Cadherin-11, Sparc-related modular calcium binding protein-2, and Pigment epithelium-derived factor are promising non-invasive biomarkers of kidney fibrosis

Abstract

Kidney fibrosis constitutes the shared final pathway of nearly all chronic nephropathies, but biomarkers for the non-invasive assessment of kidney fibrosis are currently not available. To address this, we characterize five candidate biomarkers of kidney fibrosis: Cadherin-11 (CDH11), Sparc-related modular calcium binding protein-2 (SMOC2), Pigment epithelium-derived factor (PEDF), Matrix-Gla protein, and Thrombospondin-2. Gene expression profiles in single-cell and single-nucleus RNA-sequencing (sc/snRNA-seq) datasets from rodent models of fibrosis and human chronic kidney disease (CKD) were explored, and Luminex-based assays for each biomarker were developed. Plasma and urine biomarker levels were measured using independent prospective cohorts of CKD: the Boston Kidney Biopsy Cohort, a cohort of individuals with biopsy-confirmed semiquantitative assessment of kidney fibrosis, and the Seattle Kidney Study, a cohort of patients with common forms of CKD. Ordinal logistic regression and Cox proportional hazards regression models were used to test associations of biomarkers with interstitial fibrosis and tubular atrophy and progression to end-stage kidney disease and death, respectively. Sc/snRNA-seq data confirmed cell-specific expression of biomarker genes in fibroblasts. After multivariable adjustment, higher levels of plasma CDH11, SMOC2, and PEDF and urinary CDH11 and PEDF were significantly associated with increasing severity of interstitial fibrosis and tubular atrophy in the Boston Kidney Biopsy Cohort. In both cohorts, higher levels of plasma and urinary SMOC2 and urinary CDH11 were independently associated with progression to end-stage kidney disease. Higher levels of urinary PEDF associated with end-stage kidney disease in the Seattle Kidney Study, with a similar signal in the Boston Kidney Biopsy Cohort, although the latter narrowly missed statistical significance. Thus, we identified CDH11, SMOC2, and PEDF as promising non-invasive biomarkers of kidney fibrosis.

Keywords: biomarker; biopsy; fibrosis; histopathology; kidney disease.

Figure 1.. Single-cell RNA-sequencing shows biomarker gene expression in mouse kidney fibroblasts.

Biomarker gene expression in scRNA-sequencing datasets from animals undergoing sham surgery, or at UUO day 2 (UUO2d), UUO day 7 (UUO7d), or 14 days post R-UUO (RUUO14d) were analyzed. (A) Uniform manifold approximation and projection (UMAP) plot of mouse UUO kidneys reveals 22 separate cell clusters. (B) Biomarker gene expression across cell clusters at different time points. (C) Quantification of differential gene expression at different time points shows that fibrosis biomarkers are primarily expressed in kidney fibroblasts. UUO, unilateral ureteral obstruction; R-UUO, ureter reimplantation to reverse obstruction; PT1: proximal tubule S1 segment; PT2: proximal tubule S2 segment; PT3: proximal tubule S3 segment; Prolif. PT, proliferating proximal tubule population; PEC, parietal epithelial cells; TAL, thick ascending limb of Loop of Henle; DTL: descending limb of Loop of Henle; DCT, distal convoluted tubule; PC, principal cells; IC, intercalated cells; Fib., fibroblasts type 1; Fib. 2, fibroblasts type 2; aEC, arterial endothelial cells; vEC, venous endothelial cells; gEC, glomerular endothelial cells; Mø: macrophages; DC, dendritic cells; NK cells, natural killer cells. The SERPINF1 (Serine Proteinase Inhibitor-F1) gene encodes PEDF.

Figure 2.. Single-nucleus RNA-sequencing analyses reveal biomarker gene expression in individuals with chronic kidney disease (CKD).

(A) Uniform manifold approximation and projection (UMAP) plot of 8 human kidneys from individuals with CKD reveals 16 separate cell clusters. (B) UMAP plot annotated by cell type and individual. (C) Dot plots shows quantification of gene expression across cell clusters. Podo, podocyte; PT, proximal tubule; Inj. PT, injured proximal tubule; DTL: descending limb of Loop of Henle; cTAL, cortical thick ascending limb of Loop of Henle; mTAL, medullary thick ascending limb of Loop of Henle; PC, principal cell; IC, intercalated cell; aEC, arterial endothelial cells; vEC, venous endothelial cells; gEC, glomerular endothelial cells; Fib., fibroblasts type 1; Fib. 2, fibroblasts type 2; Mono, monocyte. The SERPINF1 (Serine Proteinase Inhibitor-F1) gene encodes PEDF.

Figure 3.. Differences in plasma (A) and urine (B) CDH11/cr, SMOC2/cr, and PEDF/cr levels by grades of interstitial fibrosis and tubular atrophy (IFTA) in kidney biopsy specimens.

Boxplots show median and interquartile range (IQR) of fibrosis biomarkers on a log scale. Whiskers span data within 1.5 times of the IQR of the lower and upper quartile (25^th and 75^th percentile, respectively). P value from Kruskal Wallis test <0.001 for each biomarker, n=438 and n=602 for plasma and urinary biomarkers/cr, respectively.

Figure 4.. Associations between fibrosis biomarkers and interstitial fibrosis and tubular atrophy (IFTA) in kidney biopsy specimens.

Odds Ratios (OR) are obtained from ordinal logistic regression models using IFTA (graded as involvement of <10%, 11–25%, 26–50%, or > 50% of total cortical volume) as the dependent variable and log₂-transformed plasma biomarkers (A) and log₂-transformed urinary biomarkers/cr (B) as predictor variables. OR are expressed per change in IFTA score. Models are adjusted for age, sex, race, and eGFR. N=438 and n=602 for plasma and urine biomarkers, respectively.