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. 2022 Mar 22;7(6):e146374. doi: 10.1172/jci.insight.146374

Figure 3. Enrichment of kidney segment markers in nascent RNA.

Figure 3

Normalized counts (DESeq2) were plotted for 3 RNA pools (total kidney RNA; Hoxb7Cre RosaUprt/+ and Atp6v1b1Cre RosaUprt/+ pulldown RNA; respectively, n = 10, 21, 11 independent RNA samples). The y axis represents the ratio of counts per library size normalized for the average count of each gene across all samples. G, glomerulus; PT, proximal tubule; TALH, thick ascending limb of Henle; CD, collecting duct; PC, principal cells of the collecting duct; IC, intercalated cells of the collecting duct; α-IC, α-intercalated cells, β-IC, β-intercalated cells. Note the de-enrichment of most G, PT, and TALH markers and conversely the enrichment of PC markers by HoxB7Cre (Aqp 2, 3, 4), IC markers by Atp6v1b1Cre (Foxi1, Atp6v0d2, Atp6v1g3, Oxgr1), and collecting duct markers in both pulldowns (Rhcg, Slc4a8, Slc26a4). Some genes failed to comply with their known status as a canonical marker, suggesting low rates of RNA turnover or incomplete characterization of their distribution, including Slc9a3 (95). Alternatively, some genes are expressed in multiple segments of the kidney — Rhbg (96): connecting segment and CD; Car12 (carbonic anhydrase 12) (32): PT and CD; Car2 (carbonic anhydrase 2) (97): TALH and CD; Slc4a1 (98): RBC and CD — and consequently demonstrated mixed patterns in RNA pulldowns. HoxB7Cre pulldown vs. total RNA: adjusted P (padj) < 10–3 (range: 3.0 × 10–3 to 2.2 × 10–104) for all genes except Rhcg, Atp6v1g3, Slc4a1 (padj = NS). Atp6v1b1Cre pulldown vs. total RNA: padj < 10–3 (range: 7.7 × 10–3 to 2.07 × 10–126) for all genes except for Rhcg, Slc4a1, Atp6v1b1, Slc9a2, Aqp3, Aqp4 (padj = NS).