Abstract
Background: Although several lines of evidence suggest that renin angiotensin system (RAS) proteins are synthesized by cyst epithelium and dilated tubules, role of intrarenal RAS in the progression of otozomal dominant polycystic kidney disease (ADPKD) is not well known. We aimed to study the levels and clinical correlations of urinary angiotensinogen (UAGT) in normotensive ADPKD patients compared with age- and sex-matched healthy subjects. Methods: The study included 20 normotensive ADPKD patients (F/M: 11/9) and 20 age and sex matched healthy controls (F/M: 9/11). Diagnosis of ADPKD was made based on Ravine criteria. Twenty-four hours ambulatory blood pressure monitoring (ABPM) was performed. Serum concentrations of creatinine, Na, K, uric acid, and urinary concentrations of Na, K, uric acid, creatinine, protein and albumin were measured. UAGT were measured via commercially available ELISA kit. Results: ADPKD patients had higher urinary albumin:creatinine ratio (UAIb/UCrea) than healthy controls (p < 0.01). UAGT/UCrea levels significantly positively correlated with urinary protein: creatinine ratio (UPro/UCrea) (r = 0.785, p = 0.01), and UAIb/UCrea (r = 0.681, p = 0.01) in normotensive ADPKD patients. Conclusion: This pilot study demonstrates that UAGT levels tend to be elevated and are correlated with proteinuria and albuminuria in normotensive ADPKD patients during relatively early stages of the disease.
Introduction
ADPKD is a common, life-threatening hereditary and progressive renal tubular cystic disease resulting renal insufficiency. It is associated with hypertension, left ventricular hypertrophy, cardiac valvular defects and cerebral aneurysm that can lead increased mortality and morbidity.Citation1,Citation2 There is no effective treatment for interrupt the progression and protecting the patients from the complications of disease yet. Early diagnosis and treatment of hypertension with follow-up of disease-related complications are the main strategy for now. Determination of a prognostic biomarker that indicates the severity of disease before developing hypertension may provide the additional clinical advantage for ADPKD patients.
There are indirect but significant evidence between RAS activation and ADPKD.Citation3–7 Several studies suggest that angiotensinogen (AGT) and the other substances of RAS system are present in autosomal dominant polycystic kidneys.Citation6,Citation7 These findings implicate that intra-renal RAS may play a main role in ADPKD pathogenesis. Recently, the studies showed UAGT are a useful biomarker to reflect intrarenal RAS activity in some of renal diseases and hypertension.Citation8–13
In light of this data, we designed the present study to detect the UAGT levels and its clinical correlation in normotensive ADPKD patients compared with age- and sex-matched healthy subjects.
Subjects and methods
The study was carried out at the Ankara University School of Medicine Ibni Sina Hospital Department of Nephrology. The study protocol was approved by the Local Ethical Committees and written informed consent was obtained from all participants. Twenty normotensive adults with ADPKD (F/M: 11/9) and 20 age and sex matched healthy controls (F/M: 9/11) were included in present study. Diagnosis of ADPKD was made based on Ravine criteria.Citation14 Estimated GFR was calculated with using 4-variable Modification of Diet in Renal Disease (MDRD) formula: eGFR = 186.3 × sCr − 1.154 × Age − 0.203 × 0.742 (if woman).Citation15 Subjects with hypertension (systolic blood pressure >130 mmHg and/or diastolic blood pressure >80 mmHg according to ambulatory blood pressure monitoring), diabetes, reduced glomerular filtration rate and/or had any anti-hypertensive medication were excluded from the study.
Demographic and clinical characteristics were recorded as age, sex, height, weight and body mass index (BMI). Blood samples were obtained after at least 8 h fasting period and analyzed for serum creatinine, potassium, sodium and uric acid. Beckman coulter unicel DxC800 (Brea, CA) and Siemens/BNII equipments (Washington, DC) were used for the biochemical analysis. Creatinine level was measured with the use of the Jaffe method and uric acid level with the use of the uricase/peroxidase method. Second-morning urine samples were collected from all participants to detect the urinary sodium, potassium, uric acid, albumin, protein, and creatinine and AGT levels. Total Human AGT ELISA Assay Kit (IBL, Hamburg, Germany) was used for the measurement of urinary AGT levels. Frozen (−80 °C) urine samples were stored the night before the work day at +4 °C. Enzyme-linked immunosorbent assay (ELISA) measurements were performed at Nephrology Research Laboratory according to the manufacturer’s protocol. The results of UAGT were obtained as ng/mL. UAGT levels normalized with urinary creatinine results and UAGT/UCrea (µg/g) was used at analysis.
Twenty-four hours ambulatory blood pressure monitoring (ABPM) was performed using a Mobil-O-Graph NG (Germany) device. Blood pressure was measured every 30 min for 24 h. Measurements were considered sufficient to include if >85% of the readings were successful. Mean 24-h, day-time (06:00–22:00) and night-time (22:00–06:00) BPs were recorded in all patients.
Differences between ADPKD patients and healthy controls were tested using the 2-sample t test when normally distributed or Mann–Whitney test when not normally distributed. To investigate whether UAGT levels correlated with other study parameters, Spearman correlation coefficients were calculated. Analyses were performed using SPSS, version 15.0. p Value < 0.05 was considered significant.
Results
There were no significant differences in demographic findings and laboratory parameters between ADPKD group and controls (). Blood pressure levels and kidney function with estimated GFR were the same in both groups.
Table 1. Comparison of the demographic data and laboratory parameters in patient and control group.
ADPKD patients had higher UAIb/UCrea than healthy controls (46.1 ± 18.1 mg/g vs. 8.3 ± 1.1 mg/g, p < 0.01). UAGT/UCrea was higher in normotensive ADPKD patients than in healthy controls, but the difference was not statistically significant (19.35 ± 6.06 µg/g vs. 11.65 ± 1.66 µg/g, p > 0.05). UAGT/UCrea levels significantly positively correlated with UPro/UCrea (r = 0.785, p = 0.01), and UAIb/UCrea (r = 0.681, p = 0.01) in normotensive ADPKD patients. The results of the laboratory parameters are shown in and . Summarized of the results regarding UPro/UCrea, UAlb/UCrea and UAGT/UCrea parameters in both ADPKD and control groups are presented briefly in Figure 1.
Table 2. Non-parametric correlation analysis of the parameters in patient group.
Table 3. Non-parametric correlation analysis of the parameters in control group.
The other study parameters such as age, gender, serum creatinine, sodium (Na), potassium (K), uric acid, eGFR, BMI, systolic and diastolic blood pressures were not significantly correlated with UAGT/UCrea levels.
Discussion
In the present study, we found that UAGT/UCrea levels are significantly positive correlated with both UPro/UCrea (r = 0.785, p = 0.01) and UAIb/UCrea (r = 0.681, p = 0.01) ratios in normotensive ADPKD patients. Individual features such as race, gender, age, height, body mass index, uric acid levels, serum creatinine and potassium levels did not affect this relationship statistically. This data suggest that UAGT/UCrea levels are correlated with proteinuria and also kidney damage. But, there are several debates in this issue. It is not clear whether the source of AGT is kidneys or systemic and whether the increased UAGT levels are the initial process of kidney damage or consequence of the proteinuria.
Figure 1. Summarize of the results regarding UPro/UCrea, UAlb/UCrea and UAGT/UCrea parameters in both ADPKD and control groups. Note: ADPKD, autosomal dominant polycystic kidney disease; UCrea, urinary creatinine; UAGT, urinary angiotensinogen; UAlb, urinary albumin; UPro, urinary protein.
Although kidneys produce AGT in the proximal tubules, large amounts of serum AGT is produced and secreted by the liver.Citation16 However, systemic AGT unlikely contribute to the levels of UAGT; because of its molecular size (50–60 kDa), little or none of plasma AGT filters across the glomerular membrane. In a rat study, the human AGT was infused to the rats for determining the source of AGT. There was no detectable human AGT in the urine.Citation9 In contrast, the authors reported that there is a correlation between urinary AGT and albumin levels and also plasma AGT levels is similar to urinary AGT levels in humans. They conclude that source of urinary AGT is plasma not kidneys and urinary AGT level may be a damage marker of glomerular filtration barrier.Citation17,Citation18 However, in one study, UAGT/UCrea levels in the patients with minimal change disease (8.28 ± 3.70 µg/g) who had severe proteinuria were similar when compared with the control subjects (10.78 ± 3.42 µg/g).Citation19 Furthermore, the other study which was performed in juvenile type 1 diabetes patients and age- and sex-matched control subjects had important results to support the hypothesis that augmented UAGT levels are not a consequence of proteinuria. In this study, both groups had similar protein–creatinine and albumin–creatinine ratio statistically and these parameters were in normal range. This implicates type 1 diabetes patients were in pre-albuminuric phase of diabetic nephropathy. Interestingly, the UAGT/UCrea levels were significantly higher in those type 1 diabetes patients than in the control subjects.Citation20 More recently, Nakano et al.Citation21 reported that the majority of urinary angiotensinogen originates from the tubules rather than glomerular filtration by multipotent imaging of the glomerular permeability of angiotensinogen. All these results implicate that the source of AGT in the urine is proximal tubules not plasma and the augmented UAGT levels are not a consequence of proteinuria but it may be an initial process of the proteinuria.
As well known, renin cleaves AGT to angiotensin (ANG) I. It is produced and secreted by the juxtaglomerular apparatus cells into the renal interstitium and vascular compartment. Large amount of angiotensin converting enzyme (ACE) is present in all parts of the kidney such as proximal tubule, distal tubule and collecting ducts. So ANG II is generated throughout each nephron.Citation16 ANG II induces the reduction in single nephron glomerular filtration rate and glomerular plasma flow with constriction of both afferent and efferent arterioles. Its systemic infusion does not elicit proteinuria in both animals and humans.Citation16,Citation22,Citation23 However, sustained elevation of intrarenal ANG II induces proteinuria associated with progressive injury of the glomerular filtration barrier.Citation16,Citation24,Citation25 In addition, there are significant evidence about locally produced ANG II can induce podocyte injury directly with ANG I receptors activation.Citation16,Citation26,Citation27 This evidence implicate that the raised local ANG II and its precursor AGT level may reflect the ongoing damage of kidneys and may cause the proteinuria. Our results support this implication and showed the presence of significant relationship between proteinuria/albuminuria and urinary AGT levels.
Increased intratubular ANG II concentration leads to increase tubular sodium and water reabsorption.Citation16 This pathway may play a major role on disease progression and development of HT in ADPKD patients. Yamamoto et al. found that UAGT levels were higher in patients with CKD, low eGFR, and high urinary protein and type IV collagen excretion. They also reported that UAGT levels correlated positively with renal ANG II and type I collagen immunostaining intensities. Fibrosis in interstitium and arterial wall was detected in normotensive ADPKD patients who have normal GFR.Citation10 Similarly, the other study showed the existence of RAS components including renin, AGT, ACE, ANG II receptor and ANG II peptide in cysts and in dilated tubules of ADPKD kidneys.Citation7 In our study, urinary AGT levels tended to be high in ADPKD patients but were not significant statistically. Limited number of subjects may contribute this result.
Several studies showed positive correlation between UAGT levels and severity of HT. In one study, UAGT/Ucre ratio in primary hypertensive adolescent was significantly higher than control group.Citation28 Furthermore, the relationship between ambulatory blood pressure and UAGT levels in the patients with hypertension was studied by Zou et al. They found the positive correlation between those parameters.Citation29 In our study, UPro/UCrea and UAlb/UCrea levels were correlated with systolic blood pressure results. However, 24-h ambulatory blood pressure levels were not correlated with urinary AGT levels. Although limited number of subjects can influence these results, there can be different factors in ADPKD patients. As well known cysts can occur from different side of tubular cells in polycystic kidneys. If majority of “second hit” cells are proximal tubular cells, cysts will produce more AGT while distal tubular cells produce more renin. Our statistical findings such as very large distribution of UAGT levels and presence of high SD support this hypothesis.
Angiotensinogen had to be transformed ANG II to affect the blood pressure. The circulating concentration of AGT is abundant, 1000 times greater than the plasma ANG I and ANG II concentration. Although the concentration of plasma AGT are close to Michaelis–Menten constant (Km) and rising substrate concentration can influence the ANG I generation; renin is main determiner of ANG I and also ANG II concentration in plasma.Citation16,Citation21–33 But, in urine, this relationship may be converse. Recently, in two studies, authors implicate that urinary renin level is more correlated than UAGT level to reflect the intrarenal RAS activity. They found that concentration of urinary renin is 100- to 200-fold higher than urinary AGT.Citation17,Citation18 It can expect that small variations in UAGT levels may influence the reaction velocity and also amount of generated ANG I in the presence of high renin concentration although there is no available data about the kinetics of AGT-renin reaction in urine. The lots of factors such as urine pH, time of enzyme substrate interaction and compound of urine may change the reaction kinetics. It can speculate that AGT concentration is significant determiner for ANG I generation and also intrarenal ANG II level. ADPKD patient with cysts formed from proximal tubular cells will have more UAGT and ANG II levels and maybe will have higher blood pressure. This hypothesis may be an answer for the question that why ADPKD patients have variety of clinical presentations in this disease even the existence of same genetic mutations.
The major limitation of this cross-sectional age-and sex-matched controlled study is small sample size. This condition might influence some results as discussed earlier. There were a few patients who have increased UAGT excretion rates without the existence of albuminuria/proteinuria. These subjects might provide further data whether the increased UAGT levels is the beginning condition or not for renal injury. But, we cannot perform further statistical analysis because of the number of these subjects. Despite all these, the significant relationship between UAGT/UCrea and UPro/UCrea-UAIb/UCrea ratios was detected in normotensive ADPKD patients as first in literature. This pilot study triggered the new questions about the pathogenesis of ADPKD that need further prospective controlled studies in larger patient groups for answers.
In conclusion, the present study implicates that urinary AGT level may be a useful marker for the risk classification in ADPKD patients particularly in early stage of disease. Thereby, the patients who have higher risk for disease progression can be followed more closely and the conventional or newer treatment strategies can be used on time in clinical setting.
Declaration of interest
This study is supported by the Turkish Society of Hypertension and Renal Diseases with a research fund.
Acknowledgements
Parts of these results were presented in abstract form at the 48th ERA-EDTA Congress in Prague.
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