Enhancing Glutathione Pathway Activity for Improved Resilience to Kidney Injury

Background: Males are more susceptible to AKI than females. What underlies superior resilience and increased tolerance to kidney injury in female? Our lab's analysis identified 984 genes with sex-biased expression: proximal tubule (PT), key target in many injury scenarios emerging as source of this sex-specific variability. Excessive ROS contribute to PT damage. Female-biased genes include glutathione pathway members, Gsts, that could potentially neutralize ROS. We hypothesize that female-enriched gene expression in glutathione pathway components may enhance resilience of female PT to IRI-induced oxidative stress. If so, activating these genes in male proximal tubules is predicted to increase resilience to IRI Methods: To generate appropriate genetic tools, we identified and verified enhancers driving PT-restricted expression of HNF4a. Transgenic mice were generated that drive Gst transgene to PT and examined for effect on IRI response in male mice Results: Identifying proximal tubule-specific enhancers ensures that overexpression is from target cell type. Using ATAC-seq, we identified conserved open chromatin, 5’ and 3’ of HNF4a promoter, in mouse and human PT cells that showed strong enhancer predictions in ENCODE datasets. Transgenic lacZ reporter studies using knock-in at a safe harbor locus demonstrated PT-restricted reporter activity of human enhancers in mouse kidney. Next, we generated a transgenic line in which Gst gene that shows marked female-enriched expression in PT cells is expressed under control of human HNF4A enhancer. To examine potential protective effects of elevated Gst levels in male mice, severe IRI was performed following unilateral nephrectomy, and serum creatinine levels and GFR determined to evaluate kidney function 2 and 28 days post IRI, respectively. Preliminary data suggests protective effect of ectopic Gst expression in male kidney: despite similar level of injury as measured by creatine levels 2 days after IRI, transgenics show improved post IRI survival and higher GFR at 28 days Conclusion: Our research identified human HNF4A enhancers that will have broad utility for targeting gene expression to proximal tubule cells. Initial data addressing potential renal protective role for Gst, in male PT cells, are encouraging. Future studies will focus on earlier responses and rigorously assessing injury outcomes in transgenic and control male mouse kidneys Funding: NIDDK Support