Histologically resolved small RNA maps in primary focal segmental glomerulosclerosis indicate progressive changes within glomerular and tubulointerstitial regions

Abstract

Pathological heterogeneity is common in clinical tissue specimens and complicates the interpretation of molecular data obtained from the specimen. As a typical example, a kidney biopsy specimen often contains glomeruli and tubulointerstitial regions with different levels of histological injury, including some that are histologically normal. We reasoned that the molecular profiles of kidney tissue regions with specific histological injury scores could provide new insights into kidney injury progression. Therefore, we developed a strategy to perform small RNA deep sequencing analysis for individually scored glomerular and tubulointerstitial regions in formalin-fixed, paraffin-embedded kidney needle biopsies. This approach was applied to study focal segmental glomerulosclerosis (FSGS), the leading cause of nephrotic syndrome in adults. Large numbers of small RNAs, including microRNAs, 3'-tRFs, 5'-tRFs, and mitochondrial tRFs, were differentially expressed between histologically indistinguishable tissue regions from patients with FSGS and matched healthy controls. A majority of tRFs were upregulated in FSGS. Several small RNAs were differentially expressed between tissue regions with different histological scores in FSGS. Notably, with increasing levels of histological damage, miR-21-5p was upregulated progressively and miR-192-5p was downregulated progressively in glomerular and tubulointerstitial regions, respectively. This study marks the first genome scale molecular profiling conducted in histologically characterized glomerular and tubulointerstitial regions. Thus, substantial molecular changes in histologically normal kidney regions in FSGS might contribute to initiating tissue injury or represent compensatory mechanisms. In addition, several small RNAs might contribute to subsequent progression of glomerular and tubulointerstitial injury, and histologically mapping small RNA profiles may be applied to analyze tissue specimens in any disease.

Keywords: kidney; microRNA; omics; pathology; tRNA fragment.

Figure 1.. Approach for mapping small RNA profiles to specific histological scores in specific tissue types.

For each FFPE kidney tissue block from healthy control subjects or FSGS patients, 3 consecutive cuts were made. The first tissue section was H&E stained and each glomerular region and the surrounding tubulointerstitial (TI) region scored for histological injury by 2 blinded board-certified pathologists or nephrologists. Inconsistent scores were reviewed and scored by a third pathologist. The second and third cuts made from the FFPE tissue block were used for laser capture microdissection (LCM). First, the glomerular regions were cut, then the surrounding TI regions. Up to three regions per score per patient were pooled for small RNA library preparation and deep sequencing. Differential expression of microRNAs, 3’-tRFs, and 5’-tRFs was assessed across histological scores in glomerular and TI regions.

Figure 2.. Glomerular microRNAs show progressive changes in expression as FSGS develops and histological injury advances.

A. MicroRNAs significantly differentially expressed (FDR<0.05) between glomeruli from healthy control subjects (Control glom) and score 0, histologically normal glomeruli from FSGS patients (Glom 0). B. Glomerular microRNAs significantly differentially expressed (FDR<0.05) between score 2 (Glom 2) and score 0 (Glom 0) in FSGS patients. C. Glomerular microRNAs significantly differentially expressed (FDR<0.05) between score 2 (Glom 2) and score 1 (Glom 1) in FSGS patients. D. Clustered heatmap of all microRNAs significantly differentially expressed in at least one comparison between glomerular samples shown in panels A-C. E. Average log₂ fold change relative to healthy controls for each cluster of microRNAs shown in panel D. Dashed lines indicate first and third quartiles of each cluster. Color of lines corresponds to the brackets shown in panel D. n=9 for healthy controls, 31 for FSGS score 0, 21 for FSGS score 1, and 16 for FSGS score 2.

Figure 3.. Tubulointerstitial microRNAs show progressive changes in expression as FSGS develops and histological injury advances.

A. MicroRNAs significantly differentially expressed (FDR<0.05) between tubulointerstitial (TI) regions from healthy control subjects (Control TI) and score 0, histologically normal TI regions from FSGS patients (TI 0). B. TI microRNAs significantly differentially expressed (FDR<0.05) between score 1 (TI 1) and score 0 (TI 0) in FSGS patients. C. TI microRNAs significantly differentially expressed (FDR<0.05) between score 2 (TI 2) and score 0 (TI 0) in FSGS patients. D. TI microRNAs significantly differentially expressed (FDR<0.05) between score 2 (TI 2) and score 1 (TI 1) in FSGS patients. E. Clustered heatmap of all microRNAs significantly differentially expressed in at least one comparison between TI samples shown in panels A-D. E. Average log₂ fold change relative to healthy controls for each cluster of microRNAs shown in panel D. Dashed lines indicate first and third quartiles of each cluster. Color of lines corresponds to the brackets shown in panel D. n=10 for healthy controls, 13 for FSGS score 0, 26 for FSGS score 1, and 19 for FSGS score 2.

Figure 4.. Representative microRNAs show progressive changes in expression as FSGS develops or histological injury advances.

A. miR-21–5p in glomeruli. B. miR-146b-5p in glomeruli. C. miR-192–5p in tubulointerstitial regions. D. miR-12136 in tubulointerstitial regions. Fold changes in FSGS tissue regions with score 0, 1, or 2, relative to healthy control subjects (Healthy), are shown. * indicates significant differential expression based on small RNA deep sequencing analysis (FDR<0.05). See Figures 2 and 3 for n values. E. miRNAScope analysis of miR-21–5p. Red punctate signals in the middle row of images represent miR-21–5p detection. The score 0 and score 2 glomeruli were from the same biopsy section. H&E and miR-21–5p images were taken from adjacent sections of the biopsy specimen and were representative of three score 0 glomeruli and two score 2 glomeruli found in two patients analyzed. Scale bar, 100 μm.

Figure 5.. Glomerular tRFs are differentially expressed mainly between FSGS score 0 and healthy controls.

A. 3’-tRFs significantly differentially expressed (FDR<0.05) between glomeruli from healthy control subjects (Control glom) and score 0, histologically normal glomeruli from FSGS patients (Glom 0). B. A glomerular 3’-tRF significantly differentially expressed (FDR<0.05) between score 1 (Glom 1) and score 0 (Glom 0) in FSGS patients. C. Glomerular 5’-tRFs significantly differentially expressed (FDR<0.05) between healthy control subjects (Control glom) and score 0 from FSGS patients (Glom 0). D. Clustered heatmap of all 3’-tRFs significantly differentially expressed in at least one comparison between glomerular samples shown in panels A and B. E. Clustered heatmap of all 5’-tRFs shown in panel C. n=9 for healthy controls, 31 for FSGS score 0, 21 for FSGS score 1, and 16 for FSGS score 2.

Figure 6.. Tubulointerstitial tRFs are differentially expressed mainly between FSGS score 0 and healthy controls.

A. 3’-tRFs significantly differentially expressed (FDR<0.05) between tubulointerstitial (TI) regions from healthy control subjects (Control TI) and score 0, histologically normal TI regions from FSGS patients (TI 0). B. A TI 3’-tRF significantly differentially expressed (FDR<0.05) between score 2 (TI 2) and score 0 (TI 0) in FSGS patients. C. TI 5’-tRFs significantly differentially expressed (FDR<0.05) between healthy control subjects (Control TI) and score 0 from FSGS patients (TI 0). D. Clustered heatmap of all 3’-tRFs significantly differentially expressed in at least one comparison between TI samples shown in panels A and B. E. Clustered heatmap of all 5’-tRFs shown in panel C. n=10 for healthy controls, 13 for FSGS score 0, 26 for FSGS score 1, and 19 for FSGS score 2.

Figure 7.. MicroRNAs differentially expressed in histologically normal tissues in FSGS patients compared to healthy controls and as histological injury progresses in FSGS target shared but also different pathways.

A. Glomerular microRNA target pathways. B. Tubulointerstitial microRNA target pathways. Experimentally supported target genes for microRNAs differentially expressed in each tissue type between healthy controls and histologically normal tissue regions in FSGS (healthy vs. 0) or between FSGS tissue regions with any different histological scores (0 vs. 1 vs. 2) were retrieved and pathway enrichment analysis performed as described in Methods.

Figure 8.. miR-146b-5p is progressively upregulated, and its target gene TRAF6 progressively down-regulated, in glomeruli as FSGS develops and histological injury advances.

A. Regulatory relation between miR-146b-5p and TRAF6. TLR, toll-like receptor; TRAF6, tumor necrosis factor receptor-associated factor 6; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells. B. miR-146b-5p abundance in glomeruli assessed by qPCR. C. TRAF6 abundance in glomeruli assessed by qPCR. N = 10 healthy controls, 35 score 0 (histologically normal glomeruli in FSGS patients), and 18 score 2. *, p<0.05 vs. healthy, one-way ANOVA followed by Holm-Sidak test.