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. 2018 Sep 6;3(17):e120196.
doi: 10.1172/jci.insight.120196.

Lipocalin-2 Derived From Adipose Tissue Mediates Aldosterone-Induced Renal Injury

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Free PMC article

Lipocalin-2 Derived From Adipose Tissue Mediates Aldosterone-Induced Renal Injury

Wai Yan Sun et al. JCI Insight. .
Free PMC article

Abstract

Lipocalin-2 is not only a sensitive biomarker, but it also contributes to the pathogenesis of renal injuries. The present study demonstrates that adipose tissue-derived lipocalin-2 plays a critical role in causing both chronic and acute renal injuries. Four-week treatment with aldosterone and high salt after uninephrectomy (ANS) significantly increased both circulating and urinary lipocalin-2, and it induced glomerular and tubular injuries in kidneys of WT mice. Despite increased renal expression of lcn2 and urinary excretion of lipocalin-2, mice with selective deletion of lcn2 alleles in adipose tissue (Adipo-LKO) are protected from ANS- or aldosterone-induced renal injuries. By contrast, selective deletion of lcn2 alleles in kidney did not prevent aldosterone- or ANS-induced renal injuries. Transplantation of fat pads from WT donors increased the sensitivity of mice with complete deletion of Lcn2 alleles (LKO) to aldosterone-induced renal injuries. Aldosterone promoted the urinary excretion of a human lipocalin-2 variant, R81E, in turn causing renal injuries in LKO mice. Chronic treatment with R81E triggered significant renal injuries in LKO, resembling those observed in WT mice following ANS challenge. Taken in conjunction, the present results demonstrate that lipocalin-2 derived from adipose tissue causes acute and chronic renal injuries, largely independent of local lcn2 expression in kidney.

Keywords: Adipose tissue; Inflammation; Mouse models; Nephrology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. WT mice treated with ANS (uninephrectomy, aldosterone 200 μg/kg/day, salt 1%) exhibited increased lipocalin-2 expressions in kidney and adipose tissues.
(A) WT and Adipo-LKO mice (12 weeks old) were subjected to sham or 4-week ANS treatment. Eplerenone (EPL) was given to the WT mice 1 week after starting the ANS treatment and continued for another 3 weeks. Serum and urine samples were collected before and after the treatment for measuring lipocalin-2 levels by ELISA. (B and C) The mRNA expression of lcn2 and the protein content of lipocalin-2 were examined in kidney and epididymal adipose tissue by qPCR (B) and ELISA (C). (D) The localization of lipocalin-2 protein in kidney and adipose tissue sections was examined by IHC using an antibody specifically recognizing murine lipocalin-2. Magnification, 400×. Scale bars: 20 μm. Data are presented as mean ± SEM; *P < 0.05 vs. WT sham controls; #P < 0.05 vs. WT ANS by ANOVA with the Bonferroni post hoc test (n = 6–8).
Figure 2
Figure 2. Deficiency of lipocalin-2 protects mice from ANS-induced glomerular injuries in the kidney.
(A) WT, Adipo-LKO, and LKO mice were subjected to sham or 4-week ANS challenge as in Figure 1. At the end of treatment, urine samples were collected for measuring albumin levels as described in Methods. (B) The mRNA expression of wt1 in kidney was evaluated by qPCR and presented as fold changes against WT sham controls. (C) Morphology of the glomeruli was examined in kidney tissue sections by both H&E (upper row) and PAS (lower row) staining. Magnification, 400×. Scale bar: 20 μm. (D) The glomerular area was quantified using ImageJ software and expressed as glomerular volume (D, left). The protein levels of synaptopodin were determined by Western blotting using the tissue lysates prepared from kidney samples (D, right). Data are shown as mean ± SEM; *P < 0.05 vs. corresponding sham controls; #P < 0.05 vs. WT ANS by Mann-Whitney nonparametric Student’s t test (n = 6–8). EPL, eplerenone.
Figure 3
Figure 3. Deficiency of lipocalin-2 protects mice from ANS-induced tubular injuries in the kidney.
(A) WT, Adipo-LKO, and LKO mice were subjected to sham or ANS treatment as in Figure 1. Morphological changes of renal tubules were examined by H&E (upper row) or PAS (lower row) staining. Images of proximal tubules in the cortex are shown. Magnification, 400×. Scale bar: 20 μm. (B) Quantification of tubular cell height and diameter was performed using ImageJ software. (C) Ten random images from 1 animal were examined to calculate the average data for comparison. The mRNA expression levels of clu and kim-1 in kidney samples were measured by qPCR and presented as fold changes against WT sham controls. (D) The protein amount of Kim-1 in the 24-hour urine samples was measured by ELISA for comparison. Data are shown as mean ± SEM; *P < 0.05 vs. WT sham controls; #P < 0.05 vs. WT ANS by Mann-Whitney nonparametric Student’s t test(n = 6–8). EPL, Eplerenone.
Figure 4
Figure 4. Deficiency of lipocalin-2 protects mice from ANS-induced interstitial fibrosis and macrophage infiltration in the kidney.
(A) After sham or ANS treatment as in Figure 1, kidney tissue sections were prepared from WT, Adipo-LKO, and LKO mice. PSR staining was performed to examine fibrotic renal damage. Magnification, 400×. Scale bar: 20 μm. (B) ImageJ software was used to quantify the interstitial volume. Ten random images from 1 animal were examined to calculate the percentage values (B, left). The mRNA expression levels of col1a1 and tgfb1 in kidney tissues were examined by qPCR and shown as fold changes against WT sham controls (B, middle and right). (C) Macrophage infiltration was evaluated in kidney tissue sections by IHC staining using an antibody recognizing F4/80. Magnification, 400×. Scale bar: 20 μm. (D) The mRNA expression levels of cd68 and ccl2 in kidney tissues were examined by qPCR and presented as fold changes against WT sham controls. Data are shown as mean ± SEM; *P < 0.05 vs. WT sham controls; #P < 0.05 vs. WT ANS by Mann-Whitney nonparametric Student’s t test (n = 6–8). EPL, eplerenone.
Figure 5
Figure 5. Lipocalin-2 of nonkidney source mediated aldosterone-induced acute renal injuries.
(A) Wt1CreERT2-LKO mice were treated with vehicle or tamoxifen (33 mg/kg/day, i.p. injection for 5 days). After treatment, lcn2 expressions in kidney and epididymal adipose tissue were examined by qPCR and presented as fold changes against WT controls. (B) The 24-hour urine and serum samples were collected to measure lipocalin-2 levels by ELISA. (C ) WT, Adipo-LKO, and Wt1CreGFP-LKO mice were injected with 1 dose of either vehicle or aldosterone (2 mg/kg; s.c.) as described in the Methods. The 24-hour urine samples were collected to compare urination (C, left). qPCR and ELISA were performed to measure the mRNA expressions of lcn2 and protein amounts of lipocalin-2 in kidney (C, middle and right). (D) Renal injury markers, including clu, kim-1, and wt1 were examined by qPCR and presented as fold changes against the WT vehicle controls. Data are shown as mean ± SEM; *P < 0.05 vs. WT vehicle groups; ‡P < 0.05 vs. Wt1CreERT2-LKO with vehicle treatment; #P < 0.05 vs. WT treated with aldosterone by Mann-Whitney nonparametric Student’s t test (n = 6).
Figure 6
Figure 6. ANS treatment caused chronic renal injuries in mice with selective deletion of lcn2 alleles in kidney.
(A) Wt1CreGFP-LKO mice were subjected to sham or ANS treatment as in Figure 1. Kidney, epididymal, and s.c. adipose tissues were collected after 4-week treatment. qPCR was performed to measure the lcn2 mRNA levels. The results are shown as fold changes against kidney samples of the sham control animals (A, left). ELISA was applied to examine the lipocalin-2 protein contents in kidney and epididymal adipose tissues (A, middle and right). (B) The wet weights of each kidney were recorded and calculated as percentage ratios against body weight for comparison. (C) PAS staining was used to evaluate the glomerular and tubular injuries in kidney of sham- or ANS-treated Wt1CreGFP-LKO mice (C, top). Magnification, 400×. Scale bar: 20 μm. The glomerular area, tubular cell height, and diameter were quantified by ImageJ software as described in Methods (C, bottom). (D) The gene expression levels of injury markers, including clu, kim-1, cd68, and ccl2 were examined by qPCR and presented as fold changes against the sham controls. Data are shown as mean ± SEM; *P < 0.05 vs. sham controls by Mann-Whitney nonparametric Student’s t test (n = 6).
Figure 7
Figure 7. Acute or chronic treatment with recombinant proteins of human lipocalin-2 caused renal injuries in LKO mice.
(A) LKO mice were administered with vehicle or aldosterone (2 mg/kg; s.c.), followed by an i.p. injection with 1 type of the recombinant human lipocalin-2 proteins (10 μg/mouse), including hLcn2, C87A, R81E, K125E, or K134E. After 24 hours, the lipocalin-2 protein amount was examined in both serum and 24-hour urine samples by ELISA. (B) The kidney tissues were collected for qPCR to examine the mRNA expressions of clu and kim-1. Fold changes were calculated for comparison. (C and D) Chronic treatment was performed by i.p. injection with vehicle or 1 type of the recombinant human lipocalin-2 proteins (10 μg/mouse/day), including hLcn2, C87A, R81E, and K134E. After 4-week treatment, kidney tissues were collected and subjected to qPCR for measuring the mRNA expressions of gene markers for cellular (clu and kim-1) and inflammatory (cd68 and ccl2) injuries (C), or fibrotic tissue damages (col1a1 and tgfb1) (D, left). The mRNA expression of steroid biosynthesis enzyme hsd3b1 was measured by qPCR and the aldosterone concentrations in urine samples were measured by ELISA (D, right). Data are shown as mean ± SEM; *P < 0.05 vs. corresponding vehicle groups; ‡P < 0.05 vs. hLcn2 aldosterone group by Mann-Whitney nonparametric Student’s t test (n = 6–8).
Figure 8
Figure 8. A working model proposing that adipose-derived lipocalin-2 mediates aldosterone-induced renal injuries.
Aldosterone, a key hormonal mediator of renal disease, induces the production of lipocalin-2 from adipose tissue, which subsequently causes cellular, inflammatory, and fibrotic damages in kidney. Mineralocorticoid receptor antagonists act mainly in adipose tissue to block lipocalin-2 production, in turn alleviating aldosterone-induced renal injuries.

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