Lipoxins Regulate the Early Growth Response–1 Network and Reverse Diabetic Kidney Disease

LXs attenuate DKD. (A) Structure of endogenous LXA4 and Benzo-LXA4 and study design overview. ApoE−/− mice were rendered diabetic by STZ, and a subgroup of diabetic and nondiabetic ApoE−/− mice were administered ethanol (0.1%), LXA4 (5 μg/kg), or Benzo-LXA4 (1.7 μg/kg) twice weekly ip from weeks 1 to 10 (10-week prevention study) or weeks 1 to 20 (20-week prevention study). (B and C) Paraffin-embedded kidney sections of 20-week diabetic and control ApoE−/− mice administered ethanol (0.1%), LXA4, or Benzo-LXA4 from weeks 1 to 20 were stained by periodic acid–Schiff stain. Quantification of the glomerulosclerosis injury index is shown in the bar graph as mean±SEM (n=8–10/group; *P<0.05;**P<0.01). (D) Gene expression analysis of markers of kidney damage in kidney cortex tissue isolated from 10-week diabetic and nondiabetic ApoE−/− mice administered vehicle, LXA4, or Benzo-LXA4. Expression was normalized to 18S for gene expression analysis (n=8–10, ±SEM; *P≤0.05 versus ApoE^−/−+vehicle; φP≤0.05 versus Diabetic ApoE^−/−+Vehicle). KO, knockout; qPCR, quantitative polymerase chain reaction.

LXs reverse established DKD. (A) Study design overview. ApoE^−/− mice were rendered diabetic by STZ and diabetes was allowed to progress for 10 weeks, after which a subgroup of diabetic and nondiabetic ApoE^−/− mice were administered ethanol (0.1%), LXA₄ (5 μg/kg), or Benzo-LXA₄ (1.7 μg/kg) twice weekly ip from weeks 10 to 16. (B and C) Paraffin-embedded kidney sections of 16-week intervention study diabetic and control ApoE^−/− mice administered ethanol (0.1%), LXA₄, or Benzo-LXA₄ from weeks 10 to 16 were stained by periodic acid–Schiff stain. Quantification of the glomerulosclerosis injury index is shown in the bar graph as mean±SEM (n=8–10/group; *P<0.05; **P<0.01; ***P<0.001). (D and E) Immunohistochemical staining of collagen IV in glomeruli of paraffin-embedded kidney sections of 16-week intervention study diabetic and control ApoE^−/− mice administered ethanol (0.1%), LXA₄, or Benzo-LXA₄ from weeks 10 to 16. Quantification of staining is shown in the bar graph as mean±SEM (n=8–10/group; *P<0.05). Gene expression analysis of markers of (F) kidney fibrosis and (G) inflammation in kidney cortex tissue isolated from 16-week diabetic and nondiabetic ApoE^−/− mice administered vehicle, LXA₄, or Benzo-LXA₄ from weeks 10 to 16. Expression was normalized to 18S for gene expression analysis (n=8–10, ±SEM; *P≤0.05 versus ApoE^−/−+vehicle; ^φP≤0.05 versus Diabetic ApoE^−/−+Vehicle). KO, knockout; qPCR, quantitative polymerase chain reaction.

Transcriptome profiling identifies the networks associated with kidney disease in the diabetic ApoE^−/− mouse model. (A) Venn diagram indicating number of significant differentially expressed transcripts (FDR P value <0.05) between diabetic and control ApoE^−/− mice administered ethanol (0.1%), LXA₄, or Benzo-LXA₄ from weeks 1 to 10. (B) Renal expression levels of fn1, col4a1, grem1, vcam1, cyp2d12, trp53inp1, sulf2, mgmt, cdkn1a, and eda2r genes in control and diabetic mice administered ethanol (0.1%) (n=5–6 per group; FDR P value <0.05 for all transcripts). (C) Upstream regulators expected to be increased or decreased between control and diabetic ApoE^−/− mice administered ethanol. Regulators were identified using the IPA regulation Z-score algorithm according to gene expression changes. A positive or negative Z-score value indicates that a function is predicted to be increased (red color) or decreased (blue color), respectively. Corresponding −Log₂ P values indicate whether there is a statistically significant overlap between the dataset genes and genes that are known to be regulated by the upstream regulator. All regulators represented on the graph are significantly enriched (Z-score ≥2 or ≤−2; P<0.05). GPCRs, G-protein coupled receptors; vs, versus.

Analysis of renal transcriptome responses identifies LX-regulated transcriptional networks. (A and B) Upstream regulator analysis of transcriptome data identifies regulators predicted to be changed between diabetic ApoE^−/− mice administered ethanol versus (A) LXA₄ or (B) Benzo-LXA₄ treatment from weeks 1 to 10. Regulators were identified using the IPA regulation Z-score algorithm according to gene expression changes. A positive or negative Z-score value indicates that a function is predicted to be increased (red color) or decreased (blue color), respectively. Corresponding −Log₂ P values indicate whether there is a statistically significant overlap between the dataset genes and genes that are known to be regulated by the upstream regulator. All regulators represented on the graph are significantly enriched (Z-score ≥2 or ≤−2; P<0.05). (C and D) Heatmaps of normalized gene expression indicating transcripts displaying significant differential expression (FDR P value <0.05) between diabetic mice administered ethanol (0.1%) versus LXA₄ or Benzo-LXA₄ from weeks 1 to 10. Expression levels range from blue (low expression) to red (high expression). (E) Box plots indicating expression of genes regulated by both LXA₄ and Benzo-LXA₄ (egr1, adamtsl3, ngef, lamb3, grem1, and nr4a1). Transcript abundance across all treatment group is shown (*FDR P value <0.05). ID, identifier.