Abstract
We recently reported that urinary excretion rates of angiotensinogen (UAGT) provide a specific index of intrarenal renin-angiotensin (ANG) system (RAS) status in ANG II-dependent hypertensive rats. When this is shown to be applicable to human subjects, a diagnostic test to identify those hypertensive patients most likely to respond to an RAS blockade could provide useful information to allow a mechanistic rationale for selection of an optimized approach to treatment of hypertensive patients. However, simple and accurate methods to measure human angiotensinogen (hAGT) are unavailable. For future studies of human subjects, we developed antibodies and a sensitive and specific quantification system for hAGT using a sandwich ELISA. We raised two antibodies against hAGT: a mouse monoclonal antibody and a rabbit polyclonal antibody. The standard curve of this ELISA exhibited a high linearity (0.31–20 ng/ml). The correlation coefficient was >0.99. Plasma angiotensinogen concentrations of healthy volunteers ranged from 28 to 71 μg/ml (n = 10). The ratio of UAGT to urinary creatinine concentration ranged from 5.0 to 30 μg/g (n = 7). Intra- and interassay coefficients of variation ranged from 4.4 to 5.5% and from 4.3 to 7.0%, respectively. This ELISA system had no cross-reactivity with major proteins in proteinuric urine samples, such as human albumin, immunoglobulin, or transferrin. Moreover, the cross-reactivity of the system with angiotensin peptides was also negligible. This hAGT ELISA will be a useful tool to investigate the relationship of UAGT and reactivity to antihypertensive drugs in hypertensive patients.
Keywords: renin-angiotensin system, plasma, urine
The Renin-Angiotensin System (RAS) is well known to play an important role in blood pressure regulation and fluid and electrolyte homeostasis (18). In recent years, the focus of interest on the RAS has shifted to a main emphasis on the role of the local/tissue RAS in specific tissues (5). Emerging evidence has demonstrated the importance of the tissue RAS in the brain (1), heart (4), adrenal glands (15), and vasculature (3, 6), as well as the kidneys (18). There is substantial evidence that the major fraction of angiotensin II in renal tissues is generated locally from angiotensinogen delivered to the kidney, as well as from angiotensinogen locally produced by proximal tubule cells (7). Renin secreted by juxtaglomerular apparatus cells into the renal interstitium and vascular compartment also provides a pathway for the local generation of angiotensin I (17). Angiotensin-converting enzyme is abundant in the rat kidney and is present in the proximal and distal tubules, collecting ducts, and renal endothelial cells (2). Angiotensin I delivered to the kidney can also be converted to angiotensin II (14). Therefore, all the components necessary for generation of intrarenal angiotensin II are present along the nephron (18).
The presence of all components of the RAS in the kidney provides a great flexibility and independence in regulating intrarenal levels of angiotensin II, and the differential regulation of angiotensin peptide levels in plasma and kidney has been established (19). Recently, we reported that urinary excretion rates of angiotensinogen provide a specific index of intrarenal RAS status in angiotensin II-dependent hypertensive rats (8-11, 13). When this is shown to be applicable to human subjects, a diagnostic test to identify those hypertensive patients most likely to respond to blockade of the RAS could provide useful information to allow a mechanistic rationale for selection of an optimized approach to treatment of hypertensive patients. ELISA for human angiotensinogen has been reported (16, 21). However, simple and accurate methods to measure human angiotensinogen are unavailable. For future studies of human subjects, we developed antibodies and a sensitive and specific quantification system for human angiotensinogen using a microtiter plate-based sandwich-type ELISA.
MATERIALS AND METHODS
Protocol
The experimental protocol of this study was approved by the Institutional Review Board of Tulane University. All samples were obtained from healthy volunteers, who provided written informed consent.
Preparation of expression vectors for human angiotensinogen
The full length, except for the signal peptide (1-33 aa), of the human angiotensinogen (34-485 aa) gene (gene identification no. 73622269) was amplified by PCR using a high-fidelity Pyrococcus furiosus DNA polymerase (Promega) with sense (5′-CGG GAT CCG ACC GGG TGT ACA TAC ACC CC-3′) and antisense (5′-CGG TTG GGC GAC TCG TGT CGT GAG CTC GCC-3′) primers from the human adult liver cDNA library (Clontech). Then this fragment was inserted into pGEX4T1 expression vectors (Promega) with the glutathione S-transferase (GST) tag at the 5′-end by the restriction enzymes BamH I and Sal I.
Preparation of recombinant proteins for human angiotensinogen
The recombinant constructs were transformed to a high-efficiency expression bacterial strain (Takara). Large-scale bacterial cultures were induced with isopropyl-β-d-thiogalactopyranoside (Takara) and harvested for protein purification. GST-tagged proteins were purified using glutathione beads (Upstate) in native conditions.
Antibody preparation
We raised two antibodies for human angiotensinogen: a mouse monoclonal and a rabbit polyclonal antibody. The monoclonal antibody was raised in mouse against recombinant protein of human angiotensinogen (see above). The polyclonal antibody was raised in rabbit against synthetic oligopeptide corresponding to human angiotensinogen (72-89 aa). Both antibodies were affinity purified.
Western blot
Western blot analysis was performed as previously described (12, 20, 22) using the LI-COR Odyssey infrared imaging system.
Epitope mapping
The epitopes of these antibodies were determined by Western blot analysis using a variety of lengths of recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa).
Plate preparation
The ELISA plates were coated with the polyclonal antibody against human angiotensinogen (100 μl/well in 100 mmol/l carbonate buffer, pH 9.5) at 4°C overnight. The plates were washed with PBS and blocked with 1% bovine serum albumin (200 μl/well) in PBS containing 0.05% NaN3 at 4°C overnight. The plates were stored at 2–8°C.
Sample collections
Peripheral blood samples from healthy volunteers were collected into EDTA-containing tubes, and plasma samples were separated after centrifugation. First-morning-urine samples were also collected from healthy volunteers.
Development of sandwich ELISA
Highly purified angiotensinogen from human plasma was used as the standard. Human angiotensinogen standards (0.31–20 ng/ml diluted in ELISA buffer), plasma (1:8,000 dilution in ELISA buffer), and urine (1:8 dilution in ELISA buffer) samples (100 μl/well) were added to each well of the plates and incubated at 37°C for 1 h. Then the plates were washed a total of seven times with a washing buffer (PBS containing 0.05% Tween 20, pH 7.5). After the plates were incubated with horseradish peroxidase-labeled monoclonal antibody against human angiotensinogen (100 μl/well, 1:30 dilution in antibody solution) at 37°C for 30 min, they were washed a total of nine times with the washing buffer. Then the plates were incubated with 3,3′,5,5′-tetramethylbenzidine solution (100 μl/well) under light-protected conditions at room temperature for 30 min. The reaction was stopped by treatment of the plates with sulfuric acid (100 μl/well, 0.5 mol/l). The absorbance values were measured at 450 nm.
Measurement of urinary creatinine
The urinary creatinine concentrations were measured by an automated machine (model DCA 2000+, Bayer) with microalbumin/creatinine reagent kits (Bayer). The urinary angiotensinogen concentrations were normalized to urinary creatinine concentrations.
Statistical analysis
Statistical analysis was performed using a one-way factorial ANOVA with post hoc Scheffé's F test. Values are means ± SE. P < 0.05 was considered significant.
RESULTS AND DISCUSSION
Epitope mapping of antibodies
An equal amount (10 ng) of various lengths of recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa) was blotted on nitrocellulose membranes, and epitope mapping was done by Western blot. The polyclonal antibody against human angiotensinogen recognized GST-tagged recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa), but not mock protein or GST protein (Fig. 1A). The monoclonal antibody against human angiotensinogen recognized GST-tagged recombinant proteins of human angiotensinogen (34-485 aa), but not GST-tagged recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, and 34-393 aa), mock protein, or GST protein. An antibody against GST protein recognized GST-tagged recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa), as well as GST protein, but not mock protein. These data clearly indicate that the epitope for the polyclonal antibody against human angiotensinogen is located on the NH2 terminus of human angiotensinogen (34-124 aa) and that the epitope for the monoclonal antibody against human angiotensinogen is on the COOH terminus of human angiotensinogen (394-485 aa).
Fig. 1.
A: epitope mapping. GST, glutathione S-transferase; hAGT, human angiotensinogen; Poly and Mono, poly- and monoclonal antibodies. B: absorbance values. Polyclonal antibody-coated ELISA plates were incubated at 37°C for 1 (open bars), 8 (gray bars), or 15 (solid bars) days. C: a corresponding standard curve.
Plate stability
To address their stability, we incubated the polyclonal antibody-coated ELISA plates at 37°C for 1, 8, or 15 days. Then different concentrations of human angiotensinogen standards were applied to the plates and processed, and the absorbance values were obtained. Each concentration of human angiotensinogen standards (n = 6) exhibited similar absorbance values beyond 15 days (Fig. 1B). These data clearly indicate that the polyclonal antibody-coated ELISA plates are stable ≥15 days after antibody coating.
Standard curves
The corresponding standard curve exhibited a high linearity (0.31–20 ng/ml), with absorbance values ranging from 0.03 to 2.34 (n = 6 at each concentration; Fig. 1C). The correlation coefficient was >0.99.
Detection limit
The lowest sensitivity for this system (0.31 ng/ml) was determined using the guidelines of the National Committee for Clinical Laboratory Standards Evaluation Protocols.
Plasma and urinary angiotensinogen concentrations
Angiotensinogen concentrations in plasma obtained from 10 healthy volunteers ranged from 28 to 71 μg/ml (Fig. 2A). Angiotensinogen concentrations in urine obtained from seven healthy volunteers ranged from 7.1 to 35 ng/ml (Fig. 2B). Urinary creatinine concentrations of seven healthy volunteers were 0.93–2.2 mg/ml. The ratio of urinary angiotensinogen concentration to urinary creatinine concentration was 5.0 –30 μg/g (Fig. 2C).
Fig. 2.
A: angiotensinogen concentrations in plasma obtained from 10 healthy volunteers. Samples were applied to the ELISA system in triplicate, and averaged values were obtained. B: angiotensinogen concentrations in urine obtained from 7 healthy volunteers. Samples were applied to the ELISA system in triplicate, and averaged values were obtained. C: ratio of urinary angiotensinogen concentration to urinary creatinine concentration.
Addition and recovery test
Different known concentrations of human angiotensinogen standards were added to a human plasma sample or a human urine sample, and the added angiotensinogen concentrations were calculated as the difference between angiotensinogen concentrations in samples with and without added angiotensinogen. Recovery rates were >82.9% for plasma and >87.6% for urine (Table 1).
Table 1.
Addition-and-recovery test
| Theoretical Values, ng/ml | Measured Values, ng/ml | % |
|---|---|---|
| Human plasma | ||
| 9.29 | 7.70 | 82.9 |
| 6.79 | 6.50 | 95.7 |
| 5.54 | 4.98 | 89.9 |
| Human urine | ||
| 7.47 | 6.54 | 87.6 |
| 4.97 | 4.69 | 94.4 |
| 3.72 | 3.38 | 90.9 |
Different known concentrations of human angiotensinogen standards were added to human plasma or urine sample, and added angiotensinogen concentrations were calculated as the difference between angiotensinogen concentrations in samples with and without added angiotensinogen.
Sample stability
To address stability of samples, we performed two tests: a freeze-and-thaw test and an accelerated test. In a freeze-and-thaw test, one plasma sample was frozen and thawed 0, 1, 2, and 3 times, and plasma angiotensinogen concentrations were measured by the ELISA system. No significant differences were observed (Fig. 3A). In an accelerated test, one plasma sample was incubated at 37°C for 0, 1, 3, and 7 days, and plasma angiotensinogen concentrations were measured by the ELISA system. No differences were observed (Fig. 3B). These data clearly indicate that angiotensinogen in plasma samples is stable without preservatives or proteinase inhibitors, except EDTA.
Fig. 3.
A: freeze-and-thaw test. B: accelerated test.
Intra-assay test
Intra-assay tests were performed using three different concentrations of human angiotensinogen standards. Coefficients of variation among the wells ranged from 4.4 to 5.5% (Table 2).
Table 2.
Intra-assay test
| Measured Values, ng/ml | SD | %CV | n |
|---|---|---|---|
| 8.81 | 0.39 | 4.4 | 24 |
| 2.14 | 0.11 | 5.1 | 24 |
| 0.73 | 0.04 | 5.5 | 24 |
Intra-assay tests were performed using 3 different concentrations of human angiotensinogen standards. CV, coefficient of variation.
Interassay test
Interassay tests were performed using three different concentrations of human angiotensinogen standards. Coefficients of variation among the plates ranged from 4.3 to 7.0% (Table 3).
Table 3.
Interassay test
| Measured Values, ng/ml | SD | %CV | n |
|---|---|---|---|
| 8.77 | 0.38 | 4.3 | 32 |
| 2.14 | 0.10 | 4.7 | 32 |
| 0.71 | 0.05 | 7.0 | 32 |
Interassay tests were performed using 3 different concentrations of human angiotensinogen standards.
Specificity
To confirm cross-reactivity with other proteins, several human proteins (20 μg/ml) were applied to the ELISA system and processed. Cross-reactivity for other human proteins in this ELISA system was negligible (Table 4). In addition, downstream products of angiotensinogen (20 μg/ml) were also applied to the ELISA system. The cross-reactivity for angiotensin peptides in this ELISA system was also negligible (Table 4).
Table 4.
Specificity
| Human Proteins | Cross-Reactivity, % |
|---|---|
| Human angiotensinogen | 100 |
| Human tenascin C | <0.1 |
| Human growth-regulated oncogene-α | <0.1 |
| Human growth-regulated oncogene-β | <0.1 |
| Human angiopoietin-like 3 | <0.1 |
| Human osteopontin | <0.1 |
| Human stem cell factor | <0.1 |
| Human leptin | <0.1 |
| Human granulocyte-colony-stimulating factor | <0.1 |
| Human albumin | <0.1 |
| Human immunoglobulin | <0.1 |
| Human transferrin | <0.1 |
| Angiotensin I | <0.1 |
| Angiotensin II | <0.1 |
| Angiotensin III | <0.1 |
| Angiotensin IV | <0.1 |
| Angiotensin-(1-7) | <0.1 |
| Angiotensin-(1-9) | <0.1 |
Effect of pH of test samples on sensitivity of ELISA
As described above, plasma samples were diluted 1:8,000 and urine samples 1:8 in PBS-based dilution buffer (pH 7.0); therefore, pH of diluted plasma and urine samples should be ∼7.0. To address this issue, seven urine samples were prepared (pH 5.5–7.0, 6.3 ± 0.6). After the samples were diluted with the dilution buffer, pH values of the samples were integrated (pH 6.7–7.0, 6.8 ± 0.2). Moreover, diluted urine samples worked well with ELISA (15.9 ± 6.4 ng/ml). These data clearly indicate that the change of urine sample pH does not affect the sensitivity of ELISA.
Conclusion
We developed antibodies and a sensitive and specific quantification system for human angiotensinogen using a sandwich ELISA. This human angiotensinogen ELISA will be a useful tool to investigate the relationship between urinary angiotensinogen excretion rate and reactivity to anti-hypertensive drugs in hypertensive subjects.
ACKNOWLEDGMENTS
The authors acknowledge the excellent technical assistance of My-Linh Rauv, Duy V. Tran, and Dale M. Seth (Tulane University).
GRANTS
This study was supported by National Institutes of Health Grants R01 DK-072408, P20 RR-017659, and R01 HL-026371, the Health Excellence Fund from the Louisiana Board of Regents, and Sankyo Co., Ltd.
Footnotes
Portions of this study were presented in abstract form at the 17th Scientific Sessions of the Inter-American Society of Hypertension, Miami Beach, FL, May 6 –10, 2007.
REFERENCES
- 1.Baltatu O, Silva JA, Jr, Ganten D, Bader M. The brain renin-angiotensin system modulates angiotensin II-induced hypertension and cardiac hyper-trophy. Hypertension. 2000;35:409–412. doi: 10.1161/01.hyp.35.1.409. [DOI] [PubMed] [Google Scholar]
- 2.Casarini DE, Boim MA, Stella RC, Krieger-Azzolini MH, Krieger JE, Schor N. Angiotensin I-converting enzyme activity in tubular fluid along the rat nephron. Am J Physiol Renal Physiol. 1997;272:F405–F409. doi: 10.1152/ajprenal.1997.272.3.F405. [DOI] [PubMed] [Google Scholar]
- 3.Danser AH, Admiraal PJ, Derkx FH, Schalekamp MA. Angiotensin I-to-II conversion in the human renal vascular bed. J Hypertens. 1998;16:2051–2056. doi: 10.1097/00004872-199816121-00029. [DOI] [PubMed] [Google Scholar]
- 4.Dell'Italia LJ, Meng QC, Balcells E, Wei CC, Palmer R, Hageman GR, Durand J, Hankes GH, Oparil S. Compartmentalization of angiotensin II generation in the dog heart: evidence for independent mechanisms in intravascular and interstitial spaces. J Clin Invest. 1997;100:253–258. doi: 10.1172/JCI119529. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Dzau VJ, R Re. Tissue angiotensin system in cardiovascular medicine. A paradigm shift? Circulation. 1994;89:493–498. doi: 10.1161/01.cir.89.1.493. [DOI] [PubMed] [Google Scholar]
- 6.Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994;74:1141–1148. doi: 10.1161/01.res.74.6.1141. [DOI] [PubMed] [Google Scholar]
- 7.Ingelfinger JR, Pratt RE, Ellison K, Dzau VJ. Sodium regulation of angiotensinogen mRNA expression in rat kidney cortex and medulla. J Clin Invest. 1986;78:1311–1315. doi: 10.1172/JCI112716. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Kobori H, Harrison-Bernard LM, Navar LG. Enhancement of angiotensinogen expression in angiotensin II-dependent hypertension. Hypertension. 2001;37:1329–1335. doi: 10.1161/01.hyp.37.5.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kobori H, Harrison-Bernard LM, Navar LG. Expression of angiotensinogen mRNA and protein in angiotensin II-dependent hypertension. J Am Soc Nephrol. 2001;12:431–439. doi: 10.1681/asn.v123431. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Kobori H, Harrison-Bernard LM, Navar LG. Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production. Kidney Int. 2002;61:579–585. doi: 10.1046/j.1523-1755.2002.00155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kobori H, Nishiyama A, Harrison-Bernard LM, Navar LG. Urinary angiotensinogen as an indicator of intrarenal angiotensin status in hyper-tension. Hypertension. 2003;41:42–49. doi: 10.1161/01.hyp.0000050102.90932.cf. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Kobori H, Ozawa Y, Suzaki Y, Nishiyama A. Enhanced intrarenal angiotensinogen contributes to early renal injury in spontaneously hyper-tensive rats. J Am Soc Nephrol. 2005;16:2073–2080. doi: 10.1681/ASN.2004080676. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kobori H, Prieto-Carrasquero MC, Ozawa Y, Navar LG. AT1 receptor mediated augmentation of intrarenal angiotensinogen in angiotensin II-dependent hypertension. Hypertension. 2004;43:1126–1132. doi: 10.1161/01.HYP.0000122875.91100.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Komlosi P, Fuson AL, Fintha A, Peti-Peterdi J, Rosivall L, Warnock DG, Bell PD. Angiotensin I conversion to angiotensin II stimulates cortical collecting duct sodium transport. Hypertension. 2003;42:195–199. doi: 10.1161/01.HYP.0000081221.36703.01. [DOI] [PubMed] [Google Scholar]
- 15.Mazzocchi G, Malendowicz LK, Markowska A, Albertin G, Nussdorfer GG. Role of adrenal renin-angiotensin system in the control of aldosterone secretion in sodium-restricted rats. Am J Physiol Endocrinol Metab. 2000;278:E1027–E1030. doi: 10.1152/ajpendo.2000.278.6.E1027. [DOI] [PubMed] [Google Scholar]
- 16.Mizrahi J, Coezy E, Auzan C, Corvol P, Menard J. Monoclonal antibodies to human angiotensinogen: development of an ELISA for measurement of hepatocyte cultured cells content. Clin Exp Hypertens. 1987;9:1479–1491. doi: 10.3109/10641968709158997. [DOI] [PubMed] [Google Scholar]
- 17.Moe OW, Ujiie K, Star RA, Miller RT, Widell J, Alpern RJ, Henrich WL. Renin expression in renal proximal tubule. J Clin Invest. 1993;91:774–779. doi: 10.1172/JCI116296. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Navar LG, Harrison-Bernard LM, Nishiyama A, Kobori H. Regulation of intrarenal angiotensin II in hypertension. Hypertension. 2002;39:316–322. doi: 10.1161/hy0202.103821. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Navar LG, Prieto-Carrasquero MC, Kobori H. Renal renin-angiotensin system. In: Kastin AJ, editor. Handbook of Biologically Active Peptides. 1st ed. Elsevier; Burlington, MA: 2006. pp. 1235–1242. chapt. 170. [Google Scholar]
- 20.Ozawa Y, Kobori H, Suzaki Y, Navar LG. Sustained renal interstitial macrophage infiltration following chronic angiotensin II infusions. Am J Physiol Renal Physiol. 2007;292:F330–F339. doi: 10.1152/ajprenal.00059.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Rubin I, Lykkegaard S, Olsen AA, Selmer J, Ballegaard M. Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay. J Immunoassay. 1988;9:257–274. doi: 10.1080/01971528808053216. [DOI] [PubMed] [Google Scholar]
- 22.Suzaki Y, Ozawa Y. Kobori H. Intrarenal oxidative stress and augmented angiotensinogen are precedent to renal injury in Zucker diabetic fatty rats. Int J Biol Sci. 2007;3:40–46. doi: 10.7150/ijbs.3.40. [DOI] [PMC free article] [PubMed] [Google Scholar]