The Association between Urinary Biomarkers 3-Indoxyl Sulfate, Monocyte Chemoattractant Protein-1, Neutrophil Gelatinase-Associated Lipocalin and Klotho with Chronic Kidney Disease in Patients with Sickle Cell Disease

Introduction: Approximately one-third of patients with sickle cell disease (SCD) develop chronic kidney disease (CKD) as they age. Intestinal microbiota are reported to contribute to the development and progression of CKD. Despite an increasing understanding of the pathophysiology of SCD-related kidney disease, the role of microbiota has not been studied. Indoles, which are tryptophan metabolites produced by gut microbiota, have nephrotoxic effects that target the proximal tubules. One such indole, 3-Indoxyl Sulfate (3-IS), accumulates and causes increased tubular stress through the buildup of reactive oxygen species. It also reduces the renal expression of Klotho, an anti-aging and protective protein primarily produced in the kidneys. Although higher urinary 3-IS is observed in SCD than in iron deficiency anemia, the relationship between 3-IS and CKD in SCD has not been investigated. This single-center, longitudinal study explored the associations between CKD and urinary levels of 3-IS, Klotho, neutrophil gelatinase-associated lipocalin (NGAL, a biomarker of proximal tubule injury), and monocyte chemoattractant protein-1 (MCP-1, a biomarker of inflammation), in patients with SCD.

Methods: Participants with HbSS or HbSβ⁰ thalassemia were enrolled in a prospective cohort study to evaluate the association of select biomarkers with persistent albuminuria (PA) and progression of CKD. Exclusions were diabetic nephropathy, cancer, connective tissue diseases, other glomerular diseases, hepatitis (B or C) or HIV infections, end-stage kidney disease on dialysis, and bone marrow transplantation. Patients were enrolled during routine clinic visits, and research samples were collected at baseline and annually for up to three years. Urine levels of 3-IS, MCP-1, NGAL, and Klotho were measured using quantitative ELISA assays, normalized to urine creatinine. Glomerular filtration rate (eGFR) was estimated using the 2012 cystatin-based CKD-EPI formula. Log-transformation was applied to skewed biomarker data. Associations of urine biomarkers with albumin-creatinine ratio (ACR) and eGFR at baseline were assessed using linear regression. Multivariable analyses, adjusted for markers of hemolysis (hemoglobin, absolute reticulocyte count, indirect bilirubin, and lactate dehydrogenase), were also performed to evaluate urine biomarkers independently associated with ACR and eGFR, and backward elimination was employed for variable selection. In addition, associations of change in urine biomarkers from baseline to follow-up visits with change in eGFR were assessed via linear mixed modeling.

Results: Of 109 enrolled subjects, 37 had PA (33.9%). Subjects with PA were older, had lower hemoglobin levels, and higher systolic blood pressure. Baseline urine levels of 3-IS (β=0.65, p=0.02), MCP-1 (β=0.85, p=0.0002), and NGAL (β=0.23, p=0.07) were positively associated with ACR at baseline. After adjusting for markers of hemolysis, MCP-1 (β=0.82, p=0.03) and Klotho (β= -0.68, p=0.02) were independently associated with ACR. Baseline urine levels of 3-IS (β= -9.60, p=0.03), MCP-1 (β= -10.63, p=0.003), and NGAL (β= -7.05, p=0.0002) were inversely associated with eGFR at baseline. After adjustment for markers of hemolysis, NGAL (β= -9.5, p=0.003) remained independently associated with eGFR. Change from baseline to follow-up visits in NGAL (β= -4.20, p=0.002) and MCP-1 (β= -3.63, p=0.03) were significantly associated with changes in eGFR.

Conclusion: Our study found novel and independent associations between urinary Klotho and ACR as well as urinary NGAL and eGFR in patients with SCD. The negative association between urinary Klotho and ACR may be due to decreased renal expression of Klotho due to the nephrotoxic effects of 3-IS. Alternatively, it may be a result of decreased Klotho excretion by damaged renal tubule cells. The negative association of urinary NGAL with baseline eGFR combined with the association of change in NGAL with eGFR change suggest that it may be an important predictor of CKD progression in SCD. Further research is needed to confirm the roles of 3-IS, Klotho and tubular injury markers in the development and progression of SCD-related kidney disease.