The role of the tricellular junction protein ILDR2 in glomerulopathies: Expression patterns and functional insights

Abstract

The tricellular tight junctions are crucial for the regulation of paracellular flux at tricellular junctions, where tricellulin (MARVELD2) and angulins (ILDR1, ILDR2, or LSR) are localized. The role of ILDR2 in podocytes, specialized epithelial cells in the kidney, is still unknown. We investigated the role of ILDR2 in glomeruli and its influence on blood filtration. Western blots, single-cell RNA sequencing (scRNA-seq), and superresolution microscopy showed a strong expression of ILDR2 in podocytes that colocalized with the podocyte-specific claudin CLDN5. Co-immunoprecipitation revealed that ILDR2 interacts with CLDN5. In glomerulopathies, induced by nephrotoxic serum and by desoxycorticosterone acetate (DOCA)-salt heminephrectomy, ILDR2 was strongly up-regulated. Furthermore, Ildr2 knockout mice exhibited glomerular hypertrophy and decreased podocyte density. However, they did not develop effacement of podocyte foot processes or proteinuria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis of isolated glomeruli showed an increase in matrix proteins, such as fibronectin and collagens. This suggests a protective role of ILDR2 in glomerulopathies.

Keywords: biological sciences; cellular physiology; natural sciences; physiology.

Graphical abstract

Figure 1

ILDR2 expression in human and mouse kidney tissue (A) ILDR2 expression in glomeruli (G), outer renal cortex (OC), inner renal cortex (IC), outer renal medulla (OM), and inner renal medulla (IM). Data are represented as log2 median centered ratio. Each dot represents one sample. Data were taken from www.nephroseq.org. (B) Database analysis of Ildr2 in podos (podocytes), PECs (parietal epithelial cells), and PT (segment 1, 2, or 3 of proximal tubule) using the KidneyCellExplorer (Ransick et al., 2019) based on a single-cell RNA sequencing dataset of murine kidneys. (C) The mRNA expression of Ildr2, Ildr1, Lsr, and Marveld2 in isolated glomeruli and murine kidneys was quantified by RT-qPCR (n ≥ 3). RT-qPCR experiments were normalized to the kidney samples and Gapdh. (D) In situ hybridization of ILDR2 in human kidney tissue. ILDR2-positive mRNA spots are marked by arrowheads. Nuclei are shown in blue. Scale bar represents 20 μm. (E) Podocyte proteome data (Rinschen et al.¹⁰) showed an enrichment of ILDR2 protein level in podocytes compared to glomeruli. (F) Western blot confirmed glomerulus-specific expression of ILDR2 (n = 3). Nephrin served as positive control and total protein (visualized by Stain-Free technology) served as a loading control. ∗∗p < 0.01. Data are presented as floating bars (A, C, and E).

Figure 2

Podocytes express the tricellular tight junction-associated protein ILDR2 (A) Immunofluorescence staining for ILDR2 (green) and the podocyte-specific marker nephrin (magenta) in mouse and human kidney tissue. Nuclei were stained with Hoechst (blue). Scale bars represent 20 μm and 5 μm (magnification), respectively. (B and C) Super-resolution 3D-structured illumination microscopy (3D-SIM) revealed that ILDR2 (shown in green) localized to tricellular junctions as indicated by a co-staining with nephrin (shown in magenta) on Y-intersections of the nephrin-stained filtration slit and to bicellular junctions in a spotted manner within healthy-appearing foot processes. (D and E) Immunofluorescence staining of kidney sections of E19 embryonic mouse kidneys (D) and adult mouse kidneys (E) showed that ILDR2 broadly localized to effaced podocyte filtration slits as indicated by a linearized double ILDR2 (green) and nephrin (magenta) colocalized areas. (F) Immunogold electron microscopy showed cell-membrane-associated binding of the anti-ILDR2 antibody in podocyte foot processes. Scale bars represent 1 μm (Figures B–F).

Figure 3

ILDR2 is an integral component of podocyte bi- and tricellular junctions and forms a complex with the podocyte-specific claudin-5 (A) Co-immunoprecipitation experiments of ILDR2-myc and CLDN5-GFP followed by western blot analysis with indicated antibodies. “Input” means the sample on 10% of volume used for IP. Anti-CD9 western blot served as a (non-binding) negative control. (B) The expression of ILDR2 (green) colocalized with CLDN5 (magenta) in human nephrectomy kidney glomeruli of FFPE sections in areas of podocyte foot process effacement. Imaged by SR-SIM. Scale bars represent 500 nm.

Figure 4

ILDR2 is up-regulated in DOCA-salt/UNX and NTS-treated mice as well as in different human glomerulopathies (A–H) ILDR2 expression (shown in green) in control and NTS-treated mice (A) or uninephrectomy (UNX) and desoxycorticosterone acetate (DOCA)-salt-treated mice (E). Nephrin (magenta) served as a podocyte-specific marker. NTS injected mice (B) and DOCA-salt treated (F) mice showed significantly increased ildr2/nephrin ratio. RNAScope of Ildr2 (magenta) followed by quantification of ILDR2 positive spots per glomerulus area (1/μm²) in NTS-treated mice (C and D) and DOCA-salt-treated mice (G and H) confirmed the increased expression of Ildr2 after injury. Podocin (shown in green) served as a podocyte marker. (I) Analysis of diabetes mouse glomeruli (Nephroseq database) substantiated increased expression on mRNA levels of Ildr2 in diabetes mice (n = 5) compared to Ctrl (n = 5). (J) An increase in the Ildr2 expression was also observed in ob/ob mice (n = 18). (K) mRNA expression level of microdissected glomeruli from renal biopsies of human patients suffering from diabetic nephropathy (DN; n = 7), minimal change disease (MCD; n = 5), focal segmental glomerulosclerosis (FSGS; n = 10) and rapidly progressive glomerulonephritis (RPGN, n = 23) compared with healthy living donors (LD; n = 18). Data are represented as log2 fold change, a q-value < 0.05 was considered as significant. Red: up-regulated to LD. Data are presented as means ± SD (I), means ± SEM (J), or violin plot (B, D, F, and H); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 ns, not significant. Scale bars represent 10 μm.

Figure 5

Ildr2 knockout mice do not develop proteinuria but have altered podocyte morphology (A) Schemata of Ildr2 whole-body knockout animals by crossing animals with a floxed exon 1 of the ILDR2 gene with a CMV:Cre line. (B) Ildr2 KO verification by RT-PCR. (C) Ildr2 KO verification by RT-qPCR. Gapdh served as a reference. (D) Ildr2 KO verification by western blot. Alpha-tubulin and total protein served as a loading control. (E) Transmission electron microscopy of wild-type (WT) and Ildr2 knockout (KO) mice revealed a significantly increased glomerular basement membrane (GBM) thickness in Ildr2 KO mice compared to WT mice (thickening indicated by asterisks; dots represent individual animals). (F) 3D-SIM in Ildr2 KO animals showed no significantly reduced filtration slit density (FSD) indicative for aberrant FP architecture. SIM data of 11 WT and 9 KO animals were quantified by PEMP (at 12 months of age). Kidney sections were stained for the slit diaphragm protein podocin. z axis scales of 3D-SIM were color coded as indicated. (G) Immunofluorescence staining for DACH1 (red) and the podocyte-specific marker synaptopodin (SNP; shown in green) in Ildr2 KO and WT mice. (H and I) Model-based glomerular morphometry showed slight glomerular hypertrophy with reduced podocyte density. (J) Urinary albumin-creatinine ratio measurements indicated no significantly increased levels of proteinuria in Ildr2 KO mice in comparison to WT animals (each individual dot represents one experimental animal). (K) Ildr2 KO mice also showed no significantly reduced filtration slit density (FSD) in comparison to WT mice after NTS-induced glomerulonephritis. (L) Level of albuminuria in Ildr2 WT and KO mice at day 2, 4, 8, and 11 after NTS injection. Scale bars represent 1 μm (E, F, and K) or 50 μm (G). Data are presented as means ± SD (C, F, J, K, and L) or violin plot (H and I); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant.

Figure 6

Ildr2 KO glomeruli showed increased expression of extracellular matrix proteins, but no up-regulation of potentially compensatory angulin or tight junction proteins (A) Volcano plot displaying proteins with significantly changed abundance in Ildr2 KO glomeruliin comparison to wild-type glomeruli (n = 5). Blue: down-regulated compared to controls; red: up-regulated compared to controls. Given are proteins with a log2 fold-change ≥0.3 and p value ≤0.05. (B) Top 10 GO (Gene Ontology) CC (cellular components) clusters, which were significantly up-regulated in Ildr2 KO glomeruli. (C) Significantly up-regulated extracellular matrix proteins in Ildr2 KO mice. Data are represented as log2 fold change, a p value <0.05 was considered as significant. (D) Renal mRNA expression of Fn1, Col12a1 and Col15a1 in Ildr2 WT and Ildr2 KO mice (n = 3). (E) Histologic evaluation of glomerular damage pattern employing Periodic acid-Schiff staining (PAS). At 12 months of age, expansion of the mesangial compartment was detectable. Assessment of glomerulosclerosis (glomerular PAS+ area) confirmed glomerular damage (each dot represents one individual glomerulus (WT: n = 144; KO: n = 299); seven WT and eight KO animals were analyzed). (F) The mRNA expression of Ildr1, Lsr, Cldn1, Marveld2, and Tjp1 was not significantly regulated in Ildr2 −/− mice (n = 4). RT-qPCR experiments were normalized to wild-type mice (WT), Gapdh served as a reference. ns, not significant. (G) Immunofluorescence staining for ILDR1, LSR and CLDN1 (magenta) and the podocyte-specific marker nephrin (shown in green) in Ildr2 KO (Ildr2 −/−) and WT mice (Ildr2 +/+). (H) LC-MS/MS based relative protein level of ZO-1 (TJP1) in isolated glomeruli from Ildr2 KO and Ildr2 WT mice (n = 5). (I) Fluorescence intensity measurement of immunofluorescence stained glomeruli with specific antibodies against claudin-5 and Tjp1 (ZO-1). Data are presented as means ± SD (D, G, and I) or violin plot (E and J); p value is indicated; ∗∗∗∗p < 0.0001; n.s., not significant. Scale bars represent 50 μm (E and G) and 10 μm (I).