Estrogen Receptor α Signaling Exacerbates Immune-Mediated Nephropathies through Alteration of Metabolic Activity

Abstract

Glomerulonephritis is one of the most serious manifestations of systemic lupus erythematous (SLE). Because SLE is ≥10 times more common in women, a role for estrogens in disease pathogenesis has long been suspected. Estrogen receptor α (ERα) is highly expressed in renal tissue. We asked whether ERα expression contributes to the development of immune-mediated nephropathies like in lupus nephritis. We tested the overall effects of estrogen receptors on the immune response by immunization with OVA and induction of chronic graft-versus-host disease in female ERα-knockout mice. We used nephrotoxic serum nephritis as a model of immune-mediated nephropathy. We investigated the influence of ERα on molecular pathways during nephritis by microarray analysis of glomerular extract gene expression. We performed RNA sequencing of lupus patient whole blood to determine common pathways in murine and human nephritis. Absence of ERα protects female mice from developing nephritis, despite the presence of immune complexes and the production of proinflammatory cytokines in the kidneys and normal humoral responses to immunization. Time-course microarray analysis of glomeruli during nephrotoxic serum nephritis revealed significant upregulation of genes related to PPAR-mediated lipid metabolism and downregulation of genes in the retinol metabolism in wild-type females compared with ERα-knockout females. Similarly, RNA sequencing of lupus patient blood revealed similar expression patterns of these same pathways. During nephritis, the altered activity of metabolic pathways, such as retinol metabolism, occurs downstream of ERα activation and is essential for the progression to end-stage renal failure.

Figure 1. Absence of ERα does not impair the humoral immune response

(A) 4 weeks after OVA injections, WT and ERαKO mice developed similar levels of anti-OVA antibodies. ERα+/+ represent WT littermates of ERα−/− mice. B6 represents WT non-littermates. Data are represented as Mean ± SEM of 10 mice per group. All p-values were >0.05 (t-test). (B) After induction of cGvHD, anti-dsDNA and anti-chromatin auto-antibody levels were similar between WT and ERαKO female mice. Data are represented as Mean ± SEM of 5 mice per group (representative experiment of two experiments with a total of 10 mice per group). All p-values were >0.05 (t-test).

Figure 2. NTN Nephritis is less severe in ERαKO mice

(A) After NTN induction, blood was collected via tail vein every 2 days and blood urea nitrogen (BUN) levels were measured to assess renal disease severity. Data are represented as Mean ± SEM of 10 mice per group (pooled from two experiments). Mann Whitney-U was statistically significant at day 14 (p <0.05). (B) Renal pathology scoring was done on kidneys harvested on day 14 to assess severity of renal damage. Damage was assessed by three parameters: GN (glomerulonephritis), INT (interstitial infiltrate), and V (vasculopathy). Data are represented as Mean ± SEM of 5 mice per group. Mann Whitney-U showed statistical significance for all three parameters (p <0.05). Representative (C) H&E staining and (D) trichrome staining for fibrosis of mouse kidneys treated with NTN after 2 weeks is shown. Images taken at 400× magnification, white size bar = 50μm.

Figure 3. Mice injected with NTS have IgG and C3 deposition in glomeruli

(A) WT and ERαKO female mice demonstrated IgG deposits in the glomeruli. (B) WT and ERαKO mice show complement C3 deposition in the glomeruli. Fluorescence intensities were measured using Image J software. Data are represented as mean ± SEM of 10 mice per group. T-test used for statistical analysis showed no difference between groups.

Figure 4. ERα must be absent in both immune and renal cells to confer protection during NTN

(A) Blood urea nitrogen (BUN) of chimeric mice over 2 weeks after NTN induction is shown. Data are represented as Mean ± SEM of 5 mice per group (*p<0.05). (B) Proteinuria (mg/dL) measured over 2 weeks after NTN induction. T-test was used for statistical analysis of proteinuria, **p<0.01. (C) Renal scoring focused on the severity of glomerulonephritis, interstitial damage and infiltration. Mann-Whitney U test was used for statistical analysis of pathology scores, * p= <0.05. Data are represented as Mean ± SEM of 5 mice per group. (D) Chimeric WT and ERαKO female mice demonstrate similar levels of IgG deposition in the glomeruli. (E) Chimeric WT and ERαKO mice show similar levels of complement C3 deposition in the glomeruli. Fluorescence intensities were measured using Image J software. Data are represented as mean ± SEM of 5 mice per group. T-test used for statistical analysis shows no difference.

Figure 5. WT and ERαKO females undergo different gene expression changes over the course of NTN

(A) Gene expression changes over the course of NTN were compared for WT and ERαKO females using Transcriptome Analysis Console software by Affymetrix. The number of significantly differentially expressed genes for each comparison are located beside their respective arrows. The numbers within parentheses represent the number of genes that are up- and down-regulated. (B) Differential gene expression comparisons were completed between WT and ERαKO females for each of the underlined time points using Transcriptome Analysis Console software by Affymetrix. Significance measured by one-way ANOVA (p<0.05). The number of changing genes which are similar or dissimilar between WT and ERαKO females are represented in the Venn diagram

Figure 6. WT and ERαKO females utilize different pathways during NTN progression

Pathway association diagram displaying similarities in glomerular pathways between WT and ERαKO females over the course of NTN. t₀= day 0, t₁= 18 hours, t₂= day 6, t₃= day 14. Grey circles represent pathways that were involved during the time frame to which it is connected. Number of genes and pathways associated with WT or ERαKO females at each timepoint were determined using Transcriptome Analysis Software (TAC) by Affymetrix. Gene significance determined by ANOVA, p< 0.05.

Figure 7. Expression of 457 genes significantly differs between WT and ERαKO females at 6 days post-NTN

Differentially gene expression between WT and ERαKO females at 6 days post-NTN was calculated using TAC software, ANOVA p<0.05. Heat map of significant genes was generated by Pearson correlation in mEV software.

Figure 8. WT females have altered expression of lipid metabolism genes compared to ERαKO during NTN

(A) Heat map of PPAR signaling and retinol metabolism-associated genes found to be differentially expressed in WT females at 6 days. Significance measured by T-test (p<0.05) using MeV software. (B) qPCR validation for PPARα pathway-associated genes, Fabp1 and Cyp4a14. Data represented as ΔΔCT. Significance by t-test, ***p<0.001. (C) qPCR validation for increased Tgfb1 expression in WT females on day 6. Data represented as ΔΔCT. Significance by t-test, *p<0.05. (D) Kidney sections from WT and ERαKO females at each of the indicated time-points were stained for mesangial cells with smooth-muscle actin (SMA) and for podocytes with synaptopodin. DAPI was used to identify nuclei. The area occupied by the SMA staining was measured using Image-J. Data are represented as mean ± SEM of 5 mice per group. Significance determined by T-test, **p<0.01. (E) Representative oil red-O staining of kidney sections from WT and ERαKO females at day 14 post-NTN induction. Images taken at 400× magnification, black size bar = 50μm.

Fig. 9. PPAR- and RXR-mediated transcription can be influenced by Esr1

(A) Protein interaction map displaying Ppara-mediated lipid activation occurs downstream of ERα (Esr1). (B) Protein interaction map displaying Rxr-mediated lipid activation occurs downstream of ERα (Esr1). Interaction maps were generated using STRING protein-protein interactions software. Thickness of lines connecting proteins is representative of confidence in those interactions based on literature findings.

Fig. 10. Gene expression of whole blood reveals alterations in lipid metabolism pathways in SLE patients compared to healthy controls

Heat map displaying increased expression of PPAR signaling genes in SLE patients compared to healthy controls, while genes involved in retinol metabolism are down-regulated in patients with SLE. Significance measured by two-way T-test (p<0.05) using MeV software.