A Pilot Study to Predict Risk of IgA Nephropathy Progression Based on miR-204 Expression

Abstract

Introduction: Immunoglobulin (Ig)A nephropathy (IgAN) is the most frequently diagnosed primary glomerulonephritis worldwide. Despite the common diagnostic feature of mesangial IgA-containing immune complex deposition, the clinical course of the disease is extremely variable, with 30% of patients developing end-stage kidney disease within 20 years of diagnosis. Therefore, identifying which patients are likely to progress is paramount.

Results: In this pilot study, we found that urinary exosomal miR-204 expression was significantly reduced in IgAN compared with healthy subjects. However, there was no difference in miR-204 expression between IgAN and non-IgAN chronic kidney disease controls. Analysis of miR-204 expression in kidney biopsy cores by next-generation sequencing followed by quantitative polymerase chain reaction validation in independent cohorts demonstrated that expression of miR-204 was significantly lower in IgAN compared with thin-membrane nephropathy but not compared with membranous nephropathy. Patients with IgAN at high risk of future progression had significantly lower expression of miR-204 than those at low risk of progression. Cortical localization indicated that miR-204 was preferentially expressed in the interstitium compared with glomeruli in IgAN nonprogressors and that this distribution was lost in IgAN progressors. Receiver operating characteristic curve analysis between the 2 IgAN cohorts revealed an area under the curve of 0.82. In addition, miR-204 expression correlated with known clinicopathological prognostic risk factors. Importantly, incorporating miR-204 into the International IgAN risk prediction tool improved the diagnostic power of the algorithm to predict risk of progression.

Conclusion: Additional large-scale studies are now needed to validate the additive value of miR-204 in improving risk prediction in IgAN and more broadly in chronic kidney disease.

Keywords: IgA nephropathy; exosomes; microRNA; renal biopsy; renal progression.

Graphical abstract

Figure 1

Expression of microRNA (miR)-204 in urine exosomes. RNA was extracted from exosomes obtained from the urines of patients with immunoglobulin A nephropathy (IgAN; n = 15), chronic kidney disease (CKD) (n = 8), and healthy subjects (HS) (n = 6) and analyzed for expression of miR-204 by reverse transcription polymerase chain reaction. Dot plot shows miR-204 expression in the 3 cohorts; IgAN versus HS (P = 0.015), CKD versus HS (P = 0.049), IgAN versus CKD (not significant). Results are expressed as mean ± SEM.

Figure 2

Discovery: expression of microRNA (miR)-204 in kidney biopsy cores. miR-204 expression in kidney biopsy cores was determined by next generation sequencing. Dot plots compare expression (tags per million [TPM]) between cohorts: immunoglobulin A nephropathy (IgAN) versus thin membrane nephropathy (TMN) (P = 0.0024), IgAN versus membranous nephropathy (MN) (P = 0.017), MN versus TMN (not significant [ns]), IgAN nonprogressors (IgANnp) versus IgAN progressors (IgANp) (P = 0.0152). Results are expressed as mean ± SEM of TPM.

Figure 3

Validation: expression of microRNA (miR)-204 in kidney biopsy cores. miR-204 expression was validated by reverse transcription qualitative polymerase chain reaction in an independent set of biopsies (20 per cohort). Dot plots show miR-204 expression in each cohort: immunoglobulin A nephropathy (IgAN) versus thin membrane nephropathy (TMN) (P = 0.0024), IgAN versus membranous nephropathy (MN) (P = 0.017), MN versus TMN (not significant [ns]), IgAN progressors (IgANp) versus IgAN nonprogressors (IgANnp) (P = 0.0152). Results are expressed as means ± SEM of miR-204 expression (2^-ΔCt).

Figure 4

Correlation of microRNA (miR)204 expression with estimated glomerular filtration rate (eGFR) and urine protein/creatinine ratio (UPCR). Correlation curves show miR-204 expression versus eGFR (R = 0.64, P < 0.0001) and versus UPCR (R = –0.484, P = 0.002) in immunoglobulin A nephropathy (IgAN) (a and b). membranous nephropathy (MN) and thin membrane nephropathy (TMN) did not correlate with eGFR (c) but showed a trend (P = 0.073) with UPCR (d).

Figure 5

Relationship of microRNA (miR)204 expression to the mixed epithelial and stromal tumor (MEST)-C score. miR-204 expression was analyzed according the Oxford MEST-C score (M1 vs. M0; S1 vs. S0; E1 vs. E0, T1/2 vs. T0; all included biopsies were C0, and thus no comparison based on the C score was possible). There was no difference in miR-204 expression within the mesangial hypercellularity (M) or segmental sclerosis (S) lesions (a and c). However, a significant difference was seen within the endothelial hypercellularity (E) and interstitial fibrosis/tubular atrophy (T) lesions (b and d) (P = 0.0167 and P = 0.003, respectively).

Figure 6

Cortical location of microRNA (miR)-204 expression. miR expression was measured in glomerular and tubulointerstitial tissue obtained following laser capture microdissection. The dot blot shows expression of miR-204 in the glomeruli (G) and interstitium (I) in immunoglobulin A nephropathy nonprogressors (IgANnp), progressors (IgANp), and thin membrane nephropathy (TMN). A trend in miR-204 expression was observed between glomeruli and interstitium in IgANnp biopsies (P = 0.07) and in TMN biopsies (P = 0.07), but no significant difference was seen in IgANp biopsies.

Figure 7

microRNA (miR)-204 expression discriminates immunoglobulin A nephropathy (IgAN) patients at greater risk of progression. (a) Receiver operating characteristic (ROC) curve showing miR-204 expression in progressors (IgANp) and nonprogressors (IgANnp). Area under the curve = 0.82 (P = 0.0005), expression of <0.52 gives a sensitivity of 60% with a specificity of 90% (Table 1). (b) Combining miR-204 and miR-150 (from our previous study) increased the area under the curve to 0.88 (P < 0.001). (c) Correlation curve of miR-204 expression versus predicted 5-year risk of a 50% decline in estimated glomerular filtration rate (eGFR) or end-stage kidney disease (ESKD) calculated using the International IgAN Risk Prediction Tool. (R = –0.6758, P < 0.0001).