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. 2016 Mar;16(3):808-20.
doi: 10.1111/ajt.13521. Epub 2015 Nov 23.

Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation

M I Ashraf  1   2 , H G Schwelberger  1 , K A Brendel  3 , J Feurle  1 , J Andrassy  4 , K Kotsch  1 , H Regele  3 , J Pratschke  1   2 , H T Maier  1 , F Aigner  1   2
Affiliations

Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation

M I Ashraf et al. Am J Transplant. 2016 Mar.

Abstract

Lipocalin 2 (Lcn2) is rapidly produced by damaged nephron epithelia and is one of the most promising new markers of renal injury, delayed graft function and acute allograft rejection (AR); however, the functional importance of Lcn2 in renal transplantation is largely unknown. To understand the role of Lcn2 in renal AR, kidneys from Balb/c mice were transplanted into C57Bl/6 mice and vice versa and analyzed for morphological and physiological outcomes of AR at posttransplantation days 3, 5, and 7. The allografts showed a steady increase in intensity of interstitial infiltration, tubulitis and periarterial aggregation of lymphocytes associated with a substantial elevation in serum levels of creatinine, urea and Lcn2. Perioperative administration of recombinant Lcn2:siderophore:Fe complex (rLcn2) to recipients resulted in functional and morphological amelioration of the allograft at day 7 almost as efficiently as daily immunosuppression with cyclosporine A (CsA). No significant differences were observed in various donor-recipient combinations (C57Bl/6 wild-type and Lcn2(-/-) , Balb/c donors and recipients). Histochemical analyses of the allografts showed reduced cell death in recipients treated with rLcn2 or CsA. These results demonstrate that Lcn2 plays an important role in reducing the extent of kidney AR and indicate the therapeutic potential of Lcn2 in transplantation.

Keywords: animal models: murine, rejection: acute; basic (laboratory) research; kidney transplantation; nephrology; science.

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Figures

Figure 1
Figure 1
Kinetics of mouse renal allograft rejection, function, cell death, and lipocalin 2 (Lcn2) expression.  Kidneys from Balb/c mice were transplanted into C57Bl/6 (Bl/6) mice. Allografts were harvested at posttransplant day 3, 5, or 7, stained with hematoxylin and eosin or periodic acid–Schiff stain and analyzed for histopathology by using Banff criteria. (A) Representative hematoxylin and eosin–stained images of control kidneys from sham‐operated mice (a) and allografts harvested at day 3 (b), day 5 (c), and day 7 (d) are shown. (B) The cumulative Banff score of histological lesions of the allografts harvested at given times is presented by a box plot. Function of the renal allografts was assessed by measurement of serum creatinine (C), serum urea (D), and serum Lcn2 (E) levels. Total kidney lysates of three animals in each of the indicated groups were used to determine activation (cleavage) of caspase 3 by immunoblotting. Representative immunoblot (F) and box plot (G) are shown. Lcn2 mRNA and protein expressions were investigated by quantitative polymerase chain reaction (H) and by immunohistochemistry (I), respectively, in the kidneys of sham‐operated mice (a) and allografts harvested at day 3 (b), day 5 (c), and day 7 (d). n = 6 (allografts harvested at day 3 or 5), n = 8 (allografts harvested at day 7). Scale bars = 100 μm. *p < 0.05, **p < 0.01.
Figure 2
Figure 2
Effect of lipocalin 2 (Lcn2) on histopathology of the mouse allografts.  (A) Representative images of hematoxylin and eosin–stained sections of the kidneys from sham‐operated mice (a), C57Bl/6 (Bl/6) kidney isografts (b) and the kidney allografts Bl/6 to Balb/c (c), Bl/6 Lcn2−/− to Balb/c (d), Balb/c to Bl/6 (e), Balb/c to Bl/6 Lcn2−/− (f), Balb/c to Bl/6 (treated with recombinant Lcn2:Sid:Fe complex (rLcn2; 250 μg, perioperatively) (g), Balb/c to Bl/6 (treated with cyclosporine A [10 mg/kg body weight, daily]) (h), harvested at posttransplant day 7 are shown. Black and white arrows highlight the examples of tubulitis and periarterial lymphocytic aggregates, respectively. (B) The cumulative Banff score of the prominent histopathological lesions of the kidney allografts is presented by a box plot (n = 6, except the groups Balb/c to Bl/6 and Balb/c to Bl/6 + rLcn2 n = 8). Scale bars = 100 μm. *p < 0.05, **p < 0.01.
Figure 3
Figure 3
Amelioration of renal allograft function by peritransplant application of recombinant lipocalin 2 (rLcn2 + Siderophore + Fe).  Kidneys were transplanted syngenically from C57Bl/6 (Bl/6) to Bl/6 mice and allogenically from Bl/6 WT or Lcn2−/− to Balb/c and Balb/c to Bl/6 WT or Lcn2−/− mice. Iso‐ and allograft function was assessed by measurement of creatinine (A), urea (B), and Lcn2 (C) levels in the serum of the recipients at posttransplant day 7 (n = 6, except the groups Balb/c to Bl/6 and Balb/c to Bl/6 + rLcn2 n = 8). *p < 0.05, **p < 0.01.
Figure 4
Figure 4
Effect of recombinant lipocalin 2:siderophore:Fe complex (rLcn2) on the expression of endogenous lipocalin 2 (Lcn2) in the renal allografts.  Kidneys transplanted from C57Bl/6 (Bl/6) to Bl/6, from Bl/6 to Balb/c and from Balb/c to Bl/6 mice were harvested at posttransplant day 7, and the expression pattern of Lcn2 mRNA and protein in the grafts was determined by quantitative polymerase chain reaction (A) and by immunohistochemistry (B). Representative box plot for mRNA expression (A) and images of the Lcn2 stained (B) sections of kidneys harvested from sham‐operated mice (a), Bl/6 kidney isografts (b), Bl/6 allografts in Balb/c recipients (c), Bl/6 Lcn2−/− allografts in Balb/c recipients (d), and Balb/c allografts in Bl/6 WT (e) and Lcn2−/− (f) recipients and the Bl/6 WT recipients treated with rLcn2 (250 μg perioperatively) (g) or cyclosporine A (10 mg/kg body weight, daily) (h) are shown (n = 6). Scale bars = 100 μm. *p < 0.05, **p < 0.01.
Figure 5
Figure 5
Recombinant lipocalin 2:siderophore:Fe complex (rLcn2) protects mouse kidney allografts from transplantation‐induced apoptosis.  Cell death in the C57Bl/6 (Bl/6) isografts, Balb/c kidney allografts transplanted into Bl/6 recipients, and Bl/6 allografts transplanted into Balb/c recipients was determined at posttransplant day 7 by terminal transferase‐mediated dUTP nick end labeling (TUNEL) staining (A, B) for DNA fragmentation and by immunohistochemical analysis (C, D) and immunoblotting (E, F) for caspase 3 activation. Representative images of the TUNEL (A) and activated caspase 3 (C) stained sections of kidneys harvested from sham‐operated mice (a), Bl/6 kidney isografts (b), Bl/6 allografts in Balb/c recipients (c), Bl/6 Lcn2−/− allografts in Balb/c recipients (d), and Balb/c allografts in Bl/6 WT (e) and Lcn2−/− (f) recipients and the Bl/6 WT recipients treated with rLcn2 (250 μg perioperatively) (g) or cyclosporine A (10 mg/kg body weight, daily) (h) are shown (A, C). Quantification of the TUNEL (B) and activated caspase 3 (D)–positive cells per high‐power field are presented by box plots. (E, F) Total kidney lysates of the indicated groups were used to determine activation (cleavage) of caspase 3 by immunoblotting. Representative immunoblots of three animals in each group are shown (E). Relative activation of caspase 3 was determined using glyceraldehyde 3‐phosphate dehydrogenase as a loading control and is presented by a box plot (F). Scale bars = 100 μm. n = 6. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
Immunophenotyping of cells infiltrating mouse renal allografts.  (A) Representative images of the CD3 stained sections of kidneys harvested from sham‐operated mice (a), C57Bl/6 (Bl/6) kidney isografts (b), Bl/6 allografts in Balb/c recipients (c), Bl/6 Lcn2−/− allografts in Balb/c recipients (d), and Balb/c allografts in Bl/6 WT (e) and Lcn2−/− (f) recipients and the Bl/6 WT recipients treated with recombinant lipocalin 2:siderophore:Fe complex (250 μg perioperatively) (g) or cyclosporine A (10 mg/kg body weight, daily) (h) are shown. Representative box plots of the quantification of CD3 (B), CD4 (C), CD8 (D), and Gr‐1 (E)–positive cells per high‐power field (HPF) are shown. n = 6. Scale bars = 100 μm. *p < 0.05, **p < 0.01.
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