Journal: BMC nephrology

Article Title: Meprin β regulates osteopontin-signaling in ischemia/reperfusion-induced kidney injury.

doi: 10.1186/s12882-025-03995-7

Figure Lengend Snippet: Fig. 1 Kidney injury biomarkers and OPN expression in kidney tissue from wild type (WT) and meprin β deficient mice (βKO) at 0 h (control) and 24 h post-IR. Kidney injury was confirmed using ELISA to assess the level of kidney injury biomarker, NGAL which significantly increased in both genotypes at 24 h post-IR (A). Blood samples were collected at 0 h and 24 h post-IR injury to assess the plasma levels of NGAL in 4 mice/group. Real-time-PCR analysis showed significant increases in OPN mRNA levels in both genotypes at 24 h post-IR (B). OPN mRNA levels are expressed as fold change (FC) relative to the WT control and normalized to GAPDH mRNA and each value represents the mean ± SEM of triplicate combinations from 4 mice per group. Western blot analysis for OPN in kidney proteins from WT and meprin βKO mice showed IR-associated increased levels of the full-length OPN (66 KDa) in WT only (C). The protein bands represent samples from individual kidneys (n = 3). The relative optic densities (ODs) were calculated by normalizing the ODs of OPN to the ODs for β-tubulin in the same sample. The full-length images of western blot are presented in supplementary figure (S1). Immunohistochemical staining for OPN showed significant increase in WT and meprin βKO kidney tubules (D) and renal corpuscles of WT and meprin βKO (E). OD data were quantified (n = 3) using Image J analysis Software (ImageJ/Fiji 1.46) and analyzed for 10 non-overlapping fields from tubular and renal corpuscle sections from each kidney. Images at 60× magnification and the scale bar representing 20 μm. Immunofluorescence counterstaining of OPN (red) with either meprin β (green) in WT mice or villin (PT marker, green) in meprin β-deficient mice showed that meprin β induced expression of OPN in PTs in kidneys subjected to IR (F). DAPI was used to stain the nuclei (blue). Images at 60× magnification and the scale bar representing 20 μm. Data is expressed as mean ± SEM with P values as indicated, P ≤ 0.05 are considered statistically significant

Article Snippet: To determine localization of the proteins of interest, we used immunofluorescence counterstaining according to the previously described protocols [6, 21] with two proximal tubule markers, meprin β in WT kidney sections (Goat Anti Mouse Meprin β Subunit/MEP1B Antigen Affinity purified Polyclonal Antibody (R and D Systems, Cat # AF3300, RRID: AB_2143451) diluted 1:100 and villin in βKO kidney sections (mouse monoclonal anti-villin antibodies, Santa Cruz Biotechnology Cat# sc-58897, RRID: AB_2304475) diluted 1:200 for 1 h at room temperature.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Clinical Proteomics, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Staining, Software, Immunofluorescence, Marker

Journal: BMC nephrology

Article Title: Meprin β regulates osteopontin-signaling in ischemia/reperfusion-induced kidney injury.

doi: 10.1186/s12882-025-03995-7

Figure Lengend Snippet: Fig. 2 Caspase-3 expression in kidney tissue from WT and meprin βKO mice at 0 h and 24 h post-IR. The real-time PCR data showed that mRNA expression levels of Caspase-3 decreased in WT and increased in βKO mice at 24 h post-IR (A). Caspase-3 mRNA levels are expressed as fold change (FC) relative to the WT control and normalized to GAPDH mRNA and each value represents the mean ± SEM of triplicate combinations from 4 mice per group. Western blot analysis detected two bands of Caspase-3 (33 and 35 kDa) with significant increase in the Caspase-3 (33 kDa) band for WT kidneys only (B). The protein bands represent samples from individual kidneys (n = 3). The relative optic densities (ODs) were calculated by normalizing the ODs of Caspase-3 to the ODs for β-tubulin in the same sample. The full-length images of western blot are presented in supplementary figure (S2). Immunohistochemical staining of Caspase-3 showed significant increase in select tubules from WT kidney only (C) and renal corpuscles of both genotypes (D). OD data were quantified (n = 3) using Image J analysis Software (ImageJ/Fiji 1.46) and analyzed for 10 non-overlapping fields from tubular and renal corpuscle sections. Images at 60× magnification and the scale bar representing 20 μm. Immunofluorescence counterstaining of Caspase-3 (red) with either meprin β (green) in WT or villin (PT marker, green) in meprin β-deficient mice showed that Caspase-3 increased in both PTs and DTs of WT but not in meprin βKO mice (E). DAPI was used to stain the nuclei (blue). Images at 60× magnification and the scale bar representing 20 μm. Data is expressed as mean ± SEM with P values as indicated, P ≤ 0.05 are considered statistically significant

Article Snippet: To determine localization of the proteins of interest, we used immunofluorescence counterstaining according to the previously described protocols [6, 21] with two proximal tubule markers, meprin β in WT kidney sections (Goat Anti Mouse Meprin β Subunit/MEP1B Antigen Affinity purified Polyclonal Antibody (R and D Systems, Cat # AF3300, RRID: AB_2143451) diluted 1:100 and villin in βKO kidney sections (mouse monoclonal anti-villin antibodies, Santa Cruz Biotechnology Cat# sc-58897, RRID: AB_2304475) diluted 1:200 for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Immunohistochemical staining, Staining, Software, Immunofluorescence, Marker

Journal: BMC nephrology

Article Title: Meprin β regulates osteopontin-signaling in ischemia/reperfusion-induced kidney injury.

doi: 10.1186/s12882-025-03995-7

Figure Lengend Snippet: Fig. 3 Bcl-2 expression in kidney tissue from WT and meprin βKO mice at 0 h and 24 h post-IR. The real-time PCR data showed that mRNA expression levels of Bcl-2 significantly increased in βKO mice only at 24 h post-IR (A). Real-time-PCR analysis showed significant increases in Bcl-2 mRNA levels in both genotypes at 24 h post-IR. Bcl-2 mRNA levels are expressed as fold change (FC) relative to the WT control and normalized to GAPDH mRNA and each value represents the mean ± SEM of triplicate combinations from 4 mice per group. Western blot analysis showed a significant increase of the Bcl-2 protein level in WT at 24 h post-IR but not in βKO mice (B). The protein bands of Bcl-2 detected at 26 KDa represent samples from individual kidneys (n = 3). The relative optic densities (ODs) were calculated by normalizing the ODs of Bcl-2 to the ODs for β-tubulin in the same sample. The full-length images of western blot are presented in supplementary figure (S3). Immunostaining of Bcl-2 showed significant increase in select tubules of both genotypes (C) and renal cor puscles (D). OD data were quantified (n = 3) using Image J analysis Software (ImageJ/Fiji 1.46) and analyzed for 10 non-overlapping fields from tubular and renal corpuscle sections. Images at 60× magnification and the scale bar representing 20 μm. Representative immunofluorescence staining for Bcl-2 (red) in WT and meprin βKO mice kidney tubules showed that levels of Bcl-2 increased in both proximal and distal tubules for WT kidneys (E). Villin (green) was used as a proximal tubule marker in both genotypes because of antibody incompatibility. DAPI was used to stain the nuclei (blue). Images at 60× magni fication and the scale bar representing 20 μm. Data is expressed as mean ± SEM with P values as indicated, P ≤ 0.05 are considered statistically significant

Article Snippet: To determine localization of the proteins of interest, we used immunofluorescence counterstaining according to the previously described protocols [6, 21] with two proximal tubule markers, meprin β in WT kidney sections (Goat Anti Mouse Meprin β Subunit/MEP1B Antigen Affinity purified Polyclonal Antibody (R and D Systems, Cat # AF3300, RRID: AB_2143451) diluted 1:100 and villin in βKO kidney sections (mouse monoclonal anti-villin antibodies, Santa Cruz Biotechnology Cat# sc-58897, RRID: AB_2304475) diluted 1:200 for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Immunostaining, Software, Immunofluorescence, Staining, Marker

Journal: BMC nephrology

Article Title: Meprin β regulates osteopontin-signaling in ischemia/reperfusion-induced kidney injury.

doi: 10.1186/s12882-025-03995-7

Figure Lengend Snippet: Fig. 4 NFκB expression in kidney tissue from WT and meprin βKO mice at 0 h and 24 h post-IR. The real-time PCR data showed that mRNA expression levels of NFκB increased significantly in the βKO at 24 h post-IR but not in WT kidneys (A). NFκB mRNA levels are expressed as fold change (FC) relative to the WT control and normalized to GAPDH mRNA and each value represents the mean ± SEM of triplicate combinations from 4 mice per group. Western blot analysis detected NFκB bands at 65 kDa (B). The protein bands represent samples from individual kidneys (n = 3). The relative optic densities (ODs) were calculated by normalizing the ODs of NFκB to the ODs for β-tubulin in the same sample. The full-length images of western blot are presented in supplementary figure (S4). Immunostaining of NFκB showed a significant increase in protein levels for NFκB in select tubules of WT but not βKO (C) and staining intensity for NFκB increased significantly only in WT kidneys renal corpuscles of both genotypes (D). OD data were quantified (n = 3) using Image J analysis Software (ImageJ/Fiji 1.46) and analyzed for 10 non-overlapping fields from tubular and renal corpuscle sections. Images at 60× magnification and the scale bar representing 20 μm. Immunofluorescence counterstaining of NFκB (red) with either meprin β (green) in WT or villin (PT marker, green) in in meprin β-deficient mice showed increases in levels of NFκB in proximal tubules of WT kidneys (E). DAPI was used to stain the nuclei (blue). Images at 60× magnification and the scale bar representing 20 μm. Data is expressed as mean ± SEM with P values as indicated, P ≤ 0.05 are considered statistically significant

Article Snippet: To determine localization of the proteins of interest, we used immunofluorescence counterstaining according to the previously described protocols [6, 21] with two proximal tubule markers, meprin β in WT kidney sections (Goat Anti Mouse Meprin β Subunit/MEP1B Antigen Affinity purified Polyclonal Antibody (R and D Systems, Cat # AF3300, RRID: AB_2143451) diluted 1:100 and villin in βKO kidney sections (mouse monoclonal anti-villin antibodies, Santa Cruz Biotechnology Cat# sc-58897, RRID: AB_2304475) diluted 1:200 for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Immunostaining, Staining, Software, Immunofluorescence, Marker

Journal: Antioxidants

Article Title: The Antioxidant Carrichtera annua DC. Ethanolic Extract Counteracts Cisplatin Triggered Hepatic and Renal Toxicities

doi: 10.3390/antiox10060825

Figure Lengend Snippet: Representative photomicrographs (×400) of immuno-histochemical staining of IL-1β in liver ( A ) and kidney ( B ) tissues of the experimental groups. ( A1 ) liver tissues of normal control group, ( A2 ) liver tissues of cisplatin group, ( A3 ) liver tissues of Prophylactic C. annua - treated group, ( B1 ) kidney tissues of normal control group, ( B2 ) kidney tissues of cisplatin group, ( B3 ) kidney tissues of Prophylactic C. annua - treated group.

Article Snippet: Sections from liver and kidney tissues were stained with anti-IL-1β (Catalog number bs-0812R, Bioss Inc., Woburn, MA, USA) and anti-caspase 3 (Catalog number GTX30246, GeneTex, Irvine, CA, USA) after preparation according to the method described in [ , ].

Techniques: Staining

Journal: Antioxidants

Article Title: The Antioxidant Carrichtera annua DC. Ethanolic Extract Counteracts Cisplatin Triggered Hepatic and Renal Toxicities

doi: 10.3390/antiox10060825

Figure Lengend Snippet: ( A ) Level of IL-1β expression in liver and kidney tissues of the experimental groups. Positive proportions of IL-1β expression were increased significantly in liver and kidney of cisplatin treated rats compared to the normal control and C. annua pretreated group. ( B ) Level of caspase-3 expression in liver and kidney tissues of the experimental groups. Positive proportions of caspase-3 expression were elevated remarkably in liver and kidney of cisplatin treated rats compared to the normal control and C. annua pretreated group. Data are represented as mean ± SE and analyzed using ANOVA followed by Bonferroni’s post-hoc test at p value < 0.05, n = 15 for each group. a indicates the presence of significant difference from normal control group, b indicates the presence of significant difference from cisplatin group.

Article Snippet: Sections from liver and kidney tissues were stained with anti-IL-1β (Catalog number bs-0812R, Bioss Inc., Woburn, MA, USA) and anti-caspase 3 (Catalog number GTX30246, GeneTex, Irvine, CA, USA) after preparation according to the method described in [ , ].

Techniques: Expressing

Journal: EBioMedicine

Article Title: Renal interstitial fibroblasts coproduce erythropoietin and renin under anaemic conditions

doi: 10.1016/j.ebiom.2021.103209

Figure Lengend Snippet: Renal interstitial cells express renin in kidneys of CKD patients, and a schematic diagram of Epo and renin production in kidneys. (a) Representative images of ISH of REN (brown) in kidney sections of patients suffering from chronic tubulointerstitial nephritis. Arrowheads and dotted circles are interstitial fibroblasts positive for REN mRNA expression and glomeruli, respectively. Red and blue arrows indicate JG cells that are positive and negative for REN mRNA expression, respectively. The scale bar is 100 μm. (b) A schematic diagram of Epo and renin production in the kidney under normal, anaemic, and injured conditions. Renal interstitial fibroblasts (yellow) maintain the systemic oxygen supply under anaemic conditions through inducing Epo and renin production in response to hypoxia and hypotension, respectively. In injured kidneys, most interstitial fibroblasts are transformed into myofibroblasts (orange), which lose the ability to produce Epo and enhance renin production.

Article Snippet: Formalin-fixed paraffin-embedded kidney sections from patients suffering from chronic tubulointerstitial nephritis were purchased from OriGene (CS700464 and CS705284 for Patient 1 and Patient 2, respectively).

Techniques: Expressing, Transformation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Endogenous Notch Signaling in Adult Kidneys Maintains Segment-Specific Epithelial Cell Types of the Distal Tubules and Collecting Ducts to Ensure Water Homeostasis

doi: 10.1681/ASN.2018040440

Figure Lengend Snippet: Principal-to-intercalated cell conversion triggered by inactivation of Hes1 involves intermediary cell states and upregulation of Foxi1. (A–C) Kidneys from Hes1cKO mice given BrdU in the drinking water during 2 weeks of dox-mediated inactivation of Hes1 show an increase in the number of BrdU-labeled PCs from 0.1% to about 10% and BrdU-labeled intercalated cells from 0% to 2% (A–C). A majority of the increase in intercalated cells in the inner stripe of the OMCDs from 20% in wild-type to 40% in Hes1cKO mice (n=4 per genotype) does not involve BrdU-labeled cells (asterisks in B). In (C), * indicates significant differences by two-tailed unpaired t test when comparing percentages of Aqp4+ (P=6.5×10−5), cKit+ (P=0.001753), Aqp4+ and cKit+ (P=0.007), Aqp4− and cKit− (P=0.009), BrdU+ (P=0.04), Aqp4+ and BrdU+ (P=0.04), or cKit+ and BrdU+ (P=0.03) OMCD cells between wild-type and Hes1cKO mouse kidneys after 2 weeks of dox treatment (n=4 mice per genotype with at least 1400 OMCD cells analyzed per mouse). (D and E) Maximum intensity projections of confocal Z-stack images (optical sections) captured every 0.175 μm, using a ×60 objective on a Nikon A1 confocal microscope. (F and G) 3D ISO surface images of inner stripes of the OMCD segments generated from Z-stack images using Arivis Vision 4D. (D–G) Different views of a wild-type OMCD segment (D and F) compared with different views of a Hes1cKO OMCD segment (E and G) reveal Foxi1-expressing intercalated cells (arrowheads), Aqp4-expressing PCs (arrows), and intermediate state cells expressing both Foxi1 and Aqp4 (asterisks). The cell labeled 1 is the same cell in (E and G), and similarly the cell labeled 2 is the same cell in (E and G). (H) In the inner stripe of the OMCDs the average number of PCs per tubule length decreases significantly in Hes1cKO kidneys compared with wild-type controls. Intermediate cell types are present at significantly higher numbers in the Hes1cKO inner stripe of the OMCDs; *P<0.05, when performing unpaired t tests. A minimum of seven OMCD segments were analyzed per mouse, and three mice per genotypic group were analyzed. Scale bars, 10 μm.

Article Snippet: Stained tissue sections were imaged on a Nikon A1 confocal microscope using NIS elements software for image acquisition. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1. caption a7 Active Notch signaling occurs after development in mouse kidneys and is required for maintaining PC gene expression and function. (A) The Notch ligand Delta-like 1 (Dll1) continues to be expressed in the inner stripe of the OMCD cells adjacent to Aqp3 + cells as determined by β -galactosidase staining (red) in Dll1 +/LacZ mice. (B) The Notch reporter CBF->H2B::Venus reveals continued Notch signaling in adult mouse kidneys, including the CCDs.

Techniques: Labeling, Two Tailed Test, Microscopy, Generated, Expressing

Journal: Kidney international

Article Title: Renal tubular cell spliced X-box binding protein-1 (Xbp1s) has a unique role in sepsis-induced acute kidney injury and inflammation

doi: 10.1016/j.kint.2019.06.023

Figure Lengend Snippet: (A) RT-PCR (left panel) and qPCR (right panel) analysis showing Xbp1s transcript levels in mouse kidney is increased by lipopolysaccharide (LPS; CTR and LPS-injected, n=3 each), but not by other acute kidney injury (AKI) insults such as folic acid (FA; CTR and FA, n=3 each) or ischemia-reperfusion injury (IRI; CTR and IRI, n=4) compared to respective control. (B) RT-PCR (left panel) and qPCR (right panel) showing that Xbp1s transcript levels are unchanged in the kidney of genetic mouse models of chronic kidney disease, i.e. type II diabetes (CTR and ob/ob mice, n=3) and polycystic kidney disease [PKD; Ksp/Cre;Pkd1F/F (Pkd1, n=3) mice at postnatal day 10, P10; Pkhd1/Cre;Pkd2F/F (Pkd2, n=3) mice at postnatal day 21, P21] compared to respective control (n=3 for each condition). Mouse kidney with known Xbp1 splicing was used as positive control. (C) Xbp1s antibody co-staining with nephron segment-specific markers revealed that Xbp1s expression was induced in epithelia cells from all nephron segments in kidneys of mice treated with LPS compared to vehicle-treated controls. LTA: lotus tetragonolobus agglutinin; NKCC2: Na+-K+-Cl- cotransporter; NCC: Na+-Cl- cotransporter; DBA: dolichos biflorus agglutinin. Scale bar, 20 μm. Bars and error bars indicate mean and SEM. Student’s unpaired t-test of injury vs. control within each model. * indicates P<0.05, compared to respective control. CTR: control.

Article Snippet: Sections stained with Dolichos Biflorus Agglutinin (DBA) were incubated with biotinylated DBA (1:400; B-1035, Vector Laboratories, Burlingame, CA) at 4°C overnight, and detected by fluorescein Avidin D (1:500; A-2001, Vector Laboratories, Burlingame, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Injection, Positive Control, Staining, Expressing

Journal: Clinical science (London, England : 1979)

Article Title: Changes in cell fate determine the regenerative and functional capacity of the developing kidney before and after release of obstruction

doi: 10.1042/CS20180623

Figure Lengend Snippet: (A) Kidney sections of Six2Cre;mTmG mice showed the presence of GFP+ Six2 lineage cells all along the nephron epithelium. Obstructed kidneys with hydronephrosis score 2 displayed no obvious changes in the distribution of GFP+ tubules in contrast to an extensive loss in kidneys with severe hydronephrosis index 4 (white arrows). Release of obstruction decreased the hydronephrosis, prevented excessive tubular loss and the GFP+ nephron tubule status was similar to shams. Scale bars, 50μm (B) Atubular glomeruli were identified by the absence of Lotus tetragonolobus lectin and GFP in nephron tubules at the tubular pole (white arrows; also indicated by yellow arrows in panel A; Scale bars, 20μm) (C) Bar graph shows a significant increase in the number of atubular glomeruli in obstructed kidneys compared to sham-operated kidneys with a decrease following the release of obstruction. (D) qRT-PCR analysis for Six2 mRNA expression in 3 week age group animals revealed no significant differences (mean ± SE) between the sham (n=4) and animals that underwent obstruction (n=6) and release (n=5) surgeries (**P<0.01; NS-Non significant).

Article Snippet: Kidney sections were stained with biotinylated Lotus tetragonolobus lectin (1:50, catalog number: B1325, Vector Laboratories, Burlingame, CA, USA) for identification of proximal tubules and Lc3BII to detect autophagy in the tubular epithelium (1:200, Rabbit polyclonal, catalog number: 2775, Cell signaling).

Techniques: Quantitative RT-PCR, Expressing

Journal: BioMed Research International

Article Title: Less Vertebral Bone Mass after Treatment with Macitentan in Mice: A Pilot Study

doi: 10.1155/2019/2075968

Figure Lengend Snippet: ALP, OCN, and TRAP expression in the 5 th lumbar vertebral spongiosa Immunohistochemistry demonstrated fewer ALP(+) and OCN(+) cells (brown) but more TRAP(+) (red) cells in Treatment group compared to Control group (ALP: alkaline phosphatase, OCN: Osteocalcin, and TRAP: tartrate-resistant acid phosphatase).

Article Snippet: We incubated sections with primary antibodies to mouse Alkaline phosphatase (ALP, PA1004, Boster, Pleasanton, USA) and Osteocalcin (OCN, ab93876, Abcam, Cambridge, UK) overnight at 4°C.

Techniques: Expressing, Immunohistochemistry, Control

Journal: PLoS ONE

Article Title: Primary Cultures of Glomerular Parietal Epithelial Cells or Podocytes with Proven Origin

doi: 10.1371/journal.pone.0034907

Figure Lengend Snippet: A. Lysates of primary PECs or podocytes were subjected to immunoblotting for Pax2 expression. Primary PECs expressed significant amounts of Pax2, lower amounts were detected in primary podocytes (arrow). Lysates of endothelial cells (HUVEC or glomerular endothelial cells) were used as negative controls. B. After six passages, Pax2 was expressed in primary PECs and podocytes (arrow). Total kidney lysates were used as positive controls, endothelial cells were used as negative controls. C, C′ In immunofluorescent stainings on primary PECs and podocytes after six passages of culture, Pax2 was expressed in a nuclear fashion. Isotype-matched irrelevant antiserum was used as control (C′). D. Immunoblotting lysates of primary PECs or podocytes using ab sc192 showed that WT-1 is expressed in both cell types with significantly higher levels in primary podocytes (arrow). Lysates of endothelial cells were negative for WT1. E. After six passages, WT1 was expressed both in PECs and podocytes in a nuclear fashion (immunofluorescent staining using ab sc192). F. Parietal cells express low levels of WT1 also in vivo in mice with a mixed genetic background using ab sc846 (F, F′, arrowheads, immunohistological staining). For comparison, a strong expression of WT-1 was detected in podocytes (arrows). No WT-1 expression was noted elsewhere in the renal cortex, specifically not in proximal tubular cells (white arrowheads). G. Similarly, PECs expressed WT-1 also in other mouse genetic backgrounds (Sv129; WT-1/PAS staining) H, I. PECs (arrowheads) expressed low amounts of WT-1 also in normal Wistar rats (H) and humans (I). H′. Isotype matched controls were negative.

Article Snippet: Subsequently, the sections were incubated with rabbit polyclonal anti-WT1 antibody (180) (1∶100, sc846, Santa Cruz Biotechnology, used for immunohistochemistry on mouse kidney sections) or anti-WT1 antiserum (C-19) (1∶100, sc192, Santa Cruz Biotechnology, CA, used for immunoblotting, IF of primary cells and for immunohistochemistry on rat and human kidney sections).

Techniques: Western Blot, Expressing, Control, Staining, In Vivo, Comparison