Important aspects of urine sampling for angiotensinogen measurement: time and preservation conditions in healthy individuals.

The renin-angiotensin system (RAS) is a hormone system that regulates blood pressure and fluid/electrolyte balance (Kobori et al. 2007). In recent years, there has been growing evidence that the local/tissue RAS in various tissues including the brain (Baltatu et al. 2000), the heart (Dell’Italia et al. 1997), vasculatures (Griendling et al. 1994; Danser et al. 1998) and kidneys (Navar et al. 2002) is independently regulated from the systemic RAS. Recent basic and clinical data demonstrated that the intrarenal RAS plays an important role in the development and the progression of hypertension and renal injury (Navar et al. 2011a). Elevated intrarenal angiotensin II (Ang II) levels were observed in renal functional damage and renal tissue injury. Experimental studies demonstrated that the intrarenal Ang II levels were increased in several animal models of renal injury, including Dahl salt-sensitive rats on high salt diet (Kobori et al. 2003a; Kobori and Nishiyama 2004), spontaneously hypertensive rats (SHR) (Kobori et al. 2005) and diabetic nephropathy (Nagai et al. 2005). Angiotensinogen (AGT) is the only known substrate for renin, which is the rate-limiting enzyme of the RAS. Because of the levels of AGT are close to the Michaelis-Menten constant for renin, AGT levels can also control the RAS activity, and upregulation of AGT may lead to elevated angiotensin peptide levels (Gould and Green 1971; Brasier and Li 1996). Intrarenal AGT produced in proximal tubular cells contributes to increased intrarenal Ang II in animal models (Kobori et al. 2001). We have previously reported that urinary AGT excretion rate could be a novel biomarker for the activity of the RAS in kidney (Kobori et al. 2009, 2010). Novel sandwich ELISA for human angiotensinogen (Katsurada et al. 2007) made it possible for us to measure a large quantity of specimens over time. It was reported that the systemic RAS had a circadian rhythm. For example, plasma renin activity (PRA), Ang II, and aldosterone rose in the early morning and decreased at night (Kala et al. 1973: Kawasaki et al. 1980). It was also reported that the cardiac RAS in the SHR rat had a circadian rhythm (Naito et al. 2002). Therefore, it is necessary to confirm that urinary AGT does not have an ultradian rhythm, because it is difficult to collect early morning urine samples or 24-hour urine samples in nonhospitalized patients. In addition, it is also important to examine whether preservation conditions may affect the measured values of urinary AGT. Therefore, this study was performed to demonstrate the following two points. 1) Preservation conditions would not change the measured values of urinary AGT concentrations. 2) Urinary AGT excretion in humans would not have an ultradian rhythm.