Podocyte A20/TNFAIP3 Controls Glomerulonephritis Severity via the Regulation of Inflammatory Responses and Effects on the Cytoskeleton

Abstract

A20/Tnfaip3, an early NF-κB response gene and key negative regulator of NF-κB signaling, suppresses proinflammatory responses. Its ubiquitinase and deubiquitinase activities mediate proteasomal degradation within the NF-κB pathway. This study investigated the involvement of A20 signaling alterations in podocytes in the development of kidney injury. The phenotypes of A20Δpodocyte (podocyte-specific knockout of A20) mice were compared with those of control mice at 6 months of age to identify spontaneous changes in kidney function. A20Δpodocyte mice presented elevated serum urea nitrogen and creatinine levels, along with increased accumulation of inflammatory cells-neutrophils and macrophages-within the glomeruli. Additionally, A20Δpodocyte mice displayed significant podocyte loss. Ultrastructural analysis of A20 podocyte-knockout mouse glomeruli revealed hypocellularity of the glomerular tuft, expansion of the extracellular matrix, podocytopenia associated with foot process effacement, karyopyknosis, micronuclei, and podocyte detachment. In addition to podocyte death, we also observed damage to intracapillary endothelial cells with vacuolation of the cytoplasm and condensation of nuclear chromatin. A20 expression downregulation and CRISPR-Cas9 genome editing targeting A20 in a podocyte cell line confirmed these findings in vitro, highlighting the significant contribution of A20 activity in podocytes to glomerular injury pathogenesis. Finally, we analyzed TNFAIP3 transcription levels alongside genes involved in apoptosis, anoikis, NF-κB regulation, and cell attachment in glomerular and tubular compartments of kidney biopsies of patients with various renal diseases.

Keywords: A20; Tnfaip3; anoikis; inflammation; podocyte.

Figure 1

(A) Basal mRNA expression of A20/Tnfaip3 in human tissues. Quantitative real-time PCR analysis of prenormalized cDNA derived from poly-(A)-selected DNase-treated RNA isolated from human bone marrow and kidney. Transcript expression levels were calculated via the use of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. The data are shown as the means ± SDs. (B) Quantitative real-time PCR analysis of cDNA derived from RNA isolated from murine (C57BL/6) tissues and kidney cells/compartments as well as K5P5 cells after day 3, 7, 10, and 14 of differentiation was performed as described in the Section 2. The detected mRNA expression levels were calculated via the use of murine Gapdh as a housekeeping gene. Data are shown as the means ± SDs. (C) The data show the expression pattern of Tnfaip3 in the kidney (GSE184601). The analysis indicates high Tnfaip3 expression in monocytes (Mos), glomeruli parietal epithelial cells (PECs), proximal tubule segment 1 (PTS1), podocytes (Pods), and T lymphocytes (Tcell). The analysis indicates low Tnfaip3 expression in intercalated cell type A (ICA), distal convoluted tubule–connecting tubule (DCT-CNT), intercalated cell type A (ICA), macula densa (MD), thick ascending limb of henle in medulla (MTAL), principal cell 1 (PC1), proximal tubule segment 2 (PTS2), and pericyte (Per). (D) A20 mRNA induction following 3 h of LPS (100 ng/mL) stimulation in cultured podocytes. Total RNA was then collected to quantify gene expression via RT–PCR. The data are shown as the means ± SEMs and represent one of two independent experiments (n = 3); ** p < 0.01 and *** p < 0.001.

Figure 2

Effects of A20 knockdown and activation on proinflammatory and profibrotic factors in podocytes. A20 knockdown or control (scrambled) cells were incubated for 3 h in the presence of 100 ng/mL LPS. Total RNA was then collected to quantify gene expression via RT–PCR. The data are shown as the means ± SEMs and represent one of two independent experiments (n = 3); ** p < 0.01 and *** p < 0.001.

Figure 3

Phenotyping of A20Δpodocyte mice at the age of 6 months reveals differences in serum creatinine, BUN, and proteinuria. A20Δpodocyte mice were compared with Cre- or Flox- littermate controls. The data are shown as the means ± SDs. * p < 0.05, ** p < 0.01.

Figure 4

Phenotyping of A20Δpodocyte mice at the age of 6 months reveals differences in the infiltration of immune cells in glomerulus. Sections of kidneys from control and A20Δpodocyte mice were stained for glomerular macrophage (A), CD3+ lymphocyte, (B) and leukocyte (C) infiltration at the age of 6 months. The number of infiltrating cells was assessed in at least 15 glomeruli per kidney (n = 8–10 animals per group). The data are shown as the means ± SDs. * p < 0.05.

Figure 5

Phenotyping of A20Δpodocyte mice at the age of 6 months reveals differences in the relative mRNA expression of the indicated genes in the renal cortex. The data are shown as the means ± SDs. * p < 0.05 and ** p < 0.01.

Figure 6

(A) Representative pictures showing glomerular WT1 staining in control and A20Δpodocyte mice at the age of 6 months. The WT1-positive cells in the groups were quantified by counting WT1+ cells in 15 glomerular sections per kidney. The data are shown as the means ± SDs. *** p < 0.001 versus controls. (B) Relative mRNA expression of the indicated genes in the renal cortex of control and A20Δpodocyte mice.

Figure 7

In 6-month-old A20Δpodocyte mice, we observed hypocellularity of the glomerular tuft, expansion of the extracellular matrix (b), podocytopenia associated with foot process effacement (e), nuclear chromatin condensation (d), micronuclei (a), and podocyte detachment at the ultrastructural level. In addition to podocyte death, we detected damage to intracapillary endothelial cells with vacuolation of the cytoplasm (c) and condensation of nuclear chromatin. Lower panel left: dying podocyte with micronuclei (f); lower panel right: irregular thickening of the GBM, looking like subepithelial deposit “humps” of immune complexes.

Figure 8

We generated a Tnfaip3 knockout model via the CRISPR/Cas9 system in podocytes. The specific gRNA was designed based on the DNA sequence of the mouse Tnfaip3 gene. This gRNA guides Cas9 to cut exon 1 of Tnfaip3. Single cells were selected via cell sorting (GFP+). (A) RT–PCR screening of single clones (n = 3) identified several K5P5^A20−/− clones. (B) Changes in F-actin and cell morphology after phalloidin staining of untreated and LPS-treated podocytes (24 h stimulation). Data are presented as mean ± SD. Statistical significance is indicated as follows: *** p < 0.001.

Figure 9

(A) Podocyte metabolic activity was assessed via the MTT assay (in 0–20% FCS). (B) Relative mRNA expression of Pcna associated with cell proliferation was characterized in 2% FCS. The data are shown as the means ± SDs. * p < 0.05 and *** p < 0.001.

Figure 10

Podocytes were left untreated or exposed to LPS for 24 h, and viability was evaluated via (A) LDH assay and (B) fluorescence microscopy using PI staining and a calcein AM viability assay. (C) Podocytes were left untreated or exposed to LPS for 6 h, and viability was evaluated using PI and Annexin V flow cytometry analysis. (D) Heatmap of gene expression analysis of A20-deficient podocytes and controls that were left untreated or stimulated for 3 h with LPS. Quantitative real-time PCR analysis of selected transcripts in podocytes. The detected mRNA expression levels were calculated via the use of murine Gapdh as a housekeeping gene. The data are shown as the mean ± SDs. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 11

Heatmap depicting the expression patterns of cytoskeleton-related genes in A20-deficient podocytes and control podocytes, both untreated and stimulated with LPS for 18 h. Quantitative real-time PCR (qRT-PCR) analysis of selected transcripts in podocytes. mRNA expression levels were normalized to murine Gapdh, used as a housekeeping gene. Data are presented as mean ± SD. Statistical significance is indicated as follows: * p < 0.05 and ** p < 0.01.

Figure 12

(A) Flow cytometric analysis of integrin. (B) Immunohistochemistry for integrin (n = 6). The data are shown as the means ± SDs. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 13

The heatmap displays the expression patterns of genes in HCT116 cells subjected to A20/Tnfaip overexpression (OE) and knockout (KO). Genes are arranged vertically, and the two conditions (OE and KO) are arranged horizontally for comparison; red: upregulated genes (high expression); blue: downregulated genes (low expression).

Figure 14

Gene expression analysis of TNFAIP3 and selected genes involved in anoikis, NF-κB regulation, and cell attachment in the glomerular (A) and tubular (B) compartment of manually micro-dissected biopsies from patients with different kidney diseases. Values are expressed as log2-fold changes relative to controls (living donors/LDs). All displayed genes exhibit significant changes (p < 0.05), while non-significant genes are labeled as ns. Groups include Hypertensive Nephropathy/HT(N), Minimal Change Disease/MCD, IgA Nephritis/IgA, Rapidly Progressive Glomerulonephritis/RPGN, Systemic Lupus Erythematosus/SLE, Membranous Glomerulonephritis/MGN, Focal Segmental Glomerulosclerosis/FSGS, and Diabetic Nephropathy/DN. Red—upregulated genes, Blue—downregulated genes.