Table1_TFRC–RNA interactions show the regulation of gene expression and alternative splicing associated with IgAN in human renal tubule mesangial cells.pdf

Introduction: IgA nephropathy (IgAN) is the most common primary glomerular disease (PGD) which could progress to renal failure and is characterized by aberrant IgA immune complex deposition. Transferrin receptor1 (TFRC), an IgA receptor, is a potential RNA binding protein (RBP) which regulates expression of genes positively associated with the cell cycle and proliferation and is involved in IgAN. Molecular mechanisms by which TFRC affects IgAN development remain unclear.Methods: In this study, TFRC was overexpressed in human renal tubular mesangial cells (HRMCs) and RNA-sequencing (RNA-seq) and improved RNA immunoprecipitation sequencing (iRIP-seq) were performed. The aim was to identify potential RNA targets of TFRC at transcriptional and alternative splicing (AS) levels.Results: TFRC-regulated AS genes were enriched in mRNA splicing and DNA repair, consistent with global changes due to TFRC overexpression (TFRC-OE). Expression of TFRC-regulated genes potentially associated with IgAN, including CENPH, FOXM1, KIFC1, TOP2A, FABP4, ID1, KIF20A, ATF3, H19, IRF7, and H1-2, and with AS, CYGB, MCM7 and HNRNPH1, were investigated by RT-qPCR and iRIP-seq data analyzed to identify TFRC-bound RNA targets. RCC1 and RPPH1 were found to be TFRC-bound RNA targets involved in cell proliferation.Discussion: In conclusion, molecular TFRC targets were identified in HRMCs and TFRC found to regulate gene transcription and AS. TFRC is considered to have potential as a clinical therapeutic target.</p

Angiopoietin-Like-4, a Potential Target of Tacrolimus, Predicts Earlier Podocyte Injury in Minimal Change Disease

<div><p>Podocyte injury plays central roles in proteinuria and kidney dysfunction, therefore, identifying specific biomarker to evaluate earlier podocyte injury is highly desirable. Podocyte-secreted angiopoietin-like-4 (Angptl4) mediates proteinuria in different types of podocytopathy. In the present study, we established an experimental minimal change disease (MCD) rat model, induced by adriamycin (ADR) and resulted in definite podocyte injury, to identify the dynamic changes in Angptl4 expression. We also investigated the direct effects of tacrolimus on Angptl4 and podocyte repair. We determined that the glomerular Angptl4 expression was rapidly upregulated and reached a peak earlier than desmin, an injured podocyte marker, in the ADR rats. Furthermore, this upregulation occurred prior to heavy proteinuria and was accompanied by increased urinary Angptl4. We observed that the Angptl4 upregulation occurred only when podocyte was mainly damaged since we didn’t observe little Angptl4 upregulation in MsPGN patients. In addition, we observed the glomerular Angptl4 mainly located in injured podocytes rather than normal podocytes. Moreover, we found that tacrolimus treatment significantly promoted podocyte repair and reduced glomerular and urinary Angptl4 expression at an earlier stage with a significant serum Angptl4 upregulation. And similar results were confirmed in MCD patients. In conclusion, this study represents the first investigation to demonstrate that Angptl4 can predict podocyte injury at earlier stages in MCD and the identification of earlier podocyte injury biomarkers could facilitate the prompt diagnosis and treatment of patients with podocytopathy, as well as determination of the prognosis and treatment efficacy in these diseases.</p></div

Baseline characteristics of the enrolled patients.

<p>M, male; F, female.</p><p>Baseline characteristics of the enrolled patients.</p

Tacrolimus-treated rats exhibited ameliorated ADR-induced proteinuria and lipid metabolism disorders.

<p><b>(A)</b> Twenty-four hour urinary protein excretion in ADR rats. <b>(B)</b> Serum albumin levels in ADR rats. <b>(C)</b> Serum triglyceride levels in ADR rats. <b>(D)</b> Serum cholesterol levels in ADR rats. Con, normal rats; Untreated, ADR rats without treatment; TAC, ADR rats with tacrolimus treatment. ##P<0.05 compared with normal rats; #P<0.01 compared with normal rats; *P<0.01 compared with untreated ADR rats. The arrow indicates that tacrolimus treatment was initiated on day 14.</p

Tacrolimus promoted podocyte repair in ADR rats.

<p><b>(A)</b> Immunofluorescence of glomerular desmin in normal, tacrolimus-treated and untreated ADR rats. Scale bars = 50 μm. <b>(B)</b> Quantification of the fluorescence staining intensities of glomerular desmin in normal, tacrolimus-treated and untreated ADR rats. <b>(C)</b> Immunofluorescence of glomerular synaptopodin in normal, tacrolimus-treated and untreated ADR rats. <b>(D)</b> Quantification of the fluorescence staining intensities of glomerular synaptopodin in normal, tacrolimus-treated and untreated ADR rats. <b>(E)</b> Transmission electron microscopy of normal, tacrolimus-treated and untreated ADR rats. Foot process effacements are indicated by the black arrows. Scale bars = 2 μm. <b>(F)</b> TUNEL assay of glomeruli from ADR rats. A TUNEL-positive cell is indicated by the black arrow. Scale bars = 50 μm. <b>(G)</b> Quantification of the TUNEL assay of the glomeruli from ADR rats. <b>(H)</b> Western blot of glomerular synaptopodin and desmin expression in ADR rats. <b>(I)</b> Quantification of the western blot of glomerular synaptopodin expression in ADR rats. <b>(J)</b> Quantification of the western blot of glomerular desmin expression in ADR rats. Con, normal rats; Untreated, ADR rats without treatment; TAC, ADR rats with tacrolimus treatment. ##P<0.05 compared with normal rats; #P<0.01, compared with normal rats; *P<0.01 compared with untreated ADR rats.</p

HO-1 overexpression reduced peripheral capillary loss and inhibited the activation and proliferation of renal interstitial myofibroblasts after UUO.

<p><b>(A)</b> Representative immunofluorescence images of CD31-labeled PTCs (red) from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(B)</b> Graph showing the morphometric quantification of CD31-positive staining. <b>(C)</b> Representative immunofluorescence images of kidney sections co-labeled with PDGFRβ (green) and α-SMA (red) in the Sham and UUO groups on days 7 and 10. PDGFRβ is a specific marker of pericytes, fibroblasts and myofibroblasts, and α-SMA labels activated fibroblasts and myofibroblasts (magnification, ×400; bars = 100 μm). <b>(D)</b> Graph displaying the morphometric quantification of the PDGFRβ-positive area per high-power field. <b>(E)</b> Histogram showing the quantification of the α-SMA -positive area per high-power field. <b>(F)</b> Graph showing the morphometric quantification ratios of the double-positive/PDGFR-β-positive areas. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

The majority of glomerular Angptl4 was secreted by injured podocytes in ADR rats.

<p><b>(A)</b> Immunofluorescence of glomerular Angptl4 and desmin, an injured podocyte marker, in ADR rats on days 14, 21 and 28. <b>(B)</b> Immunofluorescence of glomerular Angptl4 and synaptopodin, a normal podocyte marker, in ADR rats on days 10 and 14. <b>(C)</b> Immunofluorescence of glomerular Angptl4 and laminin, a GBM marker, in ADR rats on day 14. <b>(D)</b> Immunofluorescence of glomerular Angptl4 and RECA-1, an endothelial cell marker, in ADR rats on days 14 and 28. <b>(E)</b> Immunofluorescence of glomerular Angptl4 and OX-7, a mesangial cell marker, in ADR rats on day 14. <b>(F)</b> Immunofluorescence of glomerular Angptl4 and RECA-1 in ADR rats with tacrolimus treatment on day 28. Scale bars: 50 μm. TAC, ADR rats with tacrolimus treatment.</p

HO-1 decreased tubulointerstitial inflammation in the UUO model.

<p><b>(A)</b> Representative immunofluorescence images of F4/80-labeled macrophages (green) from the Sham and UUO groups on days 3, 7, 10 and 14. DAPI labels the nucleus (blue). (Magnification, ×200; bars = 250 μm) <b>(B)</b> Histogram indicating the morphometric quantification of the number of F4/80-positive cells per high-power field. <b>(C-D)</b> Graph showing the mRNA expression of inflammation-related cytokines at day 7 after UUO in the TG and WT groups. <b>(E)</b> Representative ELISA for inflammatory markers (TNF-α, IL-1β, IL-6, IL-4 and IL-10) in kidney tissue homogenates of HO-1 TG mice on the 7^th day after UUO. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 reduced renal tubulointerstitial injury and fibrosis after UUO.

<p><b>(A)</b> Representative light microscopy images of HE-stained kidney sections from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(B)</b> Histogram showing the semi-quantitative determination of tubulointerstitial lesions. <b>(C)</b> Representative light microscopy images of Masson-stained and picrosirius red-stained kidney sections from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(D)</b> Representative light microscopy images of picrosirius red-stained kidney sections from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(E)</b> Histogram showing the area of picrosirius red-positive staining per high-power field in mice of the WT and TG groups after sham or UUO surgery. <b>(F)</b> Representative images of TGF-β1 immunohistochemical staining (brown) of kidney sections from the Sham and UUO groups at 3, 7, 10 and 14 days (magnification, ×200; bars = 250 μm). <b>(G)</b> Graph showing a semi-quantitative index of TGF-β1 staining. <b>(H)</b> Histogram showing TGF-β1 and CTGF mRNA levels at day 10 after UUO. The results are representative of triplicate analyses. <b>(I-J)</b> Representative Western blot (I) and quantitative data (J) for renal TGF-β1 expression 10 days after sham or UUO surgery. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 inhibited the activation of the Wnt/ β-catenin signaling pathway after renal fibrosis induced by UUO.

<p><b>(A-B)</b> Semi-quantitative real time-PCR of transcript levels of Wnt/β-catenin pathway ligands in the whole kidney 7 days after sham or UUO surgery. <b>(C)</b> Histogram showing the quantification of the β-catenin-positive area per high-power field. <b>(D)</b> Representative immunofluorescence images showing the expression and localization patterns of β-catenin, a common downstream mediator of the canonical Wnt signaling pathway, in the kidneys of the Sham and UUO groups at days 3, 7 and 10 (magnification, ×200; bars = 250 μm). Asterisks indicate β-catenin-positive tubulointerstitial cells. <b>(E-F)</b> Representative Western blot (E) and quantitative data (F) are presented for renal β-catenin expression 10 days after sham or UUO surgery. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p