Effect of urine alkalization on urinary inflammatory markers in cystinuric patients

Abstract

Background: Cystinuria is associated with a high prevalence of chronic kidney disease (CKD). We previously described a urinary inflammatory-protein signature (UIS), including 38 upregulated proteins, in cystinuric patients (Cys-patients), compared with healthy controls (HC). This UIS was higher in Cys-patients with CKD. In the present observational study, we aimed to investigate the UIS in Cys-patients without CKD and patients with calcium nephrolithiasis (Lith-patients), versus HC and the effect of urine alkalization on the UIS of Cys-patients.

Methods: UIS was evaluated by nano-liquid chromatography coupled to high-resolution mass spectrometry in adult HC, Lith-patients and non-treated Cys-patients with an estimated glomerular filtration rate >60 mL/min/1.73 m², and after a 3-month conventional alkalizing treatment in Cys-patients.

Results: Twenty-one Cys-patients [12 men, median age (interquartile range) 30.0 (25.0-44.0) years], 12 Lith-patients [8 men, 46.2 (39.5-54.2) years] and 7 HC [2 men, 43.1 (31.0-53.9) years] were included. Among the 38 proteins upregulated in our previous work, 11 proteins were also upregulated in Cys-patients compared with HC in this study (5 circulating inflammatory proteins and 6 neutrophil-derived proteins). This UIS was also found in some Lith-patients. Using this UIS, we identified two subclusters of Cys-patients (5 with a very high/high UIS and 16 with a moderate/low UIS). In the Cys-patients with very high/high UIS, urine alkalization induced a significant decrease in urinary neutrophil-derived proteins.

Conclusion: A high UIS is present in some Cys-patients without CKD and decreases under alkalizing treatment. This UIS could be a prognostic marker to predict the evolution towards CKD in cystinuria.

Keywords: cystinuria; inflammation; nephrolithiasis; urine proteomics.

Figure 1:

Differential proteomes of cystinuric patients at baseline (Cys0, n = 21) versus HC (n = 7). The proteome of the 21 Cys0-patients was compared with that of the 7 HC. Volcano plot representing the statistical comparison of the protein Label Free Quantification intensities of the Cys0 group versus the HC group. Volcano plot was established using S0 = 1 and False Discovery Rates = 0.1. The abscissa reports the fold-change in logarithmic scale (difference), the ordinate the –log₁₀ p-value. Neutrophil-derived proteins and inflammatory circulating proteins were highlighted in red crosses and red filled scares, respectively. The protein rBAT encoded by the SLC3A1 gene is highlighted with a green diamond. For the name of the protein corresponding to the gene name, refer to Supplementary data, Table S2.

Figure 2:

Heatmap of the proteins involved in the UIS among cystinuric patients at baseline (Cys0, n = 21, in blue), patients with other types of nephrolithiasis (Lith, n = 12, in pink) and HC (n = 7, in orange). The heatmap representation was performed on Perseus software (version 1.6.15.0) on z-scored Label Free Quantification intensity of the proteins with a green–red color gradient where red color represents highly abundant proteins and green low abundant proteins. Two clusters of proteins were highlighted corresponding to neutrophil-derived proteins and inflammatory circulating proteins. Moreover, individuals were clustered in four groups corresponding to different level of UIS: “strong” (red), “moderate” (orange), “low” (dark green) and “no” (light green). For the name of the protein corresponding to the gene name, refer to Supplementary data, Table S2.

Figure 3:

Heatmap of the proteins involved in the UIS among the 21 cystinuric patients at baseline (Cys0). The heatmap representation was performed on Perseus software (version 1.6.15.0) on z-scored Label Free Quantification intensity of the proteins with a green–red color gradient where red color represents highly abundant proteins and green low abundant proteins. Two clusters of proteins were highlighted corresponding to neutrophil-derived proteins and inflammatory circulating proteins. Moreover, individuals were clustered in two groups corresponding to different level of UIS: 5 patients had a very high or high inflammatory urinary signature (red) and 16 patients had a moderate or low UIS (green). For the name of the protein corresponding to the gene name, refer to Supplementary data, Table S2.

Figure 4:

Profile plots of the neutrophil-derived proteins and inflammatory circulating proteins in the 5 cystinuric patients showing a very high (n = 2) (A), or high (n = 3) (B) UIS before and after the 3-month alkalizing treatment. For the name of the protein corresponding to the gene name, refer to Supplementary data, Table S2.