Chronic Kidney Disease Predictors in Obese Adolescents Ⅲ

Biomarkers

Daily megalin excretion was estimated using Human LRP2/ Megalin ELISA assay (LS-F11978-1, BD BioSciences, Warsaw, Poland). The concentrations of NGAL, Gal-3, MMP12, and TIMP2 were estimated using Human Lipocalin-2/NGAL Quantikine ELISA Kit (R&D Systems, Minneapolis, MN), Human LGALS3/Galectin-3 ELISA Kit (RAB0661, Sigma-Aldrich, Saint Louis, MO), Human MMP-12 ELISA Kit (P39900, Invitrogen, Carlsbad, CA), and TIM2 Human ELISA Kit (P16035, ThermoFisher Scientific, Waltham, US-MA), respectively.

Finally, serum adiponectin concentration was assessed with the use of a Human Adiponectin ELISA kit (ab99968, Abcam, Cambridge, UK).

All samples from patients and controls were run in duplicate. The assays were performed according to the manufacturer's instructions. The optical density (OD) value was determined twice, using a microplate reader, set to 450 nm and 540 nm as a wavelength correction. The readings at 540 nm were subtracted from the readings at 450 nm to correct for optical imperfections in the plate. The concentrations of NGAL, Gal-3, MMP12, TIMP2, and adiponectin were calculated based on the standard curve. These standards were also assayed in duplicate.

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Statistical analysis 

All continuous variables are expressed as the mean±SD. Statistical analysis was performed through the following steps: (1) Shapiro–Wilk test was used to determine the normal distribution of data; (2) one-way analysis of variance was employed to compare "elevated GFR," "normal GFR," and "decreased GFR" study subjects and controls; (3) Bonferroni correction was used as a general alpha adjustment for all of the multiple comparisons performed in the study; (4) non-parametric continuous variables were compared using the Kruskal–Wallis test; and (5) Pearson χ2 test was employed to analyze nominal data.

The independent factors for CKD development were determined using Fisher's exact test. Generally, in Pearson correlation, a value of r greater than 0.7 was considered a strong correlation. Anything between 0.4 and 0.7 was a moderate correlation, and anything less than 0.4 was considered a weak or no correlation.

The factors potentially affecting the rate of glomerular filtration in obese adolescents ("serum uric acid concentration," "serum triglycerides concentration," "serum cholesterol concentration," "serum adiponectin concentration," "albuminuria," "urine galectin-3 concentration," "daily urine megalin excretion," and "urine NGAL concentration") were assessed using linear regression methods utilizing univariate and multivariate analyses. Regression coefficients (β) and 95% confidence interval (CI) for β were estimated for all linear models. The regression models were adjusted for age and sex.

All of the statistical analyses were performed using the Statistica 13.3 PL software package (StatSoft, Poland). A value of p<0.05 was considered statistically signifcant.

Results Clinical characteristics 

Obese study participants (BMI z-score>2), at the moment of entry into the analysis, were divided into "elevated GFR" (n = 42), "normal GFR" (n= 85), and "decreased GFR" (n=15) according to GFR values estimated by Filler formula. As compared to controls, they presented albuminuria and had a significantly lower serum adiponectin concentration (p=0.005) and higher urine Gal-3 concentration (p=0.004). "Decreased GFR" obese adolescents (as compared to controls) have been additionally reported with significantly higher serum concentrations of uric acid (p=0.004), triglycerides (p=0.037), and cholesterol (p=0.043) as well as significantly higher NGAL urine concentration and daily urine megalin excretion (p=0.005).

Obese and control adolescents did not difer in blood pressure (both ofce and during 24-h monitoring), peripheral blood morphology, and serum biochemical parameters (including concentrations of creatinine, cystatin C, urea, Na, K, Ca, Mg, fasting glucose, TSH, fT3, fT4, liver enzymes, total protein, and CRP).

None of the examined adolescents had MMP12 in urine, and the concentrations of TIMP2 were comparable in all study participants. The clinical characteristics of all adolescents are summarized in Table 1.

Correlations and multivariate analysis

As mentioned above, the group of obese adolescents was not homogeneous. It was composed of "elevated GFR," "normal GFR," and "decreased GFR" patients. Therefore, separate analyses were performed for each of the above-mentioned 3 subgroups.

In the "elevated GFR" obese study participants, we have noticed one moderate positive correlation between "urine galectin-3 concentration" and "albuminuria" (r=0.528) and one moderate negative correlation between "urine galectin-3 concentration" and "serum adiponectin concentration."

The group of "normal GFR" obese patients had a strong correlation between "urine galectin-3 concentration" and "urine NGAL concentration" (r=0.706). In the "decreased GFR" obese adolescents, three positive moderate correlations were reported. The frst one between "serum uric acid concentration" and "urine NGAL concentration" (r = 0.474), the next one between "urine NGAL concentration" and "urine megalin excretion" (r=0.524), and the last one between "serum uric acid concentration and urine megalin excretion" (r = 0.511). These results are presented in Table 2.

Estimates from linear regression models in study participants are shown in Table 3. Factors associated with obesity-related kidney damage in the period of hyperfiltration included decreased serum adiponectin concentration, albuminuria, and increased urine Gal-3 concentration. Factors associated with obesity-related kidney damage in obese adolescents with normal GFR were albuminuria, increased urine Gal-3 concentration, elevated serum cholesterol concentration, and increased urine NGAL concentration. Finally, obesity-related kidney damage followed by decreased GFR was associated with increased urine NGAL concentration, increased serum uric acid concentration, and increased urine megalin daily excretion (Table 4).

Table 2 Moderate and strong correlations in study participants

CKD development in study participants

During the follow-up period, which ranged from 6 to 30 months, 39 obese adolescents in the study group developed CKD (27.5%). Eleven of them were initially designated as "increased GFR" individuals (26.2%), 16 as "normal GFR" study participants (18.9%), and 12 were initially recognized with decreased GFR (80%).

"Elevated GFR" obese study participants, who developed CKD during the follow-up period, did not differ in peripheral blood morphology, serum creatinine, cystatin C, uric acid, urea, Na, K, Ca, Mg, total cholesterol, triglycerides, HDL, fasting glucose, TSH, fT3, fT4, liver enzymes, total protein, CRP, adiponectin as well as 24 h urine for albumin, Gal-3, megalin, NGAL, or TIMP2 values from their peers who did not develop CKD.

The majority of "normal GFR" obese study participants who developed CKD, were initially found with increased concentrations of serum uric acid (5.9±0.6 mg/dL; n=14; 87.5%), cholesterol (194±5.8 mg/dL; n=15; 93.8%), and triglycerides (172±4.9 mg/dL; n=10; 62,5%) as well as urine NGAL concentration (9.8±3.5 ng/ml; n=13; 81,2%), as compared to "normal GFR" obese adolescents who did not develop CKD.

Finally, "decreased GFR" obese adolescents who did not develop CKD within the observation period (n=3) were found initially to have normal serum uric acid concentration (4.1±0.3 mg/dL) as compared to their peers who developed CKD. Using Fisher's exact test, it was determined that "normal GFR" obese adolescents, who initially had elevated serum uric acid concentration (p<0.0001), serum triglycerides concentration (p=0.0006), serum cholesterol concentration (p<0.0001), and urine NGAL concentration (p=0.0003), developed CKD over a follow-up period of 6 to 30 months. "Decreased GFR" obese study participants who continue to have normal values of serum uric acid concentration are at lower risk of CKD development (p=0.0088).