Epigenetic regulation of genes involved in fluid‐electrolyte balance in the kidney

Abstract

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional regulators via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid‐electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus Assay for Transposase‐Accessible Chromatin‐sequencing (snATAC‐seq) and snRNA‐seq to profile kidney nuclei from adult male and female, control and littermate inducible kidney epithelial‐specific Hdac1/Hdac2 knockdown mice (iKSHdac1/2KO). Compared to littermate control mice iKSHdac1/2KO mice had significant elevations in plasma sodium (male control n=17, PNa= 143.6 ± 1.5 vs KO n=8, 147.6 ± 3.5, P=0.002, female control n=6, 141.8 ± 2.5 vs KO n=8 144.6 ± 1.8, P=0.03). iKSHdac1/2KO mice also had a significant increase in the kidney to body mass ratio (males 11.5 ±1.4 mg/g vs 13.1 ±1.5 P=0.1, females 9.7 ± 1.0 vs 12.3 ± 1.4, P=0.001) but no differences in liver to body mass ratio. Kidney histological analyses determined significant interstitial fibrosis, tubular atrophy and necrosis, dilated tubules, and protein casts in the iKSHdac1/2KO regardless of sex. Kidney structure was normal in all control mice. Survival was also significantly impacted in the iKSHdac1/2KO as all of these mice died (male mice died 27‐28 days after induction of knockdown). At 2 weeks after induction of KO, kidney nuclei from control and iKSHdac1/2KO mice were isolated and snATAC‐seq and snRNA‐seq analyses completed. We identified 26 unique clusters in both datasets that represented epithelial, endothelial, immune, stromal, and podocyte cells. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid‐electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Aqp1 expressed in the proximal tubules was reduced 60% and principal cell Aqp2 was reduced 50% in the iKSHdac1/2KO mice compared to controls. However, not all aquaporins were affected as for example, principal cell Aqp4 was not significantly different between the mice (P=0.98). Moreover, sex‐specific transcriptomic and chromatin structure were apparent in the proximal tubule clusters. Hdac1/Hdac2 knockdown resulted in different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24‐30% of chromatin accessibility agreed with transcript expression (e.g. open chromatin, increased transcript). To conclude, although chromatin structure does affect transcription, ~70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function and survival.