Discussion and Conclusion

Genetic variation has significant impact on gene expression regulation, downstream organ function, and phenotype development. Here we present the first comprehensive RNA sequencing-based gene expression analysis for genetically driven gene-expression changes in human kidney samples. While large-scale efforts are in progress to generate comprehensive eQTL analysis for the human body, kidney-specific maps have not been generated. Our project aims to fill that gap by generating kidney-specific maps.

Our results indicate that while a large percentage of genotype-driven gene-expression variation is conserved between different tissue types, kidney-specific maps have highlighted variation in gene levels that play a key role in kidney function, indicating the critical need for tissue-specific maps. In prior work, an algorithm has been developed to impute expression levels for unavailable tissue types on the basis of eQTL. However, the sample size available today does not carry enough power to detect tissue-specific eQTLs [305].

We further found that kidney eSNPs showed tissue-specificity and are enriched on kidney-specific regulatory regions: promoters, enhancers and transcription factor binding sites. We used the kidney eSNPs we discovered to interpret disease-related variants discovered in the GWAS studies. We found four major loci where genome-wide significant eSNPs and eGFR- associated GWAS SNPs directly overlapped. We also employed Bayesian methods to confirm that eSNPs and GWAS-associated SNPs are present at the same locus and contribute to both phenotypes and gene expression levels. This analysis highlighted several genes that are likely responsible for the association observed between genotype and kidney disease associated traits in large-scale GWAS. For example, Cystatin 9 (CST9), variants showed association with serum cystatin levels. Another example is PGAP3, the phospholipase that attaches GPI to proteins, which is another likely causal gene for CKD development.

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variants on chromosome 4 shown to be associated with CKD. Variants that are associated with CKD traits also altered the expression of MANBA in human kidney samples. The risk genotype was associated with lower levels of MANBA levels in human kidney samples. Interestingly, this association can be recapitulated in the fibroblast cell line in the GTEx dataset, potentially indicating that the signal could come from fibroblasts. A similar direction on gene expression was observed in patients with kidney disease and mouse models with kidney fibrosis. We have used the zebrafish model to show that lower MANBA expression is causally related to kidney disease development. Morpholino knockdown of Manba resulted in pericardial edema formation, indicating the functional role of MANBA in kidney disease development. MANBA is a lysosomal enzyme that participates in the post-translational modification of proteins by hydrolysis of the beta-D-mannose residues. Genetic deletion of Manba in mouse models was associated with neurological defects and tissue deposits such as observed in the kidney.

Recent reports highlight the role of lysosomes and lysosomal dysfunction in CKD development [306]. Lysosomes are acidic organelles that play an important role in macromolecule degradation and recycling. Fabry’s disease, cystinosis and neuronal ceroid lipofuscinosis are primary genetic diseases caused by loss-of-function mutations in lysosomal enzymes [306-308]. While many lysosomal enzyme mutations present with primary central nervous system defects, these mutations are also associated with altered kidney function and ultimately lead to CKD. Renal tubules reabsorb more than 100 liters of fluid and large amounts of organic and inorganic matter to maintain homeostasis. Renal tubule cell lysosomes seem to play an important role in recycling and uptake of urinary substances [307]. Accumulation of toxic substances have been observed in cytinosis and Fabry’s disease that likely result in tubule cell death and dysfunction, further fibroblast activation, and structural defect. Further studies will be needed to study the role of Manba in renal tubule cells.

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kidney-specific eQTL maps will likely be essential to the understanding of other traits linked to the kidney, for example metabolite levels and hypertension. Our work nicely illustrates the critical role of the kidney in xenobiotic metabolism as one of the key pathways identified by the eQTL analysis. The genetic regulation of drug metabolism-associated genes is likely important to understand drug effectiveness, toxicity and metabolism. Future studies shall aim to define these connections.

There are several limitations of our work, importantly the sample size. While 100 samples obtained from subjects with a single genetic background can identify genotype-driven gene- expression changes, they have a limited power to identify smaller gene-expression changes. Furthermore, the data was obtained from TCGA database with limited clinical characterization, including kidney function and structural information. Another limitation of this study is that we used the public dataset form TCGA where the kidney cortex has multiple cell types. We are currently implementing a new method, called multi-tissue meta-analysis (METASOFT) that was developed in GTEx [98] to identify tissue-specific eGenes. METASOFT borrows the statistical power from an additional 44 tissues to identify shared and tissue-specific eGenes. For future studies, we will need to perform cell-type-specific analyses with single-cell resolution.

Despite these potential deficiencies, the dataset will be critically important for interpretation of current and future GWAS studies and traits that are likely related to kidney function, such as metabolite levels and blood pressure-associated genes, to name a few. While proximity-based analysis has been popular in the past, our first pass eQTL analysis does not seem to support previously proposed CKD target genes, but highlights the potential role of a set of different genes and steers the field to refocus our attention. This analysis identified MANBA as a gene that has not previously been implicated in kidney disease development.