Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea.
Department of Internal Medicine, Armed Forces Capital Hospital, Gyeonggi-do, Korea.
Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute, Seoul, Korea.
Kidney Research Institute, Seoul National University, Seoul, Korea.
Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea.
Department of Internal Medicine, Keimyung University School of Medicine, Daegu, Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.
Department of Internal Medicine, Seoul National University Boramae Medical Center, Seoul, Korea.
Department of Urology, Seoul National University Hospital, Seoul, Korea.
Department of Chemistry and Nano Science, Ewha Womans University, Seoul, Korea.
Abstract
Identification of a urinary metabolite biomarker with diagnostic or prognostic significance for early immunoglobulin A nephropathy (IgAN) is needed. We performed nuclear magnetic resonance-based metabolomic profiling and identified 26 metabolites in urine samples. We collected urine samples from 201, 77, 47, 36 and 136 patients with IgAN, patients with membranous nephropathy, patients with minimal change disease, patients with lupus nephritis and healthy controls, respectively. We determined whether a metabolite level is associated with the prognosis of IgAN through Cox regression and continuous net reclassification improvement (cNRI). Finally, in vitro experiments with human kidney tubular epithelial cells (hTECs) were performed for experimental validation. As the results, the urinary glycine level was higher in the IgAN group than the control groups. A higher urinary glycine level was associated with lower risk of eGFR 30% decline in IgAN patients. The addition of glycine to a predictive model including clinicopathologic information significantly improved the predictive power for the prognosis of IgAN [cNRI 0.72 (0.28-0.82)]. In hTECs, the addition of glycine ameliorated inflammatory signals induced by tumour necrosis factor-α. Our study demonstrates that urinary glycine may have diagnostic and prognostic value for IgAN and indicates that urinary glycine is a protective biomarker for IgAN.
IgA nephropathyglomerulonephritisglycinemetabolomicsprognosis
Figures
FIGURE 1Box plots showing the creatinine‐adjusted urinary metabolite concentrations. The y‐axes indicate the creatinine‐adjusted urine concentrations (μmol/L/mmol/L Cr). The box plot shows the interquartile ranges, with the horizontal lines indicating the median values. Each dot represents the value of each study patient, and outliers over 95 percentile ranges are not presented in the figure. The blue background indicates the metabolite that was significantly low in the IgAN samples, and the yellow backgrounds indicate the metabolites that are significantly high in the urine of IgAN patients. The red background indicates that the metabolite level was significantly higher in the IgAN samples than in the disease‐control samplesFIGURE 2Association between the creatinine‐adjusted urinary metabolite concentrations and the risk of a 30% reduction in eGFR. The y‐axes indicate the unadjusted hazard ratios for risk of a 30% reduction in eGFR. The x‐axes indicate the creatinine‐adjusted urine metabolite levels. The red graphs indicate the associations between the metabolite levels and the risk of the study outcomeFIGURE 3Representative immunohistochemistry results for the enzymes related to glycine metabolism. Representative immunohistochemistry images of human kidney biopsy tissues from the IgAN (N = 12), control (N = 11), MCD (N = 3) and MN (N = 3) groups stained with primary antibodies for enzymes related to glycine metabolism. The stained intensities for T protein and H protein were lower in the IgAN patients than in the controls. The scale bars indicate 100 μm. MCD = minimal change disease, MN = membranous nephropathy. *P < .05, **P < .01FIGURE 4A, Representative Western blot results showing the expression of protein H and protein T in the hTECs treated with TNF‐α. Expression of protein H and T according to treatment with TNFα at different dosages (2.5, 5.0, 10.0 and 20.0 ng/mL) for 1 h. The experiment was performed 6 times, and a representative image is shown. Treatment with TNFα at 10 ng/mL and 20 ng/mL resulted in significantly reduced expression of protein H. The experiment was performed in triplicate, and a representative image is shown. *P < .05, **P < .01. B, Representative Western blot results showing inflammatory molecule expression according to the addition of glycine and TNF‐α induction in hTECs. Expression of inflammation‐related molecules according to the addition of glycine at different dosages (2.5 mmol/L, 5.0 mmol/L, 10.0 mmol/L and 20 mmol/L) in the cells treated with 10 ng/mL TNFα for 1 h. The addition of glycine resulted in a significant reduction in the expression levels of ICAM‐1, pp38 and pp65 compared to the results with only TNFα. The experiment was performed in quadruplicate, and a representative image is shown. *P < .05. C, Glutathione assay results. The results showed that the glutathione concentrations were increased along with the addition of glycine, reported in relative levels compared to the mean level of the control cells. Glutathione assay was performed for ten times in each condition. ** P < .01. D, Representative Western blot results of the inflammatory molecules and glycine levels according to TNF‐α induction and protein H suppression by siRNA. The suppression of protein H by siRNA under TNFα treatment resulted in a significant reduction in ICAM‐1, pp38/p38, pp65/p65 and protein H compared to the results from treatment with TNFα alone (P < .05, **P < .01 and *** P < .001). E, The glycine levels (MFI = mean fluorescent intensity) of the cell lysates were significantly increased following siRNA suppression compared to the result with scrambled RNA. Moreover, TNFα induction resulted in a further increase in glycine levels. Last, when protein H was suppressed by siRNA, the glycine levels were even higher than those in the other experiments. The experiment was performed in quadruplicate, and a representative image is shown
그림 출처: PubMed Central 공개 논문 (PMC8178259)
Conflict of interest statement
The authors declare no conflicts of interest.
그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
Journal of cellular and molecular medicine · eISSN 1582-4934
