Soluble (Pro)Renin Receptor Level in Patients with Severe Obesity Is Associated with Visceral Adiposity and Is Involved with Insulin Resistance and Renal Injury Open Access

Over the past 2 decades, the prevalence of overweight and obesity has increased dramatically, and the number of obese people, especially men, is increasing in Japan 1. Obesity is a major risk factor for many diseases such as cardiovascular disease, diabetes, and cancer 2, yet it is a difficult disease to treat. In particular, highly obese patients have more severe complications and are more difficult to treat 3. Therefore, understanding the pathogenesis of obesity and obesity-related complications is important for treatment. Adipose tissue is a central player in the pathogenesis of obesity and obesity-related organ disorders, and a vast amount of clinical and basic research has been conducted in recent years. However, many aspects of how biological changes in adipose tissue are involved in the various pathologies associated with obesity remain to be elucidated.

The (pro)renin receptor ([P]RR) is a transmembrane protein receptor consisting of 350 amino acids that interacts with V-ATPase 4. The (P)RR is encoded by the ATP6AP2 gene on the X chromosome and is expressed in many tissues including the brain, vascular endothelium, vascular smooth muscle, skeletal muscle, kidney, as well as adipose tissue 5-7. When (pro)renin or renin binds to (P)RR, the steric structure of (pro)renin changes to the active form, which promotes the activation of tissue RAS 8, 9. In addition, (P)RR stimulation by (pro)renin elicits intracellular signaling pathways independent of angiotensin II 10, 11. These effects are thought to be involved in the activation of tissue RAS under pathological conditions such as diabetes, endocrine disorders, cardiovascular diseases, and obesity, in which (pro)renin is increased 12-14. (P)RR is localized at the plasma membrane, and its large extracellular domains are cleaved by proteases to form soluble (P)RR soluble (pro)renin receptor (s[P]RR) 15. To date, many clinical studies have shown that blood s(P)RR level is an important disease biomarker. Blood s(P)RR level has been reported to be a biomarker reflecting the severity of chronic kidney disease in patients with heart failure and essential hypertension 16-18.

On the other hand, high blood s(P)RR level has also been reported in obesity 19, 20. However, most of the results are from animal models and have not been fully validated in humans. It is also unclear which body composition components (e.g., visceral fat, subcutaneous fat, skeletal muscle, etc.) are responsible for the high s(P)RR blood levels in obesity. Furthermore, there are limited reports on the changes in blood s(P)RR levels in obese patients who lose weight. Clarification of these issues is very important for understanding the role of (P)RR in the pathogenesis of obesity and obesity-related complications. In the present study, the authors examined blood s(P)RR levels and ATP6AP2 gene expression levels in visceral and subcutaneous adipose tissue (VAT, SAT) in severely obese patients who underwent laparoscopic sleeve gastrectomy (LSG), with the aim of clarifying the relationship with body composition and metabolic factors.

We retrospectively reviewed clinical data obtained between August 2011 and June 2015 at the Toho University Sakura Medical Center (Sakura City, Chiba, Japan) to identify patients who underwent LSG and were postoperatively followed-up for 12 months. In Japan, obesity is defined as body mass index (BMI) ≥25.0 kg/m²21 and the criterion for a valid indication for LSG is BMI ≥32 kg/m² with at least one obesity-related comorbidity, such as type 2 diabetes, hypertension, dyslipidemia, or BMI ≥35 kg/m², according to the guideline of the Japanese Society for the Treatment of Obesity 22. We excluded patients whose body composition were not measured and those whose stored blood samples were not sufficient for the analyzation at baseline and/or 12 months after LSG. In addition, we excluded patients whose adipose tissue samples obtained at LSG were not sufficient for the analyzation at baseline. A total of 123 LSG cases were reported during the study period. Of those, 48 were excluded due to lack of stored samples and/or body composition data at baseline, and the remaining 75 were included in the analysis of the cross-sectional survey at baseline. Six patients dropped out within 12 months after LSG. At 12 months after LSG, body composition was not measured in 8 cases, and stored blood samples were lacking in 28 cases. Overall, 42 cases were excluded, and 33 cases were ultimately included in the analysis of the longitudinal survey during the 12 month after LSG.

In the cross-sectional survey at baseline, we evaluated the following parameters: body weight (BW), BMI, visceral fat area, subcutaneous fat area, levels of aspartate transaminase (AST), alanine transaminase (ALT), γ-glutamyl transpeptidase (GTP), serum creatinine, estimated glomerular filtration rate, urine albumin-to-creatinine ratio (UACR), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting plasma glucose, hemoglobin A1c (HbA1c), serum C-peptide, as well as serum level of s(P)RR.

To determine the visceral fat area, a computed tomography scan was performed at the umbilical level with the patient lying supine. The subcutaneous fat area was calculated by subtracting the visceral fat area from the total fat area. Radiologists quantified the fat area using Ziostation 2 software version 2.9.7.1 (Ziosoft, Inc., Tokyo, Japan).

Blood samples were obtained after at least 12 h fasting in the sitting position. Within 1 h of blood collection, the serum and plasma were separated by centrifuging the specimen at 3,000 rpm for 10 min. Serum samples were used to measure the levels of HbA1c, C-peptide, AST, ALT, GTP, creatinine, estimated glomerular filtration rate. For HbA1c level measurement, blood was collected in tubes containing ethylenediaminetetraacetic acid. Stable and unstable fractions of HbA1c were measured through high-pressure liquid chromatography using an HLC-732G11 analyzer (Tosoh Bioscience, Yamaguchi, Japan). However, only the stable form data were used in the analysis. Plasma TC and TG levels were measured enzymatically using Pureauto^® S CHO-N and TG-N kits from Sekisui Medical Co., Ltd. (Tokyo, Japan) and a Hitachi 7150 analyzer (Hitachi, Ltd., Tokyo, Japan). Serum HDL-C levels were measured using a selective inhibition assay with a JCA-BM1650 auto analyzer (JEOL JAPAN Ltd., Tokyo, Japan). Serum LDL-C levels were calculated using the Friedewald formula (LDL-C = TC – TG/5 – HDL-C). Serum s(P)RR levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Immuno-Biological Laboratories IBL, Co., Fujioka, Japan) consisting of a solid-phase sandwich ELISA with antibodies highly specific for each protein 23. HOMA2-IR values were obtained by the program HOMA Calculator v2.2.3.

VAT and SAT samples were isolated during the operation form patients who underwent LSG at Toho University Sakura Medical Center. The excised material was dissected accurately to ensure only pure adipose tissue was used for subsequent steps. Ethical approval for this study was given by the local ethical committee (ethic number).

One part of freshly excised adipose tissue was immediately dissected, cut into small pieces suitable for rapid penetration by the RNAlater RNA Stabilization Reagent (Qiagen GmbH, Hilden, Germany), and submerged according to the manufacturer’s protocol. Total RNA was isolated with RNeasy Lipid Tissue Mini kit (Qiagen) according to manufacturer’s instructions. After DNase treatment, RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Qiagen GmbH). Quantitative real-time PCR was performed using Probe qPCR mix (Takara Bio Inc., Shiga, Japan). Relative mRNA expression level of ATP6AP2 was calculated using the comparative C_T method and normalized to 18S rRNA. The sequences of the primer is given as follows: ATP6AP2: forward 5′-GCT CCC AGT GAG GAA AGA GTG TAT AT-3′, reverse 5′-GCG CAA GGT GAC TGA AAG G-3′; 18S rRNA: forward 5′-CTA CCA CAT CCA AGG AAG GCA-3′, reverse 5′-TTT TTC GTC ACT ACC TCC CC-3′.

Data are expressed as mean (SD) or as median (IQR). Normal distribution was tested using the Shapiro-Wilk test. Parametric data were analyzed using Student’s t test and nonparametric data using the Wilcoxon signed rank test (paired) and Wilcoxon rank-sum test (unpaired). Fishers’ exact test was used to detect significant differences between proportions and categorical variables. Simple linear regression analysis was performed by Spearman’s rank correlation coefficient. Multiple regression analysis was performed to identify parameters contributing to the s(P)RR level at baseline and changes in the s(P)RR level for 12 months. These analyses were performed using SPSS software version 26 (IBM Corp., Armonk, NY, USA). p values <0.05 were considered significant.

Table 1 shows the baseline characteristics including background factors, anthropometric parameters, blood data, CT data, as well as the expression levels of ATP6AP2 mRNA in VAT and SAT. The mean serum s(P)RR level at baseline was 26.1 ng/mL, this value was considered higher than previously reported values in healthy subjects 24. There were no significant sex differences.

Figure 1 shows the comparison of the expression levels of ATP6AP2 mRNA between VAT and SAT. There was no significant difference in the expression level of ATP6AP2 mRNA between VAT and SAT. There were no sex differences in ATP6AP2 mRNA expression in either visceral or SAT.

Table 2 shows the correlation between serum s(P)RR level and other clinical and laboratory parameters at baseline. In the univariate analysis (A), BW, BMI, visceral fat area, serum C-peptide, HOMA2-IR, serum creatinine, UACR positively correlated with serum s(P)RR level, while subcutaneous fat area did not. Multiple regression analysis for the association between s(P)RR and variables identified as significant in the univariate analysis, identified that visceral fat area, HOMA2-IR, UACR showed the independent relationships with s(P)RR (B).

We next examined the correlation between expression levels of ATP6AP2 mRNA in VAT/SAT and other clinical and laboratory parameters at baseline (data not shown). The levels of ATP6AP2 mRNA expression in both VAT and SAT did not correlate with any variables including serum s(P)RR.

Table 3 shows clinical and laboratory variables in 33 cases at baseline, 12 months after LSG, as well as the changes during the 12 months. During the 12 months after LSG, BW, BMI, visceral fat area, subcutaneous fat area, fasting plasma glucose, HbA1c, serum C-peptide, HOMA2-IR, TG, LDL-C, AST, ALT, serum creatinine, and UACR decreased, and HDL-C increased significantly. TC and GTP did not show significant changes. Serum s(P)RR showed a significant decrease during the 12 months after LSG (from 30.0 ± 7.0 to 21.9 ± 4.3, Fig. 2).

Table 4 shows the correlation between changes in serum s(P)RR level and in other clinical and laboratory parameters during the 12 months after LSG. In the univariate analysis (A), changes in BW, BMI, visceral fat area, HbA1c, AST, and ALT positively correlated with the changes in s(P)RR. On the other hand, the changes in subcutaneous fat area did not correlate with the change in s(P)RR. Multiple regression analysis for the association between the change in s(P)RR and variables identified as significant in the univariate analysis showed that changes in visceral fat area and ALT were independently related to the change in s(P)RR (B).

The main findings of this study were that blood s(P)RR level was high in highly obese patients 24, decreased with weight loss by LSG, and was associated with visceral fat area in both pre- and postoperative changes. High blood s(P)RR level in obesity has been reported previously. Wu et al. 19 reported that blood s(P)RR levels were high in a mouse model of diet-induced obesity. Furthermore, in humans, it has been reported that adult growth hormone deficient patients with visceral fat accumulation showed high blood s(P)RR levels and that these levels were related to BMI 25. However, these reports did not sufficiently demonstrate whether obesity is associated with blood s(P)RR levels independently of other comorbid conditions. Diabetes mellitus, hypertension, and CKD are frequently associated with obesity, and s(P)RR blood levels have been reported to be high in these diseases 16, 18. Furthermore, it has been unclear which of the body composition components of obesity are associated with s(P)RR blood levels. Our results show that visceral fat mass is associated with blood s(P)RR levels independently of glucose and lipid metabolic factors, liver function, and renal function. Similar results were found in a pre- and post-bariatric surgery weight loss study, which further strengthened the possibility that visceral fat mass contributes to blood s(P)RR levels. Nishijima et al. 26 reported that blood s(P)RR levels decreased with weight loss after bariatric surgery, which is consistent with the results of this study. However, this report did not show a relationship between blood s(P)RR levels and fat distribution. To the best of our knowledge, the results of the present study are the first to show a clear association between visceral fat and blood s(P)RR levels in humans with obesity.

In the present study, we did not find any significant difference between ATP6AP2 expression levels in VAT and SAT. Achard et al. 27 reported that the levels of ATP6AP2 mRNA expression did not differ between VAT and SAT in lean and obese humans, and the result is consistent with the present study. On the other hand, in a study using an obese animal model, Tan et al. 28 reported that the expression of ATP6AP2 was higher in VAT than in SAT, which is contradictory to the result of the present study. This may be due to the fact that the study was conducted in an animal model. Interestingly, the levels of ATP6AP2 mRNA expression both in VAT and SAT did not show significant relationship with blood s(P)RR level, while visceral fat area did. In addition, the level of ATP6AP2 mRNA expression both in VAT and SAT did not correlate with the markers of glucose metabolic factors and renal function. These reports and the result of the current study suggest that the contribution of VAT to blood s(P)RR levels may involve absolute mass or post-transcriptional regulation of visceral fat rather than gene expression levels in adipose tissue. These points have not been reported so far and need to be validated by further research. In a study using the (P)RR blocker recoil region peptide (HRP), different functional changes were reported in VAT and SAT. Tan et al. 29 reported that HRP caused a redistribution of fat from VAT to SAT. Others have reported that HRP suppressed the increase in visceral fat and promoted the beiging of SAT 30. In other words, the role of (P)RR in the pathogenesis of obesity is thought to vary depending on the location of fat, and it is important to pursue this point in order to understand the pathogenesis of obesity. Although the results of this study do not address this point, further investigation is needed.

In the present study, s(P)RR levels were associated with HOMA2-IR and UACR. HOMA2-IR is a well-recognized index reflecting insulin resistance 31. It is a well-known fact that obesity causes increased insulin resistance and renal glomerulopathy 32, 33. Regarding the relationship between blood s(P)RR levels and metabolic and renal function factors, Hamada et al. 18 reported higher blood s(P)RR levels in CKD patients with type 2 diabetes and hypertension. Watanabe et al. 34 reported that blood s(P)RR levels are associated with renal damage in patients with primary aldosteronism, and Morimoto et al. 16 reported that blood s(P)RR levels are associated with the severity of renal damage in patients with essential hypertension. These reports and the results of the present study may suggest that the s(P)RR is involved in insulin resistance and renal glomerular damage associated with obesity. However, given that UACR changes before and after LSG were not associated with changes in s(P)RR and that the subjects in this study were a population with minimal renal dysfunction at baseline, the association between s(P)RR and renal impairment requires further investigation. Although the results of the present study do not address the causal relationship between high blood s(P)RR levels and insulin resistance and renal glomerulopathy, there have been a report in this regard. Wang et al. 35 reported that administration of recombinant s(P)RR to an animal model of diet-induced obesity improved obesity, insulin resistance, and renal damage. Based on the report, it is possible that s(P)RRs in blood act protectively against obesity, insulin resistance, and renal glomerulopathy. In other words, this phenomenon may be a compensatory mechanism to suppress the progression of organ damage associated with obesity. Further elucidation is needed.

Changes of s(P)RR after LSG were also independently associated with changes in ALT. Although little is known about the role of PRR in the liver, its involvement in hepatosteatosis has been reported 36. In vitro experiments using HepG2 cells showed that administration of s(P)RR upregulated SREBP-2, suggesting that s(P)RR may contribute to hepatic biosynthesis 37. Combined with the results of the present study, it is assumed that postoperative s(P)RR changes may be associated with fatty liver status. However, a causal relationship cannot be determined from the results of this study, and further investigation is needed.

The limitations of this study are as follows. First, the results from this study alone do not prove a causal relationship between visceral fat accumulation and increased blood s(P)RR levels. Second, we cannot rule out the possibility that factors not measured in this study indirectly influenced the change in blood s(P)RR levels after LSG. Third, because this study only analyzed the effects of LSG on blood s(P)RR levels, it is unclear whether the results would be similar if weight loss were achieved by other methods such as lifestyle modification. Finally, this study is an observational study with a small number of subjects.

In any case, (P)RR expression levels in adipose tissue and blood s(P)RR levels are important targets for the pathogenesis and treatment strategies of obesity and related organ disorders, and the results obtained from this study are important for a better understanding of these issues. In conclusion, we suggest that blood s(P)RR levels in obese patients may reflect the involvement of visceral adipose (P)RR in insulin resistance and renal damage mechanisms associated with obesity.

The protocol of the study was prepared and implemented in accordance with the tenets of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Toho University Sakura Medical Center (approval date; July 27, 2022, approval number; S22010). Although this was a retrospective study, we explained to individual patients for usage and release of study data and obtained written consent from each patient.

The authors declare that they have no conflicts of interest.

This article did not receive any funding.

Takashi Yamaguchi, Satoshi Morimoto, and Ichiro Tatsuno designed the original concept of this study. Takashi Yamaguchi wrote the initial draft of the manuscript. Ichiro Tatsuno reviewed and edited the manuscript. Chikahito Suda, Satoshi Morimoto, and Atsuhiro Ichihara contributed to the measurement of s(P)RR and interpretation of the data. Noriko Ishihara was in charge of the genetic analysis practice. Shoko Nakamura, Shou Tanaka, Yasuhiro Watanabe, Haruki Imamura, Masahiro Ohira, Naomi Shimizu, and Atsuhito Saiki contributed to data collection and interpretation and critically reviewed the manuscript. All the authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy of integrity of any part of the work are appropriately investigated and resolved.

Data are not publicly available due to ethical reasons. Further inquiries can be directed to the corresponding author.