Diabetes affects 220 million people worldwide, including 24 million Americans, and is the sixth leading cause of death in the United States. It is associated with increased incidence of functional and structural alterations in the kidneys, eventually leading to end-stage renal failure in many patients. Diabetic nephropathy (DN) is the most common cause of end-stage renal failure in the United States, accounting for 45% of patients starting dialysis.1,2 Type 2 diabetes mellitus (T2D) is the most common type of diabetes accounting for 90% to 95% of all diagnosed cases of diabetes and affecting 8% of the US population.3,4 Obesity has been identified as the principal risk factor associated with the rising prevalence of T2D.5 The epidemic proportions of obesity and diabetes justify the enormous effort to identify novel pathways and mechanisms involved in their prevention and treatment. Diabetes is a chronic and debilitating disease that is characterized by progressive albuminuria, declining glomerular filtration rate (GFR), functional and structural deterioration of the kidney, and increased risk of cardiovascular disease.
RENIN-ANGIOTENSIN SYSTEM CASCADE
The importance of the renin-angiotensin system (RAS) in the regulation of blood pressure (BP) and fluid and electrolyte homeostasis has been well recognized.6,7 As indicated in Figure 1, the balance between vasoconstrictor and vasodilator effects is determined by the actions of angiotensin II and angiotensin 1–7. The formation of angiotensin II is dependent on the substrate availability of angiotensinogen (AGT) and angiotensin I and the activities of renin, angiotensin-converting enzyme (ACE), ACE2, and ACE-independent enzymatic pathways including serine proteases such as chymase. Angiotensin 1–7 can be formed directly from angiotensin II hydrolyzed by ACE2 or indirectly from angiotensin I via an intermediate step of the formation of angiotensin 1–9 hydrolyzed by ACE2 and ACE in sequence. The actions of angiotensin II are determined by signaling via angiotensin II type 1 (AT1) and type 2 (AT2) receptors8 and the putative angiotensin 1–7 receptor, Mas.9,10
INTRARENAL RAS IN DIABETES
Emerging evidence has demonstrated the importance of local RAS11 in the brain,12 heart,13 adrenal glands,14 vasculature,15,16 and kidneys.6–8,17 In particular, the renal RAS is unique because all of the components necessary to generate intrarenal angiotensin II are present along the nephron in both interstitial and intratubular compartments (Fig. 2).7,10 Angiotensinogen has been localized primarily at the mRNA level,18 and immunoreactive AGT7 has been found in the proximal tubules. Detailed localization of the AGT in the proximal tubular segments was controversial; however, divergent localization of AGT mRNA and protein was reported recently.19,20 The proximal convoluted tubules and proximal straight tubules exhibit positive immunostaining for AGT (Fig. 3). Furthermore, weak expression of AGT protein was also observed in glomeruli and vasa recta, whereas the distal tubules and collecting ducts are negative.21–25 Angiotensinogen mRNA is found strongly in the proximal straight tubules. Recent evidence suggests that AGT is constitutively secreted in the proximal straight tubule as in the liver.26 Renin mRNA and renin-like activity have been demonstrated in cultured proximal tubular cells, and low concentrations of renin have been detected in proximal tubule fluid in rats27–30 Moreover, there is abundant expression of ACE mRNA31 and protein32,33 on brush border membranes of proximal tubules of human kidney. Finally, ACE is also present in proximal and distal tubular fluid but is greater in proximal tubule fluid.34 Angiotensin-converting enzyme 2 protein is found in proximal tubule cells, glomerular podocytes,35 and tunica media of renal arterioles.36 In addition, experimental studies have shown that intrarenal ACE-independent, serine-protease–dependent pathways have an increased role in the conversion of angiotensin I to angiotensin II in diabetic models, thus influencing renal hemodynamics.37
Data concerning intrarenal RAS states in diabetes are inconsistent.38–40 Although various studies support an association between RAS and DN, direct measurements have failed to establish that intrarenal angiotensin II is consistently elevated in diabetes.40 However, intrarenal AGT levels are elevated in patients with DN.41 In rodent diabetic models, renin content varies, and ACE expression has been shown to be increased or unchanged in glomeruli and vessels.39,42 In the T2D mouse kidney, proximal tubule ACE immunostaining is decreased, whereas ACE2 immunostaining is increased compared with control mice (Fig. 3).37 However, AT1 receptor protein levels were significantly elevated in renal cortex from streptozotocin-induced diabetic rats compared with control rats associated with down-regulation of AT2 receptors.39,42 The cortical collecting ducts of streptozotocin-induced diabetic kidneys displayed a striking increase in AT1 receptor immunostaining intensity relative to control kidneys (Fig. 3).39 Moreover, it was recently shown that prorenin expression is elevated in the cortical collecting ducts of type 1 diabetic (T1D) rats.43 Furthermore, studies in models of T2D show increased intrarenal angiotensin II levels and AGT mRNA levels, which are prevented by treatment with an angiotensin II receptor blocker (ARB).44 Finally, increases in renal cortical AGT (Fig. 3) and angiotensin II levels associated with increased reactive oxygen species (ROS) and renal injury have been observed in Zucker diabetic fatty obese rats compared with control lean rats.46,47
Recent studies have identified a major role for intrarenal ACE-independent formation of angiotensin II in T2D. Park et al.37 reported that afferent arteriole vasoconstriction in control kidneys that is produced by angiotensin I was significantly attenuated by ACE inhibition, but not by serine protease inhibition. In contrast, afferent arteriole vasoconstriction produced by the intrarenal conversion of angiotensin I to angiotensin II was significantly attenuated by serine protease inhibition, but not by ACE inhibition in diabetic kidneys.37 Therefore, there appears to be a switch from ACE-dependent to serine protease–dependent angiotensin II formation in the T2D kidney. It has been suggested that chymase may be responsible for serine protease–dependent angiotensin II formation in the diabetic kidney.48 It is plausible that pharmacological targeting of these serine protease–dependent pathways may provide further protection from diabetic renal vascular disease.
CLINICAL OUTCOMES FOR RAS BLOCKADE IN DN
Angiotensin II receptor blockers and ACE inhibitors retard the development and progression of renal dysfunction in human studies.49–52 Table 1 summarizes major clinical trials concerning the effects of RAS inhibition in the development and progression of renal dysfunction in both diabetic and nondiabetic renal disease. In BENEDIC (Bergamo Nergamo Nephrologic Diabetes Complications Trial),53 patients with diabetes with no history of microalbuminuria were randomized to trandolapril versus placebo over median of 3.6 years’ follow-up. Trandolapril resulted in a significant decrease in the development of microalbuminuria and limited the progression of renal dysfunction. This reduction was still significant even after adjusting for BP reduction by trandolapril. In the AASK (African American Study of Kidney Disease and Hypertension) trial,54 the reduction of microalbuminuria by ACE inhibitors was also shown in the nondiabetic renal population in which patients were randomized to ramipril versus amlodipine. The benefit of ARB therapy in patients with DN has been studied in 2 large trials. In the RENAAL (Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan) trial55 and the IDN (Irbesartan Diabetic Nephropathy) trial,56 evaluating patients with T2D with nephropathy, the addition of ARB to standard therapy resulted in improvements in all causes of mortality, progression to end-stage renal disease, and doubling of serum creatinine. In the first direct comparison of ARB with an ACE inhibitor, the DETAIL (Diabetics Exposed to Telmisartan And Enalapril) trial57 evaluated patients with T2D randomized to either enalapril or telmisartan. Telmisartan was not inferior to enalapril in the primary end point of change in baseline estimated GFR. Recently, ONTARGET (Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial)58 showed that ARBs and ACE inhibitors were equally effective in improving renal outcome (dialysis, doubling of serum creatinine, and death), and the number of events for the composite outcome was similar for telmisartan and ramipril. In the dual-therapy group, even though there was a significant reduction in proteinuria, there was an increase in adverse effects and worsening renal outcomes. The beneficial effect of ARB was also confirmed in a most recent megatrial.59,60 The ROADMAP (Randomized Olmesartan and Diabetes Microalbuminuria Prevention) trial is a randomized, double-blind, multicenter study conducted in Europe, including 4447 patients with diabetes and at least 1 additional cardiovascular risk factor, but no evidence of renal dysfunction. The participants were randomized to receive either olmesartan at 40 mg/d (n = 2232) or placebo (n = 2215), and all were allowed to take additional non-RAS antihypertensives to reach target BP (<130/80 mm Hg), until the predefined number of adjudicated microalbuminuria events occurred at a median follow-up of 3.2 years. The primary end point was time to onset of albuminuria. The results show that there was a cumulative incidence of microalbuminuria of 8.2% with olmesartan and 9.8% with placebo; the primary end point, time to onset of microalbuminuria, was delayed by 23% with olmesartan (hazard ratio, 0.77; P = 0.01), with the majority of this effect being BP independent. What is missing, however, is an actual marker to test for the efficacy of treatment. If indeed the major factor initiating the DN is an inappropriate increase in intrarenal RAS, then it would seem particularly worthwhile to have a direct means of evaluating the status of the intrarenal RAS in patients and the efficacy of treatment in reducing or arresting RAS activation in the kidneys.
URINARY AGT AS A NEW BIOMARKER OF INTRARENAL RAS STATUS IN DIABETES
Clinically, microalbuminuria is the most commonly used early marker of DN.61 Diabetic nephropathy is thought to be a unidirectional process from microalbuminuria to end-stage renal failure.62 However, recent studies demonstrate that a large proportion of DN patients revert to normoalbuminuria and that one third of them exhibit reduced renal function even in the microalbuminuria stage.63 It is claimed that urinary inflammatory markers are high in microalbuminuric T1D having diminished renal function, but not in microalbuminuric T1D patients with stable renal function. However, no single marker has been sufficient to represent the whole panel.64 Therefore, a more sensitive and more specific marker for activation of RAS in DN would be highly advantageous.
Angiotensinogen is the only known substrate for renin, which is the rate-limiting enzyme of the RAS. Because the level of AGT is close to the Michaelis-Menten constant for renin, not only renin levels but also AGT levels can control the activity of the RAS, and up-regulation of AGT levels may lead to elevated angiotensin peptide levels.65,66 Recent studies on experimental animal models and transgenic mice have documented the involvement of AGT in the activation of the RAS.67–75 Genetic manipulations that lead to overexpression of the AGT gene have consistently been shown to cause hypertension.76,77 In human genetic studies, a linkage has been established between the AGT gene and hypertension.78–81 Enhanced intrarenal AGT mRNA and/or protein levels have also been observed in multiple experimental models of hypertension and diabetes including angiotensin II–dependent hypertensive rats,25,82–86 Dahl salt–sensitive hypertensive rats,87,88 and spontaneously hypertensive rats,89 as well as in kidney diseases including DN,44,46,47,90–92 immunoglobulin A nephropathy,93,94 and radiation nephropathy.95 In addition, models of T1D and T2D and patients with metabolic syndrome also exhibit increases in intrarenal AGT and urinary AGT excretion.44–47,96,97 Thus, AGT plays an important role in the development and progression of hypertension and kidney diseases and may be particularly useful as a predictor of developing kidney disease.7,17
In rodents, urinary excretion rates of AGT provide a specific index of the intrarenal RAS status and are correlated with kidney angiotensin II levels in angiotensin II–dependent hypertensive rats (Fig. 2).25,83–86 Because of its potential importance, a direct quantitative method to measure urinary AGT using human AGT enzyme-linked immunosorbent assay was recently developed.98 Using this system, urinary excretion rates of AGT have been used as an index of intrarenal RAS status in patients with chronic kidney disease99–102 and in patients with hypertension.103,104 Recently, 2 clinical studies showed the potential of urinary AGT levels as a novel biomarker of intrarenal RAS status in diabetes mellitus.105,106
To demonstrate that the administration of an ARB interferes with the vicious cycle of high glucose–ROS–AGT–angiotensin II–AT1 receptor–ROS by suppressing ROS and inflammation, 13 hypertensive DN patients who received ARBs were recruited and evaluated before and at 16 weeks after treatment.105 Urinary AGT, albumin, 8-hydroxydeoxyguanosine, 8-epi-prostaglandin F2α, monocyte chemoattractant protein 1 (MCP-1), interleukin 6, and interleukin 10 were assessed. Angiotensin II receptor blocker treatment reduced the BP and urinary levels of AGT, albumin, 8-hydroxydeoxyguanosine, 8-epi-prostaglandin F2α, MCP-1, and interleukin 6, while increasing urinary interleukin 10 levels. The reduction of urinary AGT correlated with the reduction of BP and urinary levels of albumin, 8-hydroxydeoxyguanosine, 8-epi-prostaglandin F2α, MCP-1, and interleukin 6 and the increased urinary interleukin 10 levels. These results suggest that the mechanisms by which ARBs exert their renoprotective effect may involve the suppression of intrarenal AGT levels in association with reduced anti-inflammatory and antioxidant effects in patients with T2D (Fig. 4).105
To determine if urinary AGT levels can be dissociated from urinary albumin or protein excretion rates in T1D juveniles, early-phase studies were performed in control and diabetic juveniles.106 Of the 55 juveniles recruited, 34 were patients with T1D, and 21 were sex- and age-matched control subjects. Because the primary focus of the study was comparison between characteristics of normoalbuminuric patients with T1D and those of control subjects, 6 microalbuminuric patients with T1D (urinary albumin-creatinine ratio >30 mg/g) were excluded. Consequently, 49 urine and plasma samples were analyzed. None of them received treatment with RAS blockade. Neither urinary albumin-creatinine ratios nor urinary protein-creatinine ratios were significantly increased in these patients with T1D compared with control subjects, suggesting that these patients were in their premicroalbuminuric phase of DN. However, urinary AGT-creatinine ratios were significantly increased in these patients compared with control subjects (12.1 ± 3.2 vs 4.2 ± 0.7 μg/g, P = 0.0454). Importantly, the AGT increase was not observed in plasma (26.3 ± 1.3 vs 29.5 ± 3.3 μg/mL, P = 0.3148) (Fig. 5). These data indicate that urinary AGT levels are increased in T1D subjects and that increased urinary AGT levels precede the increased urinary albumin levels, suggesting a possibility that urinary AGT levels serve as a very sensitive early marker of intrarenal RAS activation and may be one of the earliest predictors of DN in patients with diabetes.106
The complicated and pleiotropic actions of an activated RAS in pathogenesis of DN continue to receive recognition from emerging and ongoing studies. Clearly, the use of ARBs and ACE inhibitors has become common practice in treating patients with diabetes. Because RAS activation plays such a central role in the development and progression of DN, there has been extensive interest in the potential hope for reduction in morbidity and mortality by using agents that block 1 or more steps in the RAS. Accordingly, the assessment of urinary AGT as an early biomarker of the status of the intrarenal RAS may be of substantial importance. It may be particularly helpful in serving as a means to determine efficacy of the treatment to reduce intrarenal angiotensin II levels.
1. Joss N, Paterson KR, Deighan CJ, et al.. Diabetic nephropathy: how effective is treatment in clinical practice? QJM. 2002; 95: 41–49.
2. Mokdad AH, Ford ES, Bowman BA, et al.. Prevalence of obesity, diabetes, and obesity-related health risk factors, 2001. JAMA. 2003; 289: 76–79.
3. Bays HE, Bazata DD, Clark NG, et al.. Prevalence of self-reported diagnosis of diabetes mellitus and associated risk factors in a national survey in the us population: SHIELD (Study to Help Improve Early evaluation and management of risk factors Leading to Diabetes). BMC Public Health. 2007; 7: 277.
4. National Diabetes Information Clearinghouse. A Service of the National Institute of Diabetes and Digestive and Kidney Diseases. The National Institutes of Health, 2007.
5. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among us adults: findings from the third national health and nutrition examination survey. JAMA. 2002; 287: 356–359.
6. Mitchell KD, Navar LG. Intrarenal actions of angiotensin II in the pathogenesis of experimental hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press; 1995: 1437–1450.
7. Kobori H, Nangaku M, Navar LG, et al.. The intrarenal renin-angiotensin system: from physiology to the pathobiology of hypertension and kidney disease. Pharmacol Rev. 2007; 59: 251–287.
8. Carey RM, Siragy HM. The intrarenal renin-angiotensin system and diabetic nephropathy. Trends Endocrinol Metab. 2003; 14: 274–281.
9. Santos RA, Simoes e Silva AC, Maric C, et al.. Angiotensin-(1–7) is an endogenous ligand for the G protein–coupled receptor MAS. Proc Natl Acad Sci U S A. 2003; 100: 8258–8263.
10. Navar LG, Prieto-Carrasquero MC, Kobori H. Chapter 1: molecular aspects of the renal renin-angiotensin system. In: Re RN, DiPette DJ, Schiffrin EL, et al., eds. Molecular Mechanisms in Hypertension. Oxfordshire, UK: Taylor & Francis Medical; 2006: 3–14.
11. Dzau VJ, Re R. Tissue angiotensin system in cardiovascular medicine. A paradigm shift? Circulation. 1994; 89: 493–498.
12. Baltatu O, Silva JA Jr, Ganten D, et al.. The brain renin-angiotensin system modulates angiotensin II–induced hypertension and cardiac hypertrophy. Hypertension. 2000; 35: 409–412.
13. Dell’Italia LJ, Meng QC, Balcells E, et al.. 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.
14. Mazzocchi G, Malendowicz LK, Markowska A, et al.. 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.
15. Danser AH, Admiraal PJ, Derkx FH, et al.. Angiotensin I-to-II conversion in the human renal vascular bed. J Hypertens. 1998; 16: 2051–2056.
16. Griendling KK, Minieri CA, Ollerenshaw JD, et al.. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.
17. Navar LG, Harrison-Bernard LM, Nishiyama A, et al.. Regulation of intrarenal angiotensin II in hypertension. Hypertension. 2002; 39: 316–322.
18. Ingelfinger JR, Zuo WM, Fon EA, et al.. In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system. J Clin Invest. 1990; 85: 417–423.
19. Kamiyama M, Garner MK, Farragut KM, et al.. The establishment of a primary culture system of proximal tubule segments using specific markers from normal mouse kidneys. Int J Mol Sci. 2012; 13: 5098–5111.
20. Kamiyama M, Farragut KM, Garner MK, et al.. Divergent localizations of angiotensinogen mRNA and protein in proximal tubular segments of normal rat kidney. J Hypertens. 2012; 30: 2365–2372.
21. Terada Y, Tomita K, Nonoguchi H, et al.. Pcr localization of angiotensin II receptor and angiotensinogen mRNAs in rat kidney. Kidney Int. 1993; 43: 1251–1259.
22. Richoux JP, Cordonnier JL, Bouhnik J, et al.. Immunocytochemical localization of angiotensinogen in rat liver and kidney. Cell Tissue Res. 1983; 233: 439–451.
23. Darby IA, Congiu M, Fernley RT, et al.. Cellular and ultrastructural location of angiotensinogen in rat and sheep kidney. Kidney Int. 1994; 46: 1557–1560.
24. Darby IA, Sernia C. In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys. Cell Tissue Res. 1995; 281: 197–206.
25. 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.
26. Pohl M, Kaminski H, Castrop H, et al.. Intrarenal renin angiotensin system revisited: role of megalin-dependent endocytosis along the proximal nephron. J Biol Chem. 2010; 285: 41935–41946.
27. Leyssac PP. Changes in single nephron renin release are mediated by tubular fluid flow rate. Kidney Int. 1986; 30: 332–339.
28. Yanagawa N, Capparelli AW, Jo OD, et al.. Production of angiotensinogen and renin-like activity by rabbit proximal tubular cells in culture. Kidney Int. 1991; 39: 938–941.
29. Henrich WL, McAllister EA, Eskue A, et al.. Renin regulation in cultured proximal tubular cells. Hypertension. 1996; 27: 1337–1340.
30. Moe OW, Ujiie K, Star RA, et al.. Renin expression in renal proximal tubule. J Clin Invest. 1993; 91: 774–779.
31. Sibony M, Gasc JM, Soubrier F, et al.. Gene expression and tissue localization of the two isoforms of angiotensin I converting enzyme. Hypertension. 1993; 21: 827–835.
32. Schulz WW, Hagler HK, Buja LM, et al.. Ultrastructural localization of angiotensin I–converting enzyme (EC 22.214.171.124) and neutral metalloendopeptidase (EC 126.96.36.199) in the proximal tubule of the human kidney. Lab Invest. 1988; 59: 789–797.
33. Vio CP, Jeanneret VA. Local induction of angiotensin-converting enzyme in the kidney as a mechanism of progressive renal diseases. Kidney Int Suppl. 2003; 64: S57–S63.
34. Casarini DE, Boim MA, Stella RC, et al.. Angiotensin I–converting enzyme activity in tubular fluid along the rat nephron. Am J Physiol. 1997; 272: F405–F409.
35. Ye M, Wysocki J, William J, et al.. Glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensin-converting enzyme: implications for albuminuria in diabetes. J Am Soc Nephrol. 2006; 17: 3067–3075.
36. Soler MJ, Ye M, Wysocki J, et al.. Localization of ACE2 in the renal vasculature: amplification by angiotensin II type 1 receptor blockade using telmisartan. Am J Physiol Renal Physiol. 2009; 296: F398–F405.
37. Park S, Bivona BJ, Kobori H, et al.. Major role for ACE-independent intrarenal ANG II formation in type II diabetes. Am J physiol Renal Physiol. 2010; 298: F37–F48.
38. Anderson S. Physiologic actions and molecular expression of the renin-angiotensin system in the diabetic rat. Miner Electrolyte Metab. 1998; 24: 406–411.
39. Harrison-Bernard LM, Imig JD, Carmines PK. Renal AT1 receptor protein expression during the early stage of diabetes mellitus. Int J Exp Diabetes Res. 2002; 3: 97–108.
40. Zimpelmann J, Kumar D, Levine DZ, et al.. Early diabetes mellitus stimulates proximal tubule renin mrna expression in the rat. Kidney Int. 2000; 58: 2320–2330.
41. Kamiyama M, Urusihara M, Morikawa T, et al.. Augmented intrarenal angiotensinogen mRNA expression parallels renal dysfunction in patients with type 2 diabetes [abstract]. Hypertension. 2010; 56: e135.
42. Wehbi GJ, Zimpelmann J, Carey RM, et al.. Early streptozotocin–diabetes mellitus downregulates rat kidney AT2 receptors. Am J Physiol Renal Physiol. 2001; 280: F254–F265.
43. Kang JJ, Toma I, Sipos A, et al.. The collecting duct is the major source of prorenin in diabetes. Hypertension. 2008; 51: 1597–1604.
44. Nagai Y, Yao L, Kobori H, et al.. Temporary angiotensin II blockade at the prediabetic stage attenuates the development of renal injury in type 2 diabetic rats. J Am Soc Nephrol. 2005; 16: 703–711.
45. Delete in proof.
46. 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.
47. Miyata K, Ohashi N, Suzaki Y, et al.. Sequential activation of the reactive oxygen species/angiotensinogen/renin-angiotensin system axis in renal injury of type 2 diabetic rats. Clin Exp Pharmacol Physiol. 2008; 35: 922–927.
48. Lorenz JN. Chymase: the other ACE? Am J Physiol Renal Physiol. 2010; 298: F35–F36.
49. Hansson L, Lindholm LH, Niskanen L, et al.. Effect of angiotensin-converting-enzyme inhibition compared with conventional therapy on cardiovascular morbidity and mortality in hypertension: the Captopril Prevention Project (CAPPP) randomised trial. Lancet. 1999; 353: 611–616.
50. Efficacy of atenolol and captopril in reducing risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 39. UK prospective diabetes study group. BMJ. 1998; 317: 713–720.
51. Schmieder RE. Endothelial dysfunction: how can one intervene at the beginning of the cardiovascular continuum? J Hypertens Suppl. 2006; 24: S31–S35.
52. Parving HH, Lehnert H, Brochner-Mortensen J, et al.. Irbesartan in Patients with Type 2 Diabetes and Microalbuminuria Study G. The effect of irbesartan on the development of diabetic nephropathy in patients with type 2 diabetes. N Engl J Med. 2001; 345: 870–878.
53. Ruggenenti P, Fassi A, Ilieva AP, et al.. Bergamo Nephrologic Diabetes Complications Trial I. Preventing microalbuminuria in type 2 diabetes. N Engl J Med. 2004; 351: 1941–1951.
54. Agodoa LY, Appel L, Bakris GL, et al.. African American Study of Kidney Disease and Hypertension Study G. Effect of ramipril vs amlodipine on renal outcomes in hypertensive nephrosclerosis: a randomized controlled trial. JAMA. 2001; 285: 2719–2728.
55. Brenner BM, Cooper ME, de Zeeuw D, et al.. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med. 2001; 345: 861–869.
56. Lewis EJ, Hunsicker LG, Clarke WR, et al.. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med. 2001; 345: 851–860.
57. Barnett AH, Bain SC, Bouter P, et al.. Angiotensin-receptor blockade versus converting-enzyme inhibition in type 2 diabetes and nephropathy. N Engl J Med. 2004; 351: 1952–1961.
58. Mann JF, Schmieder RE, McQueen M, et al.. Renal outcomes with telmisartan, ramipril, or both, in people at high vascular risk (the ONTARGET study): a multicentre, randomised, double-blind, controlled trial. Lancet. 2008; 372: 547–553.
59. Haller H, Ito S, Izzo JL, et al.. Olmesartan for the delay or prevention of microalbuminuria in type 2 diabetes. N Engl J Med. 2011; 364: 907–917.
60. Ingelfinger JR. Preemptive olmesartan for the delay or prevention of microalbuminuria in diabetes. N Engl J Med. 2011; 364: 970–971.
61. Coresh J, Selvin E, Stevens LA, et al.. Prevalence of chronic kidney disease in the united states. JAMA. 2007; 298: 2038–2047.
62. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The diabetes control and complications trial research group. N Engl J Med. 1993; 329: 977–986.
63. Perkins BA, Ficociello LH, Silva KH, et al.. Regression of microalbuminuria in type 1 diabetes. N Engl J Med. 2003; 348: 2285–2293.
64. Wolkow PP, Niewczas MA, Perkins B, et al.. Association of urinary inflammatory markers and renal decline in microalbuminuric type 1 diabetics. J Am Soc Nephrol. 2008; 19: 789–797.
65. Gould AB, Green D. Kinetics of the human renin and human substrate reaction. Cardiovasc Res. 1971; 5: 86–89.
66. Brasier AR, Li J. Mechanisms for inducible control of angiotensinogen gene transcription. Hypertension. 1996; 27: 465–475.
67. Ding Y, Davisson RL, Hardy DO, et al.. The kidney androgen-regulated protein promoter confers renal proximal tubule cell–specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice. J Biol Chem. 1997; 272: 28142–28148.
68. Kimura S, Mullins JJ, Bunnemann B, et al.. High blood pressure in transgenic mice carrying the rat angiotensinogen gene. EMBO J. 1992; 11: 821–827.
69. Fukamizu A, Sugimura K, Takimoto E, et al.. Chimeric renin-angiotensin system demonstrates sustained increase in blood pressure of transgenic mice carrying both human renin and human angiotensinogen genes. J Biol Chem. 1993; 268: 11617–11621.
70. Bohlender J, Menard J, Ganten D, et al.. Angiotensinogen concentrations and renin clearance: implications for blood pressure regulation. Hypertension. 2000; 35: 780–786.
71. Smithies O. Theodore cooper memorial lecture. A mouse view of hypertension. Hypertension. 1997; 30: 1318–1324.
72. Merrill DC, Thompson MW, Carney CL, et al.. Chronic hypertension and altered baroreflex responses in transgenic mice containing the human renin and human angiotensinogen genes. J Clin Invest. 1996; 97: 1047–1055.
73. Kobori H, Ozawa Y, Satou R, et al.. Kidney-specific enhancement of Ang II stimulates endogenous intrarenal angiotensinogen in gene-targeted mice. Am J Physiol Renal Physiol. 2007; 293: F938–F945.
74. Sachetelli S, Liu Q, Zhang SL, et al.. Ras blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney. Kidney Int. 2006; 69: 1016–1023.
75. Lavoie JL, Lake-Bruse KD, Sigmund CD. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in the renal proximal tubule. Am J Physiol Renal Physiol. 2004; 286: F965–F971.
76. Smithies O, Kim HS. Targeted gene duplication and disruption for analyzing quantitative genetic traits in mice. Proc Natl Acad Sci U S A. 1994; 91: 3612–3615.
77. Kim HS, Krege JH, Kluckman KD, et al.. Genetic control of blood pressure and the angiotensinogen locus. Proc Natl Acad Sci U S A. 1995; 92: 2735–2739.
78. Inoue I, Nakajima T, Williams CS, et al.. A nucleotide substitution in the promoter of human angiotensinogen is associated with essential hypertension and affects basal transcription in vitro. J Clin Invest. 1997; 99: 1786–1797.
79. Jeunemaitre X, Soubrier F, Kotelevtsev YV, et al.. Molecular basis of human hypertension: role of angiotensinogen. Cell. 1992; 71: 169–180.
80. Zhao YY, Zhou J, Narayanan CS, et al.. Role of C/A polymorphism at -20 on the expression of human angiotensinogen gene. Hypertension. 1999; 33: 108–115.
81. Ishigami T, Umemura S, Tamura K, et al.. Essential hypertension and 5′ upstream core promoter region of human angiotensinogen gene. Hypertension. 1997; 30: 1325–1330.
82. Schunkert H, Ingelfinger JR, Jacob H, et al.. Reciprocal feedback regulation of kidney angiotensinogen and renin mRNA expressions by angiotensin II. Am J Physiol. 1992; 263: E863–E869.
83. Kobori H, Prieto-Carrasquero MC, Ozawa Y, et al.. AT1 receptor mediated augmentation of intrarenal angiotensinogen in angiotensin II–dependent hypertension. Hypertension. 2004; 43: 1126–1132.
84. Kobori H, Nishiyama A, Harrison-Bernard LM, et al.. Urinary angiotensinogen as an indicator of intrarenal angiotensin status in hypertension. Hypertension. 2003; 41: 42–49.
85. Kobori H, Harrison-Bernard LM, Navar LG. Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production. Kidney Int. 2002; 61: 579–585.
86. Kobori H, Harrison-Bernard LM, Navar LG. Enhancement of angiotensinogen expression in angiotensin II–dependent hypertension. Hypertension. 2001; 37: 1329–1335.
87. Kobori H, Nishiyama A. Effects of tempol on renal angiotensinogen production in Dahl salt–sensitive rats. Biochem Biophys Res Commun. 2004; 315: 746–750.
88. Kobori H, Nishiyama A, Abe Y, et al.. Enhancement of intrarenal angiotensinogen in Dahl salt–sensitive rats on high salt diet. Hypertension. 2003; 41: 592–597.
89. Kobori H, Ozawa Y, Suzaki Y, et al.. Enhanced intrarenal angiotensinogen contributes to early renal injury in spontaneously hypertensive rats. J Am Soc Nephrol. 2005; 16: 2073–2080.
90. Anderson S, Jung FF, Ingelfinger JR. Renal renin-angiotensin system in diabetes: functional, immunohistochemical, and molecular biological correlations. Am J Physiol. 1993; 265: F477–F486.
91. Singh R, Singh AK, Leehey DJ. A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat glomeruli. Am J Physiol Renal Physiol. 2005; 288: F1183–F1190.
92. Leehey DJ, Singh AK, Bast JP, et al.. Glomerular renin angiotensin system in streptozotocin diabetic and zucker diabetic fatty rats. Transl Res. 2008; 151: 208–216.
93. Kobori H, Katsurada A, Ozawa Y, et al.. Enhanced intrarenal oxidative stress and angiotensinogen in IgA nephropathy patients. Biochem Biophys Res Commun. 2007; 358: 156–163.
94. Takamatsu M, Urushihara M, Kondo S, et al.. Glomerular angiotensinogen protein is enhanced in pediatric IgA nephropathy. Pediatr Nephrol. 2008; 23: 1257–1267.
95. Kobori H, Ozawa Y, Suzaki Y, et al.. Young scholars award lecture: intratubular angiotensinogen in hypertension and kidney diseases. Am J Hypertens. 2006; 19: 541–550.
96. Kamiyama M, Zsombok A, Kobori H. Urinary angiotensinogen as a novel early biomarker of intrarenal renin-angiotensin system activation in experimental type 1 diabetes. J Pharmacol Sci. 2012; 119: 314–323.
97. Thethi T, Kamiyama M, Kobori H. The link between the renin-angiotensin-aldosterone system and renal injury in obesity and the metabolic syndrome. Cur Hyperten Rep. 2012; 14: 160–169.
98. Katsurada A, Hagiwara Y, Miyashita K, et al.. Novel sandwich elisa for human angiotensinogen. Am J Physiol Renal Physiol. 2007; 293: F956–F960.
99. Yamamoto T, Nakagawa T, Suzuki H, et al.. Urinary angiotensinogen as a marker of intrarenal angiotensin II activity associated with deterioration of renal function in patients with chronic kidney disease. J Am Soc Nephrol. 2007; 18: 1558–1565.
100. Kobori H, Ohashi N, Katsurada A, et al.. Urinary angiotensinogen as a potential biomarker of severity of chronic kidney diseases. J Am Soc Hypertens. 2008; 2: 349–354.
101. Nishiyama A, Konishi Y, Ohashi N, et al.. Urinary angiotensinogen reflects the activity of intrarenal renin-angiotensin system in patients with IgA nephropathy. Nephrol Dial Transplant. 2011; 26: 170–177.
102. Urushihara M, Kondo S, Kagami S, et al.. Urinary angiotensinogen accurately reflects intrarenal renin-angiotensin system activity. Am J Nephrol. 2010; 31: 318–325.
103. Kobori H, Alper AB, Shenava R, et al.. Urinary angiotensinogen as a novel biomarker of the intrarenal renin-angiotensin system status in hypertensive patients. Hypertension. 2009; 53: 344–350.
104. Kobori H, Urushihara M, Xu JH, et al.. Urinary angiotensinogen is correlated with blood pressure in men (Bogalusa Heart Study). J Hypertens. 2010; 28: 1422–1428.
105. Ogawa S, Kobori H, Ohashi N, et al.. Angiotensin II type 1 receptor blockers reduce urinary angiotensinogen excretion and the levels of urinary markers of oxidative stress and inflammation in patients with type 2 diabetic nephropathy. Biomark Insights. 2009; 4: 97–102.
106. Saito T, Urushihara M, Kotani Y, et al.. Increased urinary angiotensinogen is precedent to increased urinary albumin in patients with type 1 diabetes. Am J Med Sci. 2009; 338: 478–480.