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LCN2 expression evaluated by ( a ) immunohistochemistry (scale bar, 100 μm), ( b ) quantitative reverse transcriptase–PCR and ( c ) western blotting in kidneys in WT1 +/mut mice at 3 ( n =5), 4 ( n =6) and 6 ( n =7) weeks after birth and their control littermates ( n =5). LCN2 overexpression occurred before apparition of tubular lesions. ( d , e ) LCN2 protein (upper panel) and mRNA (lower panel) abundance in ( d ) mIMCD-3 cells and ( e ) in mouse renal primary cultured cells exposed to 1% albumin (Alb) for 24 h ( n =3). ( f ) Representative western blotting (upper panel) and quantification (lower panel) of LCN2 protein abundance in mIMCD-3 cells exposed to 1% albumin for 24 h, treated with NAC, Cd 2+ or Gd 3+ ( n =5). ( g ) LCN2 protein expression in mouse renal primary cultured cells exposed to 1% albumin or heat-denatured (denatured) 1% albumin for 24 h ( n =3). ( h ) CHOP and LCN2 protein, and ( i ) Lcn2 mRNA expression in mice injected with thapsigargin (Thap), tunicamycin (Tuni) or the vehicle (Ctrl). ( n =5 per group; scale bar, 100 μm; insert scale bar 10 μm). ( j ) LCN2 expression in mIMCD-3 cells exposed to Thap (left panel) or Tuni (right panel) for 24 h ( n =3). The different apparent size of LCN2 protein due to the inhibition of LCN2 glycosylation by Tuni is worth noting. ( k ) LCN2 protein (upper panel) and mRNA abundance (lower panel) in mIMCD-3 cells transiently transfected with different constructs (control, ATF4, ATF6, CHOP, unspliced XBP1 (XBP1u) and spliced XBP1 <t>(XBP1p)).</t> ( n =3). ( l ) Luciferase activity in mIMCD-3 cells co-transfected with different transcription factors and a Lcn2 promoter luciferase reporter ( n =3). ( m ) LCN2 protein and mRNA expression in wild-type ATF4 +/+ and ATF4 −/− MEFs exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). ( n ) LCN2 mRNA expression in ATF4 −/− MEFs transfected with empty vector (EV) or ATF4 construct and exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). Statistical analysis: one way analysis of variance followed by Tukey–Kramer test; Mann–Whitney test when only two groups are compared; * P <0.05, ** P <0.01, *** P <0.001 versus controls.
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LCN2 expression evaluated by ( a ) immunohistochemistry (scale bar, 100 μm), ( b ) quantitative reverse transcriptase–PCR and ( c ) western blotting in kidneys in WT1 +/mut mice at 3 ( n =5), 4 ( n =6) and 6 ( n =7) weeks after birth and their control littermates ( n =5). LCN2 overexpression occurred before apparition of tubular lesions. ( d , e ) LCN2 protein (upper panel) and mRNA (lower panel) abundance in ( d ) mIMCD-3 cells and ( e ) in mouse renal primary cultured cells exposed to 1% albumin (Alb) for 24 h ( n =3). ( f ) Representative western blotting (upper panel) and quantification (lower panel) of LCN2 protein abundance in mIMCD-3 cells exposed to 1% albumin for 24 h, treated with NAC, Cd 2+ or Gd 3+ ( n =5). ( g ) LCN2 protein expression in mouse renal primary cultured cells exposed to 1% albumin or heat-denatured (denatured) 1% albumin for 24 h ( n =3). ( h ) CHOP and LCN2 protein, and ( i ) Lcn2 mRNA expression in mice injected with thapsigargin (Thap), tunicamycin (Tuni) or the vehicle (Ctrl). ( n =5 per group; scale bar, 100 μm; insert scale bar 10 μm). ( j ) LCN2 expression in mIMCD-3 cells exposed to Thap (left panel) or Tuni (right panel) for 24 h ( n =3). The different apparent size of LCN2 protein due to the inhibition of LCN2 glycosylation by Tuni is worth noting. ( k ) LCN2 protein (upper panel) and mRNA abundance (lower panel) in mIMCD-3 cells transiently transfected with different constructs (control, ATF4, ATF6, CHOP, unspliced XBP1 (XBP1u) and spliced XBP1 (XBP1p)). ( n =3). ( l ) Luciferase activity in mIMCD-3 cells co-transfected with different transcription factors and a Lcn2 promoter luciferase reporter ( n =3). ( m ) LCN2 protein and mRNA expression in wild-type ATF4 +/+ and ATF4 −/− MEFs exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). ( n ) LCN2 mRNA expression in ATF4 −/− MEFs transfected with empty vector (EV) or ATF4 construct and exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). Statistical analysis: one way analysis of variance followed by Tukey–Kramer test; Mann–Whitney test when only two groups are compared; * P <0.05, ** P <0.01, *** P <0.001 versus controls.

Journal: Nature Communications

Article Title: Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2

doi: 10.1038/ncomms10330

Figure Lengend Snippet: LCN2 expression evaluated by ( a ) immunohistochemistry (scale bar, 100 μm), ( b ) quantitative reverse transcriptase–PCR and ( c ) western blotting in kidneys in WT1 +/mut mice at 3 ( n =5), 4 ( n =6) and 6 ( n =7) weeks after birth and their control littermates ( n =5). LCN2 overexpression occurred before apparition of tubular lesions. ( d , e ) LCN2 protein (upper panel) and mRNA (lower panel) abundance in ( d ) mIMCD-3 cells and ( e ) in mouse renal primary cultured cells exposed to 1% albumin (Alb) for 24 h ( n =3). ( f ) Representative western blotting (upper panel) and quantification (lower panel) of LCN2 protein abundance in mIMCD-3 cells exposed to 1% albumin for 24 h, treated with NAC, Cd 2+ or Gd 3+ ( n =5). ( g ) LCN2 protein expression in mouse renal primary cultured cells exposed to 1% albumin or heat-denatured (denatured) 1% albumin for 24 h ( n =3). ( h ) CHOP and LCN2 protein, and ( i ) Lcn2 mRNA expression in mice injected with thapsigargin (Thap), tunicamycin (Tuni) or the vehicle (Ctrl). ( n =5 per group; scale bar, 100 μm; insert scale bar 10 μm). ( j ) LCN2 expression in mIMCD-3 cells exposed to Thap (left panel) or Tuni (right panel) for 24 h ( n =3). The different apparent size of LCN2 protein due to the inhibition of LCN2 glycosylation by Tuni is worth noting. ( k ) LCN2 protein (upper panel) and mRNA abundance (lower panel) in mIMCD-3 cells transiently transfected with different constructs (control, ATF4, ATF6, CHOP, unspliced XBP1 (XBP1u) and spliced XBP1 (XBP1p)). ( n =3). ( l ) Luciferase activity in mIMCD-3 cells co-transfected with different transcription factors and a Lcn2 promoter luciferase reporter ( n =3). ( m ) LCN2 protein and mRNA expression in wild-type ATF4 +/+ and ATF4 −/− MEFs exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). ( n ) LCN2 mRNA expression in ATF4 −/− MEFs transfected with empty vector (EV) or ATF4 construct and exposed to 1% albumin or 0.5 μM thapsigargin (Th) for 24 h ( n =3). Statistical analysis: one way analysis of variance followed by Tukey–Kramer test; Mann–Whitney test when only two groups are compared; * P <0.05, ** P <0.01, *** P <0.001 versus controls.

Article Snippet: The following plasmids were used: ATF4 (Addgene Inc., ID 21845), ATF6 (Addgene Inc., ID 11975), CHOP (Addgene Inc., ID 21898), XBP1u (Addgene Inc., ID 21832) and XBP1p (Addgene Inc., ID 21833). pcDNA3.1 vector was used as a control.

Techniques: Expressing, Immunohistochemistry, Reverse Transcription, Western Blot, Control, Over Expression, Cell Culture, Quantitative Proteomics, Injection, Inhibition, Glycoproteomics, Transfection, Construct, Luciferase, Activity Assay, Plasmid Preparation, MANN-WHITNEY