Macrophage-Derived Iron-Bound Lipocalin-2 Correlates with Renal Recovery Markers Following Sepsis-Induced Kidney Damage

Abstract

During the course of sepsis in critically ill patients, kidney dysfunction and damage are among the first events of a complex scenario toward multi-organ failure and patient death. Acute kidney injury triggers the release of lipocalin-2 (Lcn-2), which is involved in both renal injury and recovery. Taking into account that Lcn-2 binds and transports iron with high affinity, we aimed at clarifying if Lcn-2 fulfills different biological functions according to its iron-loading status and its cellular source during sepsis-induced kidney failure. We assessed Lcn-2 levels both in serum and in the supernatant of short-term cultured renal macrophages (MΦ) as well as renal tubular epithelial cells (TEC) isolated from either Sham-operated or cecal ligation and puncture (CLP)-treated septic mice. Total kidney iron content was analyzed by Perls' staining, while Lcn-2-bound iron in the supernatants of short-term cultured cells was determined by atomic absorption spectroscopy. Lcn-2 protein in serum was rapidly up-regulated at 6 h after sepsis induction and subsequently increased up to 48 h. Lcn-2-levels in the supernatant of TEC peaked at 24 h and were low at 48 h with no change in its iron-loading. In contrast, in renal MΦ Lcn-2 was low at 24 h, but increased at 48 h, where it mainly appeared in its iron-bound form. Whereas TEC-secreted, iron-free Lcn-2 was associated with renal injury, increased MΦ-released iron-bound Lcn-2 was linked to renal recovery. Therefore, we hypothesized that both the cellular source of Lcn-2 as well as its iron-load crucially adds to its biological function during sepsis-induced renal injury.

Keywords: CLP; iron; lipocalin-2; macrophages; renal tubular epithelial cells.

Figure 1

Cecal ligation and puncture (CLP)-induced sepsis promotes kidney damage. (a) Schematic overview of the experimental setup of the CLP-model. Sham-operated animals were used as controls. Arrows indicate the time-points of harvesting both blood (3, 6, 9, 24, and 48 h) and kidneys (24 and 48 h) for further processing. (b) Histologic analysis of tissue damage applying PAS staining. Pictures are representative of 5 animals in each group. (c,d) Analysis of kidney injury markers (c) blood urea nitrogen (BUN) (n = 5 animals per group; one-way ANOVA followed by Tukey’s multiple comparison test) and (d) SrCrea (n = 5 animals per group; one-way ANOVA followed by Tukey’s multiple comparison test). (e) Measurement of IL-6 in serum applying cytometric Bead Array (CBA) (n = 6 animals for 3, 6, and 9 h; n = 5 animals per group for 24 and 48 h; Kruskal-Wallis test followed by Dunn’s post-hoc test). (f) Analysis of Lcn-2 protein expression in serum using ELISA (n = 6 animals for 3, 6, and 9 h; n = 5 animals per group for 24 and 48 h; one-way ANOVA followed by Tukey’s multiple comparison test). * p < 0.05, ** p < 0.01.

Figure 2

Lcn-2 is expressed from different sources during CLP-induced renal injury progression. (a) Schematic representation for the isolation of both macrophages (MΦ) and tubular epithelial cells (TEC). (b–d) ELISA of Lcn-2 protein expression in the supernatant of short-term cultured and freshly isolated (b) peritoneal MΦ, (c) TEC, and (d) renal MΦ. n = 6 isolated primary peritoneal MΦ, TEC, and renal MΦ per group, *** p < 0.001; One-way ANOVA followed by Tukey’s multiple comparison test.

Figure 3

Renal injury markers correlate with Lcn-2 from TEC and peritoneal MΦ during sepsis. (a–e) Correlation between injury markers BUN (a,b,d) and SrCrea (c,e) with (a) each other or (b,c) Lcn-2 protein expression in TEC, or (d,e) with Lcn-2 expression from peritoneal MΦ (separated by time points after CLP treatment, 24 h and 48 h respectively). Both R² and the p-value for each correlation are indicated in the individual graph.

Figure 4

Iron distribution in renal tissue during CLP-induced sepsis. (a) Iron accumulation in renal tissue restricted to the lumen of kidney tubules (blue colored areas) at 24 h after CLP detected by Perls’ staining. At the 48 h time-point, only brownish-stained hemosiderin deposits can be observed, which are mainly found in infiltrates. Pictures are representative for 5 animals in each group. (b,c) Measurements of total iron amounts in the supernatants of short-term cultivated and freshly isolated (b) renal MΦ and (c) TEC using AAS. (d,e) Lcn-2 was immunoprecipitated in the supernatants of short-term cultivated and freshly isolated (d) renal MΦ and (e) TEC, and Lcn-2-bound iron was quantified by AAS. For (b–e) n = 6 isolated primary peritoneal MΦ, TEC, and renal MΦ per group, ** p < 0.01; (b–d): one-way ANOVA followed by Tukey’s multiple comparison test. (e): Kruskal-Wallis test followed by Dunn’s post-hoc test.

Figure 5

Renal recovery correlates with both total iron as well as Lcn-2-bound iron from renal MΦ. mRNA expression of (a) PCNA and (b) Stathmin relative to the housekeeping gene RPS27a. n = 5 animals per group. ** p < 0.01, *** p < 0.001; one-way ANOVA followed by Tukey’s multiple comparison test. (c–h) Correlation between (c,e,g) PCNA or (d,f,h) Stathmin with either (c,d) Lcn-2 protein expression, (e,f) Lcn-2-bound iron, or (g,h) total iron measured in the supernatant of renal MΦ at 48 h after CLP treatment (all values from this time-point were included; R² and p-values are depicted in the individual graph).

Figure 6

Patient data. Analysis of Lcn-2 expression in the blood of septic patients from publicly available datasets. Data are presented as box- and whisker plots of Lcn-2 expression in the serum. White and gray rectangles represent interquartile range, line in the middle of each rectangle represents the median value. Lines extending from the interquartile range mark the 5th and 95th percentile values, and the individual open circles represent values of each single patient. Analysis of (a) Lcn-2 expression in the whole blood transcriptome of survivors and non-survivors of sepsis from Parnell et al. (GSE54514) [22]. (b) Lcn-2 expression in PAXgene blood collected for leucocyte RNA isolation and gene expression analysis) from Sutherland et al. (GSE28750) [23]. *** p < 0.001.