Fig 1: The phenotype of MD-NGFR–KO mice.(A) Increased frequency of MD cell Ca2+ transients in MD-NGFR–KO vs. WT mice. Single MD cell (left) and whole-MD recording (center, original/smoothed) of G5F transients in WT (green) and MD-NGFR–KO mice (red). GFR changes (normalized to baseline before induction/treatment) in WT treated with vehicle (control) or NGF and MD-NGFR–KO mice (right); n = 4–5. (B) Reduced MD cell connectivity and sensitivity in MD-NGFR–KO vs. WT mice. Left: Functional MD cell–to-cell connectivity map of all 21 (WT) and 18 (MD-NGFR–KO) individually numbered MD cells. Red line connecting individual cell pairs indicates Pearson’s r > 0.35. Red and blue cell color indicates hub and lone cells, respectively. Center: Heatmap of each MD cell pair’s Pearson’s coefficient in 2-color gradient, as in scale. Right: Effect of i.v. gastrin on MD cell Ca2+ (G5F) in WT vs. MD-NGFR–KO mice; n = 4–8 (average of 4–5 MD cells/animal). (C–E) Renin cell density (C), endothelial injury and podocyte number (D), and renal pathology (E) in WT mice treated with vehicle (control) or NGF,and MD-NGFR–KO mice. n = 4–5 (average of 5 glomeruli/animal). Renin, PLVAP, KIM1 immunofluorescence (red) images and PAS-stained kidney tissue sections, and summary of respective cell numbers, labeling density (tissue fibrosis index), and albuminuria (ACR). Cell nuclei are labeled blue with DAPI, tissue autofluorescence (C, green) is shown for tissue morphology. Scale bar: 50 μm (C–E). (F) Representative immunoblots and summary of MD-specific protein expression in renal cortical homogenates, including CCN1, CCN3, and CXCL14 in WT vs. MD-NGFR–KO mice; n = 4. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using Student’s t test (B and D–F) or ANOVA followed by Dunnett’s test (A and C).
Fig 2: Treatment with MD biologicals improves kidney function in CKD.(A) Illustration of therapeutic study design for testing the effects of MD biologicals (human recombinant CCN1 and LS-conditioned MDgeo cell culture media) using the adriamycin (ADR) model of glomerulosclerosis in BALB/c mice. (B) Time course of the absolute (left) and relative (normalized to baseline before treatment, right) changes in GFR followed in the same mice measured by the MediBeacon noninvasive transcutaneous method. Note the significant improvement of GFR returning to normal baseline levels (the red dotted line represents mean ± SEM [gray shaded area], measured at baseline) in the MD treatment group indicating functional regression of FSGS pathology; n = 6–8. (C) Time course of albuminuria (albumin/creatinine ratio [ACR]) changes followed in the same mice measured by ELISA. Note the significant improvement in albuminuria in the CCN1 and MD treatment groups in contrast to the PBS and DMEM-F12 controls; n = 6–8. Data represent mean ± SEM. *P < 0.05, ****P < 0.0001, 2-way (mixed-effect) ANOVA with Tukey’s test (B, left, and C), 1-way ANOVA followed by Dunnett’s test (B, center), or t test (B, right).
Fig 3: CCN1 expression in the kidney in patients with normal kidney function or CKD.(A) Immunofluorescence labeling (red, left) and quantification (right) of MD cell markers CCN1 (top), NOS1 (center), and COX2 (bottom) in human kidney sections. Note the strong CCN1 expression exclusively in cells of the macula densa (MD; red arrows) in controls and mostly absent labeling in kidney tissue samples from patients with CKD, in contrast to the pattern in NOS1 and COX2 labeling; n = 6 (average of 5 MDs/sample). Nuclei are labeled blue with DAPI; green is tissue autofluorescence. G: glomerulus. Scale bars: 25 μm. (B) Intrarenal CCN1 (CYR61), NOS1, and PTGS2 (COX2) mRNA expression in kidney biopsies (tubulointerstitial compartment) from living donor (LD), tumor nephrectomy (TN), and patients with CKD with various etiologies from the ERCB. LD (n = 31), TN (n = 4), diabetic nephropathy (DN, n = 17), minimal change disease (MCD, n = 14), thin basement membrane disease (TMD, n = 6), arterial hypertension (HTN, n = 20), IgA nephropathy (IgAN, n = 25), focal segmental glomerulosclerosis (FSGS, n = 17), lupus nephritis (SLE, n = 32), membranous glomerulonephropathy (MGN, n = 18), and vasculitis (RPGN, n = 21). Differential expression comparison between LD and each disease subtype was performed using t test. (C) The association of urinary CCN1 levels with kidney function. Comparison of urinary CCN1 in individuals acting as controls (n = 11) and patients with CKD (n = 29) (left) and the positive correlation between urinary CCN1 excretion and eGFR in patients with CKD; n = 18, log2-transformed urinary CCN1/creatinine ratios are shown (right). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using Student’s t test.
Fig 4: MD cell secretome analysis.(A) Workflow of the generation and characterization of the MDgeo cell line and its culturing (conditioning) in control (normal salt [NS]) and low salt (LS) conditions. Differentiated mMDgeo cells show an epithelial cobblestone-like pattern, while semiconfluent MDgeo cells feature long axon-like processes. Scale bar: 25 μm. (B) Mass spectrometry (left) and CCN1 ELISA (right) analysis of the LS-conditioned MDgeo cell culture medium. Mass spectrum plot representing the detected MD-derived secreted proteins in the MDgeo cell culture medium as indicated. LS, low-salt medium. Data represent mean ± SEM. *P < 0.05 with t test, n = 4–6. (C) Immunohistochemistry validation of the expression of top enriched mouse MD-specific genes or their homologous isoforms in the human kidney. Data are from the Human Protein Atlas (HPA) where indicated. Image available from https://www.proteinatlas.org/ENSG00000012171-SEMA3B/tissue/kidney Scale bar: 50 μm.
Fig 5: Manipulation of MD Wnt signaling alters glomerular structure and function.(A) Representative fluorescence images of frozen kidney sections (left) and quantification (center) of Wnt activity (GFP fluorescence [F] intensity, green) from mice with nuclear TCF/Lef:H2B-GFP reporter in control, LiCl (as positive control), LS, and LS+ACEi conditions; n = 4–6 (average of 5 MDs/animal). Intense GFP labeling in MD cells. Right: TCF4 immunohistochemistry in human kidney (data from the Human Protein Atlas [HPA]). Image available from https://www.proteinatlas.org/ENSG00000196628-TCF4/tissue/kidney Intense labeling in MD cells (arrows). G, glomerulus. (B) Illustration of the applied Cre/lox-based breeding strategies to generate inducible MD Wnt gain-of-function (MD-Wntgof) and loss-of-function (MD-Wntlof) mouse models. (C) Top: Renal histological (representative H&E images [left]) and functional (glomerular filtration rate [GFR, right]) features of MD-Wntgof and lof mice 2 months after tamoxifen induction; n = 4–5 (average of 5–10 glomeruli/animal). Note the enlarged or smaller cortical glomeruli in MD-Wntgof and lof mice, respectively, compared with control WT mice, with extracellular (mesangial) matrix accumulation in MD-Wntlof mice. Bottom: Representative immunofluorescence images (left) and statistical summary (right) of WT1+ (red) and CD34+ (green) cell number. Note the high cell density at the macula densa (MD) cell base (arrows). Yellow (in WT1) and orange (in CD34) colors represent the autofluorescence of red blood cells. Scale bars: 25 μm. Nuclei are labeled blue with DAPI. (D) Altered expression of MD-specific proteins in renal cortical homogenates, including CCN1 and SEMA3C; n = 4. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ANOVA followed by Dunnett’s test.
Supplier Page from Abcam for Mouse CYR61 ELISA Kit