Reduction in Urinary Chemokine (C-C Motif) Ligand 2 (CCL2) After Surgery-Induced Weight Loss

Kidney dysfunction, a deleterious effect of obesity, is now recognized as a relevant health risk. Chemokine (C-C Motif) Ligand 2 (CCL2) is one of the critical chemokines that play a vital role in the development of obesity-related metabolic disease. We aim to measure the changes in urinary CCL2 in our patients before and after their bariatric procedure and examine the correlation between CCL2 and renal function. A prospective cohort study was conducted at our teaching university hospital. Ethics approval was obtained from our institutional review board. Patients with a BMI of ≥37.5 kg/m2 with no history of renal disease were included. They underwent single anastomosis gastric bypass (SAGB), Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG), all performed via laparoscopic approach. Venous blood and urine samples were obtained preoperatively and six months after surgery. A total of 58 patients were recruited, with SG being performed in 74.1% of patients. At six-months follow-up, median (IQR) body weight reduced from 101.35 kgs (20.25) to 76.95 kg (24.62) p < 0.001. The mean (SD) estimated glomerular filtration rate (eGFR) improved from 96.26 ± 14.97 to 108.06 ± 15.00 mL/min/1.73 m2, p < 0.001. The median (IQR) urinary CCL2 levels reduced from 15.2 pg/ml (10.77) to 4.30 pg/ml (4.27) p < 0·001. There is a significant correlation between the reduction of BMI and the reduction of urinary CCL2 (r = −0.220, p = 0.048). We also found a significant correlation between the reduction of urinary CCL2 with the reduction of urine ACR (r = −0.240, p = 0.035). Urinary CCL2 is a promising biomarker that can be used to assess improvement in renal function in obese patients after bariatric surgery.


Results
A total of 58 patients were recruited, in which 68.9% (n = 40) were women. The mean (SD) age was 38.79 ± 8.40 years. The majority of patients were of Malay ethnicity (86.2%). The median (IQR) body weight before surgery was 101.35 kgs (20.25) with a median (IQR) BMI of 45.0 kg/m 2 (7.655). The mean (SD) eGFR and median(IQR) urine ACR before surgery was 96. 26 (14.97) and 2.5 (3.7), respectively. About 24% of our patients had a diabetic range of HbA1c level (≥6.5%) before surgery. The median(IQR) HbA1c level was 5.7% (0.95). Baseline characteristic of patients with anthropometric measurement prior to bariatric surgery are summarised in Table 1.
At baseline, 25 patients (43.1%) fell into the category of microalbuminuria (urine ACR between 30 to 300), and only one patient had macroalbuminuria (urine ACR > 300.0). At six-months follow-up, a significant reduction of urine ACR was evident, p < 0.001, with the number of patients with microalbuminuria reduced to only three (5.17%). The baseline median (IQR) of urinary CCL2 for patients without microalbuminuria was 16.16 (15.73) and 15.21(8.50) for patients with microalbuminuria. Using the Mann Whitney U test, there was no significant correlation between both groups (z = −0.088, p = 0.93). Interestingly, the sole patient with macroalbuminuria did not have the highest level of urinary CCL2. The change in baseline parameters before and after surgery are illustrated in Table 2.
There was no significant difference in the baseline and renal function parameters between SG, SAGB, and RYGB. SG was performed for the majority of our patients (74.1%). The changes in baseline and renal parameters six months after surgery are summarised in Table 2.
Overall, there is a significant correlation between the reduction of BMI and the reduction of urinary CCL2 (r = −0.220, p = 0.048). There is also a significant correlation between the reduction of urinary CCL2 with the reduction of urine ACR (r = −0.240, p = 0.035). However, there is no significant correlation between the reduction of urinary CCL2 with the difference in eGFR or serum creatinine before and after surgery. There was no significant difference in the reduction of urinary CCL2 seen between SG, RYGB and SAGB as well.

Discussion
This study demonstrates a significant reduction of urinary CCL2 after bariatric surgery. CCL2 is an inflammatory cytokine that is involved in many inflammatory activities in the human body. This biomarker is one of the most frequently studied for various inflammatory-induced diseases, including obesity-related kidney disease 11,13 . High levels of serum CCL2 in obese patient results from high expression and production of this chemokine from excessive subcutaneous and visceral adipose tissue among obese patients 8 . A high level of urinary CCL2 was present in our patients, and this level reduced significantly following bariatric surgery. Surgically-induced weight loss may ameliorate renal inflammatory cascade and improve ORG 12 . Urinary CCL2 has been proposed as a marker of renal injury following the evidence showing a positive correlation between urinary MCP-1 with albuminuria, mesangial proliferation, and interstitial fibrosis 13 . Renal tubular epithelial cells express CCL2 in response to a variety of pro-inflammatory stimuli 14 . This local production of CCL2 in the kidney will lead to higher excretion of this cytokine in the urine. A reduction in excessive adipose tissue following bariatric surgery will cause a decreased CCL2 production, which subsequently eliminates the stimulus for local production of CCL2 from the kidneys 12 .
Our study also demonstrates a significant correlation between urinary CCL2 with urine ACR, a marker of renal damage. CCL2 produced in the renal tubular cells is released into the urine in proportion to the degree of renal damage 14,15 . This CCL2 increases inflammatory molecules on mesangial and tubular cells and induces tubule-intestinal injury via recruitment and activation of macrophage 16 . Persistent stimulation will ultimately lead to in situ renal fibrosis and, subsequently, renal damage 14,15 Thus, urinary CCL2 levels following bariatric surgery may be used as a surrogate marker to the ongoing renal inflammatory process.
Our study has several limitations. We did not include patients with established kidney disease as bariatric surgery in patients with advanced kidney disease carries a higher complication rate 17 . However, a systematic review concluded that bariatric surgery might prevent a decline in renal function by improving glomerular hyperfiltration and reducing albuminuria 18 . More studies are needed to elucidate whether progression to end-stage renal disease can be halted with bariatric surgery.
We also failed to show a significant correlation between an improvement in eGFR with a reduction in urinary CCL2, in contrast to a previous study 12 . It is possible that calculating eGFR using a creatinine-based formula may lead to an overestimation of renal function. A majority of our patients (63.8%) have an eGFR ≥ 90 mL/ min/1.73 m 2 at the beginning of the study, and this increased to 91.4% six months after surgery. The observed decrease in serum creatinine levels after bariatric surgery might reflect the inevitable decrease in lean body mass rather than an actual improvement in renal function 19 .
Of note, cystatin C is an endogenous substance produced continuously and freely filtered in the glomerulus without tubular secretion or reabsorption. Its excretion in the kidney is not affected by body mass or weight loss 20 . The use of cystatin C to measure the glomerular filtration rate may give a more accurate reading. However, studies have shown that cystatin C has a direct relationship with fat mass, and a higher BMI seems to be associated with a higher cystatin C 21,22 . Hence, the use of creatinine or cystatin C based formula both poses their limitations.
Urinary CCL2 as a marker of renal injury in the obese population, has mostly been studied in the western population. To date, only one paper had studied this inflammatory marker and its relation to renal injury in the obese Asian population 10 , and it only included patients of the male gender. A more extensive prospective study evaluating the use of urinary CCL2 to assess improvements in renal function in obese Asian patients with established renal disease is needed.

Conclusion
Urinary CCL2 is a promising biomarker that can be used to assess improvement in renal function in the obese population after bariatric surgery, as creatinine-based formula often leads to an overestimation of renal function. A reduction in urinary CCL2 after bariatric surgery correlates well with urine ACR and the reduction of BMI, and this reduction is also uniform across genders. In the obese population with early-stage renal disease, bariatric surgery may be beneficial in ameliorating the existing renal inflammation and prevent a decline in renal function.

Methods
This is a prospective cohort study assessing the change in urinary CCL2 levels before and after bariatric surgery, conducted at a teaching university hospital from June 2016 to June 2018. The study was conducted following the principles of the Declaration of Helsinki. Ethics approval was obtained before the start of this study from our institutional review board, Universiti Kebangsaan Malaysia Research Ethics Committee (reference number: FF-2017-035). This study was also registered with the Thai Clinical Trials Registry with the number TCTR20190825002. Written informed consent was taken prior to study commencement. All data collected were kept confidential, and patients were allowed to refuse participation in the study. Data presented do not identify individuals.
Purposive sampling method was employed. We included patients of both genders aged 18-65 years, with a BMI of ≥37.5 kg/m 2 with no history of renal disease who are planned for bariatric surgery. Patients who received steroid therapy within six months prior to the surgery, patients with malignancy, patients who are pregnant, and patients with stage 3 A kidney disease and beyond (defined as an eGFR < 60 mL/min/1.73 m 2 ) were excluded.
A multidisciplinary team performed a thorough assessment, and the final bariatric procedure to be performed is decided, taking into consideration the patient's choice as well. These procedures include SAGB, RYGB, and SG, all performed via laparoscopic approach by a consultant upper gastrointestinal surgeon with more than five years' experience. Venous blood and midstream urine samples were taken preoperatively and six months after the surgery. Midstream urine was collected into sterile containers in the morning. The biochemical evaluation was performed for serum creatinine and HbA1c, urinary CCL2, and urine ACR. Serum creatinine and HbA1c were measured using standard laboratory methods. (2020) 10:790 | https://doi.org/10.1038/s41598-020-57763-8 www.nature.com/scientificreports www.nature.com/scientificreports/ EGFR was calculated using the MDRD 4 variable formula 23 . An abnormal glomerular filtration rate is defined as eGFR < 90 mL/min per 1.73 m 2 based on the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (KDOQI) 24 . All urine samples were centrifuged at 1600g for 10 min at 4 °C, and the supernatant was stored at −80 °C until further analysis. The reading of urine albumin was expressed as urine albumin-creatinine ratio (ACR) 17 .
The level of urinary CCL2 was measured using specific enzyme-linked immunoassay (ELISA) kits (Minneapolis, Minn, USA, R&D Systems). Dilution of urine samples was performed according to the manufacturer's instructions. Samples were then incubated for two hours at room temperature. After washing the plates, a conjugate was added to erase any unbound substances and was left alone for an hour. A substrate solution was then added after washing the plates. Stop solution was added after incubation for 20 min at room temperature. The absorbance was then read at 450 nm with the correction wavelength set at 540 nm. All samples were assayed in duplicate. Urine CCL2 levels were expressed as pictogram per ml (pg/ml).
The study planned as a continuous response variable based on a previous study 25 . The difference in the response is normally distributed with a standard deviation of 25. If true difference in the mean response is 13, we will need 58 subjects to be able to reject the null hypothesis that this response difference is zero with probability (power) of 0.8, and confidence interval of 95%. Type I error probability associated with this test of this null hypothesis is 0.05 26 .
Variables with normal distribution were expressed as mean ± SD, whereas non-parametric variables were expressed as median (IQR). The significance of differences before and after bariatric surgery was evaluated with a paired t-test for parametric data and the Mann Whitney U test for non-parametric data. For comparisons among the LAGB, LRYGB, and LSG groups, the Kruskal Wallis one-way test was used. Correlation between variables was performed using the Pearson Bivariate test. All statistical analyses were performed using SPSS (version 24.0; SPSS, Chicago, IL). Statistical significance was set at p < 0.05.