1Department of Medicine, Veterans Affairs Hospital, Hines, Illinois, and Loyola University Stritch School of Medicine, Maywood; and 2Hektoen Institute for Medical Research, Chicago, Illinois
Submitted 23 April 2003 ; accepted in final form 26 January 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
diabetes; angiotensin peptides; angiotensin(19)
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(19) 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.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male Sprague-Dawley rats (200300 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.
Preparation of Glomerular Extracts
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
Antibodies to angiotensinogen were prepared using renin substrate tetradecapeptide [angiotensinogen(114)] (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(114), 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(114) as the standard and the levels of angiotensinogen in samples were calculated from the standard curve.
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 1114 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.
Measurement of ANG I and ANG II Levels in Glomerular Extracts
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 05 min 90% A:10% B, 532 min 60% A: 40% B, 3240 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.
|
|
Measurement of ACE Levels in Glomerular Extracts
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).
Analysis of ANG II Formation from Exogenous ANG I or ANG(19) Using HPLC
Formation of ANG II from exogenous ANG I or ANG(19) 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(28) [ANG III], ANG(38) [ANG IV], ANG(19), and ANG(17) were purchased from Bachem Biosciences; breakdown products of ANG I [ANG(210), ANG(310), ANG(410), ANG(510)] and ANG(19) [ANG(29), ANG(39), and ANG(49)] 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(28) and ANG(38) were found to be overlapping with elution times of ANG(510) and ANG(410), respectively (Table 1). Glomerular extracts were incubated with 104 M of exogenous ANG I or ANG(19) 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(19). The results are presented as the ratio of ANG peptides to exogenous ANG I or ANG(19). In some experiments, glomerular extracts were prepared in PBS containing EDTA to inhibit peptidases, and conversion of exogenous ANG I or ANG(19) to ANG II was analyzed by HPLC.
|
Statistics
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 <0.05 was considered to be significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 < 0.0001, n = 7). Body weights of diabetic rats were significantly lower compared with control rats (STZ: 243 ± 18 g vs. control: 330 ± 6 g; P < 0.001, n = 7). In STZ-diabetic rats, kidney weights increased significantly compared with controls. The kidney weight to body weight ratio was 11 ± 0.7 g/kg in STZ rats compared with 6.6 ± 0.1 g/kg in control rats (P < 0.001, n = 7).
Effect of Diabetes on Glomerular Angiotensinogen, ANG I, and ANG II
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).
|
Effect of Diabetes on Glomerular ACE
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
First, to test whether exogenous ANG I was stable in incubation buffer (PBS), ANG I (104 M) was incubated in PBS buffer at 37°C for a period of 14 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 (104 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(19) and ANG(17). The identity of another peak recorded at 19 min could not be established with certainty because both ANG(28) and ANG(510) 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(19)/ANG I ratio in diabetic rats suggesting increased formation of ANG(19) from exogenous ANG I and a decrease in ANG(17)/ANG I ratio, suggesting a decrease in formation of ANG(17) from exogenous ANG I in diabetic rats (Fig. 4A).
|
|
Preincubation of glomerular extracts with captopril (104 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(17)/ANG I or ANG(19)/ANG I ratios was observed in either control or STZ diabetic glomerular extracts (data not shown).
Role of ANG(19) in ANG II Formation in Glomerular Extracts
The possibility of ANG II formation from the intermediary angiotensin peptide ANG(19) was examined by HPLC. Glomerular extract samples from control and STZ-diabetic rats were incubated with exogenous ANG(19) (104 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(19) to ANG(17) and ANG II. In STZ glomerular extracts, the ANG(17)/ANG(19) ratio was significantly decreased compared with control extracts (Table 3). In contrast, the ANG II/ANG(19) ratio was significantly increased in STZ rat glomerular extracts compared with control glomerular extracts (Table 3).
|
|
Effect of Enzyme Inhibitors on Conversion of ANG(19) to ANG II in Glomerular Extracts
Although not much data are available on the putative carboxypeptidase enzyme involved in the conversion of ANG(19) to ANG II, the role of ACE and chymase in ANG II formation from exogenous ANG(19) was tested. Glomerular extracts from control or STZ rats were preincubated with captopril (104 M) for 30 min at 37°C followed by incubation with exogenous ANG(19) for 1 h and analysis by HPLC. Captopril treatment produced a significant decrease in the formation of ANG(17) and ANG II from exogenous ANG(19) in both STZ and control rats (Table 3). However, the ANG(17)/ANG(19) ratio remained lower and the ratio of ANG II/ANG(19) 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(19) had no effect on the conversion of ANG(19) to ANG II and ANG(17) in either STZ or control rats (data not shown).
Conversion of Exogenous ANG II in Glomerular Extracts
In separate experiments, when STZ and control glomerular extracts were incubated with exogenous ANG II, generation of ANG(16) (identified based on its elution time) and two other unidentified peaks were observed (Fig. 6). No significant differences were observed in the ANG(16)/ANG II ratio between the two groups (STZ: 0.078 ± 0.01 vs. control: 0.065 ± 0.01; P = NS, n = 3).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The conversion of ANG I to ANG(19) 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(19) to ANG II points to the existence of yet another unidentified carboxypeptidase in the rat glomeruli that can convert ANG(19) 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(19) 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(19) in either STZ or control rats. These results indicate that chymases are probably not involved in the conversion of ANG(19) to ANG II, at least in the glomeruli. Also, chymostatin did not affect conversion of ANG(19) to ANG(17) in either STZ or control glomerular extracts. On the other hand, captopril (ACE inhibitor) produced a significant inhibition of ANG(17) generation from exogenous ANG(19) in both STZ and control glomerular extracts, indicating that ACE is involved in the conversion of ANG(19) to ANG(17). In addition, captopril inhibited conversion of exogenous ANG(19) to ANG II by >50% in both STZ and control glomerular extracts. This result was surprising, as ANG(19) 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(19) ratio in both STZ and control glomerular extracts, the ANG II/ANG(19) ratio remained higher in STZ glomerular extracts in the presence of captopril. These results emphasize that the conversion of exogenous ANG(19) to ANG II, at least in part, employs an enzyme other than ACE and its activity is stimulated in diabetic rat glomeruli.
|
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.
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 2030% 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(19) 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(19) have yielded interesting information on other ANG peptides whose roles are being increasingly recognized. One such finding was the decreased formation of ANG(17) from both exogenous ANG I and ANG(19). ANG(17) has important physiological functions that are often opposed to those of ANG II (8). ANG(17) 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(17) has antiproliferative effects (10) and could oppose the effects of ANG II on matrix metabolism.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |