Urinary mRNA Signatures as Predictors of Renal Function Decline in Patients With Biopsy-Proven Diabetic Kidney Disease

Abstract

The clinical manifestations of diabetic kidney disease (DKD) are more heterogeneous than those previously reported, and these observations mandate the need for the recruitment of patients with biopsy-proven DKD in biomarker research. In this study, using the public gene expression omnibus (GEO) repository, we aimed to identify urinary mRNA biomarkers that can predict histological severity and disease progression in patients with DKD in whom the diagnosis and histologic grade has been confirmed by kidney biopsy. We identified 30 DKD-specific mRNA candidates based on the analysis of the GEO datasets. Among these, there were significant alterations in the urinary levels of 17 mRNAs in patients with DKD, compared with healthy controls. Four urinary mRNAs-LYZ, C3, FKBP5, and G6PC-reflected tubulointerstitial inflammation and fibrosis in kidney biopsy and could predict rapid progression to end-stage kidney disease independently of the baseline eGFR (tertile 1 vs. tertile 3; adjusted hazard ratio of 9.68 and 95% confidence interval of 2.85-32.87, p < 0.001). In conclusion, we demonstrated that urinary mRNA signatures have a potential to indicate the pathologic status and predict adverse renal outcomes in patients with DKD.

Keywords: biomarker; diabetic kidney disease; mRNA; renal pathology; urine.

Figure 1

Flowchart of participant selection. We first screened 155 patients with diabetic kidney disease (DKD) whose diagnoses were confirmed by kidney biopsy. Among these, 83 patients with availability of urine samples were enrolled in this study. We also recruited 19 patients exhibiting both DKD and non-diabetic renal disease (NDRD) and 32 healthy individuals as control groups. Urinary cell pellets from the participants were collected and analyzed for measurement of the levels of DKD-specific mRNA candidates selected based on the metanalysis of the public GEO repository. DKD, diabetic kidney disease; NDRD, non-diabetic renal disease; PCR, polymerase chain reaction.

Figure 2

Urinary levels of diabetic kidney disease-specific mRNA candidates in healthy controls, patients with combined diabetic kidney disease and non-diabetic renal disease, and those with isolated diabetic kidney disease. The levels of selected mRNA biomarker candidates whose expressions are significantly altered between the different groups are shown. (A, B) mRNA candidates up-regulated (A) and down-regulated (B) in DKD via GEO profiling. mRNA levels are measured by quantitative real-time polymerase chain reaction and are expressed as log-transformed delta-delta cycle threshold (ΔΔCt) after an adjustment by 18S rRNA and controls. Five mRNAs among those listed in (CX3CR1, HOPX, COMP, APOLD1, and CYP27B) are not illustrated in this figure as these mRNAs failed to pass the quality control process. ^* p < 0.05, ^** p < 0.005, up-regulated vs. control; ^† p < 0.05, ^†† p < 0.005, down-regulated vs. control. DKD, diabetic kidney diseases; NDRD, non-diabetic renal disease; GEO, gene expression omnibus.

Figure 3

Association between pathologic classifications and urinary mRNA levels in patients with diabetic kidney disease. (A–C) The levels of significantly altered urinary mRNAs according to (A) glomerular classification, (B) interstitial fibrosis and tubular atrophy, and (C) interstitial inflammation scores in patients with diabetic kidney disease are shown. Levels of each mRNA are expressed as log-transformed delta-delta cycle threshold (ΔΔCt) after adjusting for 18S rRNA and controls. ^* p < 0.05, ^** p < 0.005, up-regulated vs. glomerular class II or IFTA score of 1; ^† p < 0.05, down-regulated vs. interstitial inflammation score of 0.

Figure 4

Renal survival of patients with diabetic kidney disease according to their clinicopathologic features. The renal survival of patients with diabetic kidney disease according to (A) stages of chronic kidney disease (CKD), (B) the amount of proteinuria, (C) glomerulonephritis classification score, (D) interstitial fibrosis and tubular atrophy (IFTA) score, (E) interstitial inflammation score, (F) arterial hyalinosis score, and (G) arteriolosclerosis score are shown. P-values were calculated by log-rank test. CKD, chronic kidney disease; uPCR, urinary protein-to-creatinine ratio; IFTA, interstitial fibrosis and tubular atrophy.

Figure 5

Renal survival of patients with diabetic kidney disease according to compartmental mRNA signatures. (A, B) The renal survival of patients with diabetic kidney disease according to the tertiles of (A) glomerular and (B) tubulointerstitial mRNA signatures are shown. Each signature was generated from the integration of mRNAs differentially expressed in corresponding compartments (NNMT, THBS2, SPON2, COL3A1, COL1A1 for glomerular signature and LYZ, C3, FKBP5, G6PC for tubulointerstitial signature). p < 0.001 for both comparisons by log-rank test.