Abstract: TH-OR13
Alternative Splicing in CKD
Session Information
- CKD Mechanisms: Prediction, Propagation, and Prevention
November 02, 2023 | Location: Room 119, Pennsylvania Convention Center
Abstract Time: 04:48 PM - 04:57 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Mohandes, Samer, University of Pennsylvania, Philadelphia, Pennsylvania, United States
- Susztak, Katalin, University of Pennsylvania, Philadelphia, Pennsylvania, United States
Background
Alternative splicing (AS) is a process that can lead to variable genotype expression. The primary forms are exon skipping, intron inclusion, alternative 5’ and 3’ splice sites. The role of alternative splicing of VEGF has been shown to play an important role in chronic kidney disease (CKD) development, however a comprehensive assessment of AS events in CKD has not been undertaken yet.
Methods
Human kidney tissue (n=410) was collected from nephrectomies of healthy subjects and patients with diabetic or hypertensive CKD. Clinical demographics information was collected via honest broker and histopathology was analyzed in an unbiased manner. RNA was extracted from the tubular compartment and sequenced. RNA-seq reads were aligned to the human genome using STAR v 2.7.3. Outliers identified by Mahalanobis distance. Aligned BAM files were sorted and indexed using Samtools v1.17 and junctions were extracted using Regtools v 1.0. Differential splicing for was analyzed separately using leafcutter v 0.2.7 and adjusted for age, sex, race and RNA quality indices (RNA integrity number, 5’-3’bias, %ribosomal genes, total reads).
Results
When comparing healthy samples to those with eGFR of less than 60 cc/min/1.73m2 (CKD stage 3-5), we identified 31 intron cluster alternative splicing events at an adjusted p-value of 0.05. 26 of these were previously annotated. Pathway analysis using gene ontology (GO) terms showed enrichment for cell death and metabolic pathway. When comparing samples with less than 10% fibrosis to those with greater than 10% fibrosis, we identified 239 alternative differential splicing clusters. 233 of these were previously annotated. Pathway analysis using GO terms demonstrated these genes were enriched in the metabolic and immune pathways.
Conclusion
Large number of differential splicing events were observed in CKD compared to controls with enrichment in the metabolic and immune pathway genes.
| Comparison | Samples (CKD vs control) | No. of Significant Differentially Spliced Genes | Top Genes | Top pathway enrichment by GO terms |
| GFR <60 ml/min/1.73m2 | 146 vs 264 | 31 | RAB11FIP3 RPS24 AP2S1 FIS1 RPS3A | Regulation of cell death Negative regulation of cell death Generation of precursor metabolites and energy Cell death Homeostatic process |
| Interstitial Fibrosis >10% | 170 vs 240 | 239 | RPS24 RAB11FIP3 RACK1 TPM1 GLS | Small molecule metabolic process Organic acid metabolic process Positive regulation of immune system Positive regulation of leukocyte migration Organonitrogen compound |