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. 2021 Jan 27;108(2):357–367. doi: 10.1016/j.ajhg.2021.01.008

Figure 2.

Figure 2

TRIM8 is expressed in podocytes of mammalian kidneys and localizes to nuclear bodies

(A) TRIM8 mRNA (z-score) was predominantly expressed by podocytes (red arrows) from single-cell mRNA sequencing data.26,28,29 In human fetal kidneys from 12 to 19 weeks gestation (left), TRIM8 expression was highest in the mature podocyte cluster, marked by expression of NPHS1, NPHS2, SYNPO, and WT1, relative to other developmental cell-type and nephron segment clusters. Trim8 expression in E14.5 mouse kidneys (middle) was, similarly, highest in the podocyte cluster (Nphs1, Nphs2, Synpo, Wt1) with lower expression in one cap mesenchyme cluster and two tubular segment clusters. In adult mouse glomeruli (right), Trim8 mRNA was predominantly expressed in podocytes relative to other clusters. CAP MES, cap mesenchyme; CNT DIST, distal connecting tubule; COLL DUCT, collecting duct; CORT CD, cortical collecting duct; CORT STRO, cortical stroma; DIST COM, distal comma shaped body; EARL PODO, early podocyte; ENDO, endothelium; IMM, immune cells; LOH, Loop of Henle; LOH DIST, distal Loop of Henle; MAT PODO, mature podocyte; MED CD, medullary collecting duct; MED STRO, medullary stroma; NEPH PROG, nephron progenitor; NEPH STRO, nephrogenic stroma; PAR EPI, parietal epithelial cell; PODO, podocyte; PRET AGG, pretubular aggregate; PROX TUB; proximal tubule; RBC, red blood cells; SS Body, mid S-shaped body; STRO, stroma; TUB, tubule; URET TIP, ureteric tip.

(B) Immunohistochemistry of adult human kidney tissue demonstrates immunoreactivity for TRIM8 within the nuclei, as well as the cytoplasm, of individual glomerular podocytes (arrowheads). There is also staining of the adjacent tubular epithelial cells. Scale bar: 50 μm.

(C) A human podocyte cell line was transfected with N-terminal GFP-tagged wild-type TRIM8 or TRIM8 mutant constructs based on NS patient variants c.1375C>T and c.1231C>T. Cells were imaged by confocal microscopy. Representative images of GFP-tagged protein and DAPI localization are shown, revealing that wild-type TRIM8 localizes to nuclear bodies (NBs) (red arrows) while patient mutants exhibit pan-nuclear staining overlapping with DAPI signal. Scale bars: 7 μm.

(D) The presence of TRIM8 NBs in (C) is determined from the sum of three independent experiments, as the percentage of 100 GFP-positive cells with NB localization versus those with pan-nuclear staining. Wild-type TRIM8 localized to NBs in 94% of GFP-positive cells (94/100). In contrast, mutated TRIM8 localized diffusely to the nucleoplasm in 100% (100/100) and 99% (99/100) of cells for the c.1375C>T and c.1231C>T constructs, respectively.

(E) A human podocyte cell line was transfected with N-terminal GFP-tagged wild-type TRIM8. IF and confocal microscopy was performed for endogenous Gemini body marker SMN1 (top) or Cajal body marker p80 Coilin (p80, bottom), demonstrating these bodies frequently abut a GFP-TRIM8 NB (white arrow). Representative images are shown. Scale bars: 7 μm.

(F) The percentage of endogenous SMN1-positive Gemini bodies or p80 Coilin-positive Cajal bodies that are abutted by a GFP-TRIM8 nuclear body, as in (E), is determined from the sum of three independent experiments. 72.5% of Gemini bodies (58/80 NB in 38 transfected cells) and 56.4% of Cajal bodies (57/101 NB in 21 transfected cells) abutted a TRIM8 NB.