeruli. A possible explanation for these results could be increased conversion of ANG I into ANG II and other ANG peptides in the diabetic rat glomeruli as suggested by our HPLC experiments with glomerular extracts. An increase in ANG II levels in diabetic glomeruli has been suggested indirectly by previous studies showing downregulation of glomerular ANG II receptors in diabetic rats (3, 24). Ours is the first report showing increased ANG II levels in the diabetic glomeruli by direct measurement, lending further credence to the hypothesis that increased ANG II activity plays an important role in the development of diabetic glomerulosclerosis.! F/ c5 Z; p; g$ g5 A% f

In the present study, Western blot analysis demonstrated the presence of ACE in glomerular extracts. A distinct band of 75-kDa molecular mass was observed in glomerular extracts from STZ or control rats that is very similar to ACE (68 kDa) present in the cytosol of cultured mesangial cells (2). However, ACE protein levels were found to be similar in STZ and control rats. These observations are in agreement with the results of ELISA studies that indicated no differences in immunoreactive ACE levels between the two groups. Moreover, captopril inhibited only 20–30% conversion of exogenous ANG I to ANG II in STZ and control glomerular extracts. These results suggest the role of other enzymatic pathways (non-ACE) that may be responsible for the increased formation of ANG II in the presence of exogenous ANG I. One such pathway may be the conversion of ANG I to ANG II via intermediary ANG(1–9) peptide as shown by HPLC analysis of the glomerular extracts.

Our studies of the converting activities of diabetic glomerular extracts in the presence of exogenous ANG I and ANG(1–9) have yielded interesting information on other ANG peptides whose roles are being increasingly recognized. One such finding was the decreased formation of ANG(1–7) from both exogenous ANG I and ANG(1–9). ANG(1–7) has important physiological functions that are often opposed to those of ANG II (8). ANG(1–7) has diuretic and natriuretic effects (4), inhibits oxygen consumption in the rat proximal tubule (11), and attenuates ANG II-induced vasoconstriction, suggesting that it may be an endogenous antagonist of ANG II (17). In addition, ANG(1–7) has antiproliferative effects (10) and could oppose the effects of ANG II on matrix metabolism.
6 {! c- G6 v. w* p4 d+ j$ o
In conclusion, these findings support the hypothesis that diabetes is associated with stimulation of the glomerular RAS. Glomerular ANG II levels are increased by an increase in angiotensinogen substrate and by conversion of ANG I to ANG II via non-ACE mechanisms. Future studies are needed to further characterize the non-ACE mechanisms for ANG II formation so that effective inhibition of ANG II forming activities can be achieved in diabetes.

GRANTS
$ s% u& Y" o* @' M7 Z3 P4 T; _
This study was supported by a grant from the Department of Veterans Affairs.

ACKNOWLEDGMENTS& ?+ j6 F( {  t* a
. w9 Q+ I* u7 k( k' r( V) K
Portions of this work were presented at the meeting of the American Society of Nephrology, Philadelphia, PA, November 2002 and appear in abstract form in J Am Soc Nephrol 13: 533A, 2002.& ~4 A; s1 s% c% ^8 }  q4 h

FOOTNOTES4 |' c# f' n% o+ t2 ~
9 J, s% g3 @9 [; m: n- y5 i3 h
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.: z. t/ w# D- V4 Z6 j  ~0 E
4 r$ g$ t. k4 i+ Z) d/ Z9 T
REFERENCES

Anderson S, Jung FF, and Inglefinger JR. Renal renin-angiotensin system in diabetes: functional, immunohistochemical, and molecular biological correlations. Am J Physiol Renal Fluid Electrolyte Physiol 265: F477–F486, 1993.

Andrade MC, Quinto BM, Carmona AK, Ribas OS, Boim MA, Schor N, and Casarini DE. Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture. J Hypertens 16: 2063–2074, 1998.

Ballerman BJ, Skorecki KL, and Brenner BM. Reduced glomerular angiotensin II receptor density in early-untreated diabetes mellitus in the rat. Am J Physiol Renal Fluid Electrolyte Physiol 247: F110–F116, 1984.

Benter IF, Ferrario CM, Morris M, and Diz DI. Antihypertensive actions of angiotensin(1–7) in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 269: H313–H319, 1995.

Correa-Rotter R, Hostetter TH, and Rosenberg ME. Renin and angiotensinogen gene expression in experimental diabetes mellitus. Kidney Int 41: 796–804, 1992.

Crackower MA, Sarao R, Oudlt GY, Yagil C, Kozieradzki I, Scanga SE, Oliveira-dos-Santos AJ, Costa J, Zhang L, Pei Y, Scholey J, Ferrario CM, Manouklan AS, Chappel MC, Backx PH, Yagil Y, and Penninger JM. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417: 822–828, 2002.
* c& u, M0 H, h
Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart RE, and Acton S. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ Res 87: e1–e9, 2000.9 k6 d1 o8 Y& C0 F$ x: |

Ferrario CM and Iyer SN. Angiotensin-(1–7): a bioactive fragment of the renin-angiotensin system. Regul Pept 78: 13–18, 1998.1 q' f+ \9 f1 o6 q
- T& V  I' E  f; {; ]9 Q0 `
Fox J, Guan S, Hymel AA, and Navar G. Dietary Na and ACE inhibition effects on renal tissue angiotensin I and II and ACE activity in rats. Am J Physiol Renal Fluid Electrolyte Physiol 262: F902–F909, 1992.

Freeman EJ, Chisolm GM, Ferrario CM, and Tallant EA. Angiotensin(1–7) inhibits vascular smooth muscle cell growth. Hypertension 28: 104–108, 1996.0 u1 W0 V" V* N6 G5 _

Handa RJ, Ferrario CM, and Strandhoy JW. Renal action of angiotensin(1–7): in vivo and in vitro studies. Am J Physiol Renal Fluid Electrolyte Physiol 270: F141–F147, 1996.
; g2 R7 ~% X! ^, d. y5 V
Kalinyak JE, Sechi LA, Griffin CA, Don BR, Tavangar K, Kraemer FB, Hoffman AR, and Schambelan M. The renin-angiotensin system in streptozotocin-induced diabetes mellitus in the rat. J Am Soc Nephrol 4: 1337–1345, 1993.

Kohzuki M, Yasujima M, Kanazawa M, Yoshida K, Fu LP, Obara K, Saito T, and Abe K. Antihypertensive and renal-protective effects of losartan in streptozotocin diabetic rats. J Hypertens 13: 97–103, 1995.2 i! K; m/ ^" x4 w. c" [! K
5 ^  _  ]: t1 V1 L3 B
Leehey DJ, Singh AK, Alavi N, and Singh R. Role of angiotensin II in diabetic nephropathy. Kidney Int 58: S93–S98, 2000., ^. f# Z* _% o1 i/ l2 b1 r0 I/ \

Lewis EJ, Hunsicker LG, Bain RP, Rohde RD, and Collaborative Study Group. The effect of angiotensin-converting enzyme inhibition on diabetic nephropathy. N Engl J Med 329: 1456–1462, 1993.! m0 R9 O+ d/ F# T
! D8 M# Z# g% |7 _' D
Remuzzi A, Fassi A, Sangalli F, Malanchini B, Mohamed EI, Bertani T, and Remuzzi G. Prevention of renal injury in diabetic MWF rats by angiotensin II antagonism. Exp Nephrol 6: 28–38, 1998.2 K, T. A! z) N# D" @+ f

Santos RAS, Campagnole-Santos MJ, and Andrade SP. Angiotensin-(1–7): an update. Regul Pept 91: 45–62, 2000." r7 m, z( @9 v) \  o

Seikaly MG, Arant BS Jr, and Seney FD Jr. Endogenous angiotensin concentrations in specific intrarenal fluid compartments in the rat. J Clin Invest 86: 352–357, 1990.6 y2 [0 q9 m0 R* ~2 f
% B& Q8 N) H% v# u: i' H
Singh R, Alavi N, Singh AK, and Leehey DJ. Role of angiotensin II in glucose-induced inhibition of mesangial matrix degradation. Diabetes 48: 2066–2073, 1999.
0 J( k" G' \9 W$ l% s# h
Singh R, Singh AK, Alavi N, and Leehey DJ. Mechanism of increased angiotensin II levels in glomerular mesangial cells cultured in high glucose. J Am Soc Nephrol 14: 873–880, 2003.3 S' k" ]0 }3 @+ u

Tipnis SA, Hooper NM, Hyde R, Karran E, Christie G, and Turner AJ. A human homolog of angiotensin-converting enzyme. J Biol Chem 275: 33238–33243, 2000.* F, d6 b! G! k; k4 P9 M# U2 g

Vickers C, Hales P, Kaushik V, Dick L, Garin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, Acton S, Patane M, Nichols A, and Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277: 14838–14843, 2002.% M2 n0 M; x1 q: x6 A

Wehbi GJ, Zimpelmann J, Carey RM, Levine DZ, and Burns KD. Early streptozotocin-diabetes mellitus down regulates rat kidney AT2 receptors. Am J Physiol Renal Physiol 280: F254–F265, 2001.
5 d7 G! I* ?; V9 V' M& z
Wilkes BM. Reduced glomerular angiotensin II receptor density in diabetes mellitus in the rat: time course and mechanism. Endocrinology 210: 1291–1298, 1987.(Rekha Singh, Ashok K. Sin)

收藏0 分享0 顶0 踩0