Lipocalin 2 is essential for chronic kidney disease progression in mice and humans

Mechanisms of progression of chronic kidney disease (CKD), a major health care burden, are poorly understood. EGFR stimulates CKD progression, but the molecular networks that mediate its biological effects remain unknown. We recently showed that the severity of renal lesions after nephron reduction varied substantially among mouse strains and required activation of EGFR. Here, we utilized two mouse strains that react differently to nephron reduction — FVB/N mice, which develop severe renal lesions, and B6D2F1 mice, which are resistant to early deterioration — coupled with genome-wide expression to elucidate the molecular nature of CKD progression. Our results showed that lipocalin 2 (Lcn2, also known as neutrophil gelatinase–associated lipocalin [NGAL]), the most highly upregulated gene in the FVB/N strain, was not simply a marker of renal lesions, but an active player in disease progression. In fact, the severity of renal lesions was dramatically reduced in Lcn2^–/– mice. We discovered that Lcn2 expression increased upon EGFR activation and that Lcn2 mediated its mitogenic effect during renal deterioration. EGFR inhibition prevented Lcn2 upregulation and lesion development in mice expressing a dominant negative EGFR isoform, and hypoxia-inducible factor 1α (Hif-1α) was crucially required for EGFR-induced Lcn2 overexpression. Consistent with this, cell proliferation was dramatically reduced in Lcn2^–/– mice. These data are relevant to human CKD, as we found that LCN2 was increased particularly in patients who rapidly progressed to end-stage renal failure. Together our results uncover what we believe to be a novel function for Lcn2 and a critical pathway leading to progressive renal failure and cystogenesis.

Regardless of the initial insult, human chronic kidney disease (CKD) is characterized by progressive destruction of the renal parenchyma and the loss of functional nephrons, which ultimately lead to end-stage renal failure (ESRF). CKD represents a worldwide concern: in the United States, 102,567 patients began dialysis in 2003 (341 patients/million per year) (1), and similar rates were found in developing countries and in particular ethnic groups (2). However, these numbers are a small fraction of the millions of patients who are thought to have some degree of renal impairment. In the United States, the prevalence of chronically reduced kidney function is 11% of adults (3). Understanding the pathophysiology of CKD progression is, therefore, a key challenge for medical planning.

The mechanisms of CKD progression are poorly understood. It has been shown that reduction of the number of functional nephrons triggers molecular and cellular events promoting compensatory growth of the remaining ones (4). In some cases, this compensatory process becomes pathological, with the development of renal lesions and ESRF. Although the pathophysiology of compensation and progression is complex, unregulated proliferation of glomerular, tubular, and interstitial cells may promote the development of glomerulosclerosis, tubular cysts, and interstitial fibrosis (5–7). The molecular programs that control this cascade of events are largely unknown.

Attempts to dissect the molecular basis of CKD have been facilitated by the development of several experimental models of renal deterioration. Among these, the remnant kidney model is a mainstay, since nephron reduction characterizes the evolution of most human CKD. Consequently, this model recapitulates many features of human CKD, including hypertension, proteinuria, and glomerular and tubulointerstitial lesions. Over the last 50 years, this model has led to the discovery of critical pathways and, more importantly, to the design of therapeutic strategies to slow the progression of CKD, such as the widely clinically used renin-angiotensin inhibitors (8).

More recently, studies in various mouse strains have highlighted the importance of genetic factors in the evolution of experimental nephron reduction (9–11). We previously showed that the course and extent of renal lesions following nephron reduction vary significantly between two mouse strains: whereas FVB/N mice develop severe lesions, (C57BL/6 × DBA2)F1 (hereafter referred to as B6D2F1) mice undergo compensation alone (12). Moreover, we observed that the development of renal lesions paralleled the extent of cell proliferation (12). In fact, once the compensatory growth is achieved, a second wave of cell proliferation occurs only in the FVB/N strain. Hence, this model offers a powerful tool to unravel the transcriptional programs and the critical mediators that are selectively activated long after nephron reduction to drive deterioration of the remaining nephrons.

In the present study, we performed an unbiased profiling of gene expression in the kidneys of the FVB/N and B6D2F1 mouse strains, 2 months after nephron reduction, when renal lesions develop and the second wave of cell proliferation is ongoing. We identified a critical mediator of progressive renal failure, namely the carrier protein lipocalin 2 (Lcn2, or neutrophil gelatinase-associated lipocalin [NGAL], siderocalin, 24p3, uterocalin), and uncovered what we believe to be a novel function of Lcn2. Moreover, we elucidated a unique molecular pathway by which activation of EGFR results in Lcn2 expression, which then stimulates tubular proliferation and cystogenesis.

Gene profiling. To elucidate the molecular pathways of CKD progression, we performed unbiased profiling of gene expression in remnant kidneys of two mouse strains that react differently to nephron reduction. Using microarrays containing 5,579 cDNAs, we found 70 genes whose expression levels differed significantly 2 months after nephron reduction (P < 0.05). Among these transcripts, 44 were upregulated (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI42004DS1) and 26 were downregulated (Supplemental Table 2) in damaged FVB/N kidneys as compared with well-preserved kidneys from B6D2F1 mice. Grouping these results by gene ontology category, we observed a range of functions for the 70 transcripts, although many of the downregulated mRNAs (38%) regulated metabolic processes (Supplemental Figure 1). The gene undergoing maximal transcriptional induction (9.95-fold change, P = 0.008) in the FVB/N lesion-prone strain was Lcn2.

Lcn2 correlates with lesion progression in mouse and human with CKD. Lcn2 is a member of the lipocalin superfamily (13), a family of proteins that transport hydrophobic molecules such as retinoids, fatty acids, and organic chelators of iron (14). Real-time RT-PCR confirmed that Lcn2 mRNA increased 10-fold 2 months after nephron reduction in FVB/N but not in B6D2F1 mice, while it was almost undetectable in control animals (Supplemental Figure 2). In situ hybridization and immunohistochemistry corroborated these observations and showed a marked increase in Lcn2 mRNA and protein in damaged kidneys of FVB/N mice (Figure 1, A and B). Lcn2 was predominantly found in proximal tubules and in a few ascending limbs of Henle’s loops and collecting ducts (Supplemental Figure 3A). High magnification revealed that Lcn2 was mainly located in cytoplasmic granules at the subapical zone (Supplemental Figure 3B). By combining in situ hybridization and immunohistochemistry on serial sections, we found that a proportion of Lcn2 must have derived from the glomerular filtrate, since in some proximal tubules Lcn2 mRNA staining was negative while anti-Lcn2 staining was markedly positive (in situ^– and antibody⁺). On the other hand, the majority of proximal epithelia that had undergone dilation and cystic transformation displayed both Lcn2 message and antibody staining (in situ⁺ and antibody⁺), indicating not only endocytosis of filtered protein but ongoing local synthesis and secretion of Lcn2 (Supplemental Figure 3C). Renal Lcn2 mRNA (Figure 1C) and protein (Figure 1D) levels correlated with the intensity of tubular damage (r = 0.87, P < 0.001 and r = 0.74, P < 0.01 for mRNA and protein, respectively). In addition, we observed that renal Lcn2 protein content significantly correlated with Lcn2 excretion (r = 0.99, P < 0.01) (Figure 1E), suggesting that the kidney is the major source of urinary Lcn2.

Figure 1

Lcn2 is overexpressed after nephron reduction in mice and correlates with lesion progression. (A and B) Lcn2 expression evaluated by (A) in situ hybridization (original magnification, ×100) and (B) immunohistochemistry (×200) in kidneys from sham-operated (control) and 75% Nx FVB/N mice 2 months after surgery. Representative images for each group (n ≥ 6) are shown. (C) Correlation between renal Lcn2 mRNA expression evaluated by real-time RT-PCR and tubular lesion score in FVB/N mice, 2 months after Nx (r = 0.87, P < 0.001). (D) Correlation between renal Lcn2 protein expression evaluated by Western blot and tubular lesion score in FVB/N mice, 2 months after Nx (r = 0.74, P < 0.01). (E) Urinary Lcn2 excretion significantly correlates with renal Lcn2 protein expression. Left panel: Western blot of renal and urinary Lcn2 protein in control (C) and Nx FVB/N mice. Protein extracts from kidney of Nx Lcn2^–/– mice was used as negative control (C–). Right panel: Correlation between renal Lcn2 production and urinary Lcn2 excretion evaluated by Western blot (r = 0.99, P < 0.01).

A time course analysis of Lcn2 expression and renal morphology revealed that the increase in both Lcn2 mRNA and protein levels preceded the development of renal lesions 4 weeks after nephron reduction (Supplemental Figure 4). Moreover, we confirmed that Lcn2 upregulation was associated with the progressive development of tubular dilations in another experimental model of CKD, the juvenile cystic kidney (jck) mice (Figure 2, A and B). Of note, these mice develop a form of polycystic kidney disease similar to human autosomal dominant polycystic kidney disease (ADPKD) (15). Last, in patients with ADPKD, who are similar to our model in displaying severe and progressive tubular dilations, LCN2 immunoreactivity was markedly increased, particularly in cysts (Figure 2C). Urinary LCN2 was most prominent in fast progressors toward ESRF rather than in slow progressors (see Methods) (496 ± 146 vs. 152 ± 52 ng/mg creatinine, P < 0.01) (Figure 2D), and it inversely correlated with residual estimated glomerular filtration rate (eGFR) (r = –0.77, P < 0.0001) (Figure 2E) and microalbuminuria (r = 0.72, P < 0.0001). Interestingly, LCN2 expression was also increased in renal tubules of kidneys from patients with either congenital nephron deficit, a pathological condition very similar to nephron reduction, or IgA nephropathy, the most common primary form of CKD (Supplemental Figure 5). Our findings in mice and humans together with recent works (16–18) suggested that Lcn2 might participate in the pathogenesis of cysts and CKD.

Figure 2

Lcn2 is overexpressed in polycystic kidney disease in mice and humans and correlates with CKD progression. (A and B) Lcn2 expression evaluated by (A) immunohistochemistry and (B) Western blot in kidneys from wild-type (control) and jck mice, 3 weeks after birth. Representative images and blots for each group (n ≥ 4) are shown. Original magnification, ×200. (C) LCN2 staining in kidneys from controls (n = 9) and patients with ADPKD (n = 9). Original magnification, ×100. (D) Urinary LCN2 excretion in patients with slow progression (eGFR decline <4.5 ml/min/1.73 m² per year) as compared with fast progressors (eGFR decline >4.5 ml/min/1.73 m² per year) toward ESRF. (E) Urinary LCN2 excretion inversely correlates with eGFR in patients with ADPKD (r = –0.77, P < 0.0001). Data are mean ± SEM; n = 4–6 for mice and n = 87 for ADPKD patients. Mann-Whitney U test: *P < 0.05, control versus jck mice; **P < 0.01 slow versus fast progressors.

Lcn2 gene inactivation prevents lesion development and cyst formation. To determine the role of Lcn2 in progressive CKD, we performed 75% nephron reduction (Nx) in Lcn2^–/– mice (19). To this end, we first introduced the Lcn2 mutated allele in the lesion-prone (FVB/N) background. The Lcn2^–/– FVB/N mice reproduced normally and had no apparent phenotype under physiological conditions (data not shown). As expected, 2 months after nephron reduction, wild-type mice developed severe renal lesions, mainly consisting of glomerulosclerosis, tubular atrophy, and cystic dilation, mild interstitial fibrosis, and multifocal mononuclear cell infiltration (Figure 3A). However, the frequency and severity of renal lesions were dramatically reduced in Lcn2^–/– mice. Quantification showed that Lcn2^–/– mice had considerably fewer glomerular, tubular, and interstitial lesions as compared with wild-type littermates (Figure 3A). Notably, there were fewer tubular dilations and no cysts in Lcn2^–/– mice. Consistent with these findings, renal function was better preserved in Lcn2^–/– mice as compared with wild-type littermates, 2 months after nephron reduction. Serum creatinine and blood urea nitrogen were 5 ± 0.5, 18 ± 2.6, and 11 ± 0.6 μmol/l (P < 0.01) and 29 ± 1, 109 ± 15, and 65 ± 4 mg/dl (P < 0.01) in control, Nx Lcn2^+/+, and Nx Lcn2^–/– mice, respectively (Supplemental Figure 6, A and B). As expected, mean arterial blood pressure significantly increased in wild-type mice as compared with control animals (135 ± 7.5 and 116 ± 3.4 mmHg, P < 0.05) 2 months after nephron reduction. The increase was of the same magnitude in Lcn2^–/– mice (143 ± 2.2 mmHg). Development of renal lesions was accompanied by severe proteinuria in wild-type mice (6.16 ± 1.21 vs. 0.003 ± 0.001 mg/d in Nx and control mice, respectively, P < 0.001), whereas proteinuria was substantially decreased in Lcn2^–/– animals (3.30 ± 1.03 mg/d, P < 0.05) (Supplemental Figure 6C). Of note, Lcn2 inactivation did not change the course of nephron reduction in lesion-resistant C57BL/6 mice (Figure 4).

Figure 3

Lcn2 deficiency protects from the development of renal lesions. (A) Morphology and lesion scores of kidneys from control, 75% Nx Lcn2^+/+, and Lcn2^–/– FVB/N mice, 2 months after nephron reduction. Original magnification, ×200, ×600, and ×400 for tubular, glomerular, and interstitial lesions, respectively. (B) Morphology and cystic surface of kidneys from control, jck/Lcn2^+/+, and jck/Lcn2^–/– mice, 3 weeks after birth. Original magnification, ×200. Because no differences were detected between wild-type and mutant control mice, results for only 1 group are shown. Data are mean ± SEM; n = 4–6, 10–12, and 5–6 for control, Nx, and jck mice, respectively. ANOVA followed by Tukey-Kramer test: **P < 0.01, ***P < 0.001, control versus Nx or jck mice; ^##P < 0.01, ^###P < 0.001, Lcn2^+/+ versus Lcn2^–/–.

Figure 4

Lcn2 inactivation does not affect the response to nephron reduction in the lesion-resistant C57BL/6 mice. Morphology of kidneys from control, 75% Nx Lcn2^+/+, and Lcn2^–/– C57BL/6 mice, 2 months after surgery. Original magnification, ×200, ×600, and ×400 for tubular, glomerular, and interstitial lesions, respectively. Because no differences were detected between wild-type and mutant control mice, results for only 1 group are shown. n = 4–6 and 8–10 for control and Nx mice, respectively.

To confirm the beneficial effect of Lcn2 gene inactivation in renal deterioration and cyst formation, we bred Lcn2^–/– mice with the jck mice. Notably, the severity of renal lesions was substantially reduced in double mutant jck/Lcn2^–/– mice (Figure 3B). Quantification showed that the tubular dilation score was significantly lower in double mutant mice as compared with jck littermates 3 weeks after birth (Figure 3B). Collectively, these results demonstrated that Lcn2 is an effector of renal damage during CKD progression.

Iron accumulation does not account for progressive renal dysfunction. We next aimed at elucidating the mechanisms underlying the lesion-promoting effect of Lcn2. Lcn2 might act through iron mobilization (20). In fact, abnormal levels of iron accumulate in kidneys during CKD, where it may participate in the deterioration process (21, 22). Perls staining confirmed that iron content increased in damaged tubules 2 months after nephron reduction. However, iron accumulation was similar in remnant kidneys of Lcn2^–/– mice as compared with wild-type littermates (Supplemental Figure 7). More importantly, chelation of iron by desferrioxamine (DFO) (Figure 5A) unexpectedly worsened renal disease in FVB/N mice (Figure 5B). In particular, tubular dilations were more severe and diffuse in mice treated with DFO 2 months after nephron reduction. Notably, Lcn2 mRNA and protein expression were dramatically increased in kidneys of DFO-treated animals as compared with vehicle-treated counterparts (Figure 5, C and D). Proliferation of tubular cells was also significantly enhanced 2 months after nephron reduction in DFO-treated mice (Figure 5E). Hence, whereas iron deposited in the proximal tubules does not account for renal deterioration in our model, the experiments with DFO clearly show that manipulating Lcn2 levels is tightly correlated with hyperproliferation and progressive damage.

Figure 5

DFO increases Lcn2 expression and worsens renal lesions after nephron reduction. (A) Perls staining of kidneys from control and 75% Nx mice either treated or not with DFO. Original magnification, ×200. (B) Morphology of kidneys from control, DFO-treated control, Nx, and Nx DFO-treated mice, 2 months after surgery. Original magnification, ×200. (C and D) Lcn2 expression evaluated by (C) in situ hybridization (×100) and (D) immunohistochemistry (×200) in kidneys from control and Nx mice treated or not with DFO, 2 months after surgery. (E) DFO enhances cell proliferation after nephron reduction. PCNA staining (black arrows) and quantification of PI in kidneys from control and Nx mice, either treated or not with DFO. Original magnification, ×400. Because no differences were detected between wild-type and DFO-treated control mice, results for only 1 group are shown. Data are mean ± SEM; n = 4–10. ANOVA followed by Tukey-Kramer test: **P < 0.01, ***P < 0.001, control versus Nx mice; ^##P < 0.001, Nx versus Nx DFO-treated mice.

Lcn2 is a target of EGFR signaling. It is known that cell proliferation contributes to the development of renal lesions, and particularly to cystogenesis (23). Previous studies have suggested that Lcn2 can be induced by a number of growth factors that stimulate tubular cell proliferation (24). Among these, EGFR is of particular interest, since it is critical in the evolution of CKD (25). We therefore hypothesized that Lcn2 could act downstream of EGFR and mediate its growth effects. To investigate this hypothesis, we first treated renal tubular mIMCD-3 cells with EGF. Western blot analysis revealed that Lcn2 protein levels were markedly increased after addition of EGF (Figure 6A). Quantitative RT-PCR showed that Lcn2 mRNA levels paralleled the increase of the protein in EGF-treated cells (Figure 6B), indicating that the Lcn2 gene is transcriptionally regulated by EGFR. To validate these findings in vivo, we took advantage of a line of transgenic mice that overexpresses a dominant negative EGFR isoform (EGFR-M) selectively in proximal tubular cells (26). Inhibition of EGFR prevented the increase in Lcn2 mRNA in remnant kidneys of transgenic mice, 2 months after nephron reduction (Figure 6C). Consistently, the severity of renal lesions was substantially reduced in EGFR-M mice as compared with wild-type littermates (Figure 6D).

Figure 6

Lcn2 is a transcriptional target of EGFR. Lcn2 protein (A) and mRNA (B) expression in mIMCD-3 cells, 24 hours after EGF treatment. (C and D) Overexpression of a dominant negative EGFR isoform abolishes renal Lcn2 synthesis and prevents lesion development after nephron reduction. (C) Lcn2 mRNA expression visualized by in situ hybridization (original magnification, ×100) and (D) renal morphology (PAS, ×200) of kidneys from control and 75% Nx wild-type and EGFR-M mice, 2 months after surgery. Data are mean ± SEM; n = 2–3 and 6–10 for in vitro and in vivo experiments, respectively. Wilcoxon test: *P < 0.05, vehicle- versus EGF-treated cells.

Hif-1α is a critical intermediate between EGFR and Lcn2. We next tried to identify the factors that account for Lcn2 transcription upon EGFR activation. The observation that DFO dramatically stimulated Lcn2 expression after nephron reduction suggested that hypoxia-inducible factors (HIFs) might play a role. In fact, by inhibiting Fe²⁺-dependent prolyl hydroxylases, DFO stabilizes Hif-1α and Hif-2α (27). Interestingly, our results showed that Hif-1α protein levels increased in damaged kidneys of FVB/N mice 2 months after nephron reduction (Figure 7A). Since previous studies have shown that hypoxia may develop in damaged kidneys in CKD (28), we analyzed renal oxygenation 2 months after nephron reduction. Pimonidazole hypoxia probe failed to detect any positive tubules in remnant kidneys of FVB/N mice, with the exception of those located in the surgical scars (Figure 7B), demonstrating that hypoxia did not account for Hif-1α overexpression in our experimental model of CKD. In vitro experiments confirmed that EGF stimulated Hif-1α expression in renal mIMCD-3 cells. In fact, Hif-1α protein levels markedly increased upon EGF stimulation (Figure 7C). Hif1a mRNA levels, determined by real-time RT-PCR, changed neither in vivo after nephron reduction nor in vitro upon EGF treatment (data not shown), suggesting that Hif-1α is induced via a post-transcriptional mechanism. In addition, we observed that the increase in Hif-1α was specific, since the expression of Hif-2α changed neither in remnant kidneys nor in EGF-stimulated cells (Supplemental Figure 8). More importantly, we showed that Hif-1α silencing by siRNA partially inhibited Lcn2 expression both under basal conditions and, mainly, upon EGF stimulation in mIMCD-3 cell lines (Figure 7D), indicating that Hif-1α is a critical intermediate in EGFR-induced Lcn2 overexpression.

Figure 7

Hif-1α is a critical intermediate between EGFR and Lcn2. (A) Hif-1α protein expression and quantification in control and 75% Nx mice, 2 months after surgery. (B) Pimonidazole immunostaining in control and Nx mice, 2 months after surgery. Postischemic kidneys (2 hours clamping of renal pedicle) were used as positive hypoxic control. Original magnification, ×200. (C) Hif-1α protein expression and quantification in mIMCD-3 cells, 24 hours after EGF treatment. (D) Hif-1α silencing partially overcomes EGFR-induced Lcn2 upregulation. Hif-1α and Lcn2 protein expression in either mock- (Control) or Hif-1α siRNA–transfected (siHif-1α) mIMCD-3 cells, treated or not with EGF. Data are mean ± SEM; n = 2–6 and 6–10 for in vitro and in vivo experiments, respectively. Mann-Whitney U test: *P < 0.05, control versus Nx mice; Wilcoxon test: *P < 0.05, vehicle versus EGF-treated cells.

Lcn2 mediates the proliferative effect of EGFR. To next investigate whether Lcn2 mediated the mitogenic effect of EGFR, we established mIMCD-3 cell lines expressing Lcn2 shRNAs. Quantitative RT-PCR and Western blots revealed that Lcn2 mRNA was depleted by 96% (Figure 8A), whereas the protein was undetectable (Figure 8B) in Lcn2-silenced cells. Interestingly, Lcn2 silencing completely abolished cell proliferation after the addition of EGF at different experimental time points (Figure 8C). Similar results were obtained by using different clones and a second shRNA targeting Lcn2 (data not shown). We found consistent results in our mouse model in vivo. In fact, Lcn2 gene deletion prevented the increase in tubular cell proliferation 2 months after nephron reduction, as reflected in a significantly lower level of proliferating cell nuclear antigen–positive (PCNA-positive) tubular cells in remnant kidneys of Lcn2^–/– mice as compared with wild-type littermates (Figure 8D). Notably, Lcn2 gene inactivation did not inhibit the increase in cell proliferation in glomeruli (Figure 8E). These results were confirmed using an antibody directed against Ki-67, a protein selectively expressed in proliferating cells (Supplemental 9). Thus, it appears that Lcn2 is an essential mediator of the mitogenic effect of EGF in renal tubular cells.

Figure 8

Lcn2 mediates the proliferative effect of EGFR. (A and B) Stable Lcn2 shRNA (shLcn2) inhibits Lcn2 expression in mIMCD-3 cell lines. mRNA (A) and protein (B) expression in shLcn2 cells as compared with scrambled shRNA cells (Control). (C) Cell proliferation assay (left panel) and cell counting (right panel) in scrambled shRNA and Lcn2 shRNA mIMCD-3 cells, treated or not with EGF. (D) PCNA staining (black arrows) and quantification of tubular proliferation in kidneys from control, 75% Nx Lcn2^+/+, and Lcn2^–/– mice, 2 months after surgery. Original magnification, ×400. (E) PCNA staining (black arrow) and quantification of glomerular proliferation in kidneys from control, Nx Lcn2^+/+, and Lcn2^–/– mice, 2 months after surgery. Original magnification, ×600. Because no differences were detected between wild-type and mutant control mice, results for only 1 group are shown. Data are mean ± SEM; n = 3–4 and 6–10 for in vitro and in vivo experiments, respectively. ANOVA followed by Tukey-Kramer test: **P < 0.01, control versus shLcn2 cell lines; *P < 0.05, **P < 0.01, control versus Nx mice; ^#P < 0.05, Nx Lcn2^+/+ versus Nx Lcn2^–/– mice.

The dual effect of Lcn2 inactivation on apoptosis. Tubular growth reflects the balance between cell proliferation and cell loss by apoptosis. Both EGFR and Lcn2 have been implicated in the control of apoptosis (25, 29). TUNEL analysis revealed an increase in apoptosis in both tubules (Figure 9A) and glomeruli (Figure 9B) of wild-type mice as compared with control animals 2 months after nephron reduction. The number of TUNEL-positive cells was significantly reduced in Lcn2^–/– mice in both glomerular and tubular structures (Figure 9, A and B). However, Lcn2 silencing did not significantly affect the number of apoptotic tubular mIMCD-3 cells, regardless of the presence of EGF (Figure 9B).

Figure 9

Impact of Lcn2 inactivation on apoptosis. (A) TUNEL assay and quantification of TUNEL-positive tubular cells (white arrows) in kidneys from control, 75% Nx Lcn2^+/+, and Lcn2^–/– mice, 2 months after surgery. Original magnification, ×400. (B) TUNEL assay and quantification of TUNEL-positive glomerular cells (white arrows) in kidneys from control, Nx Lcn2^+/+, and Lcn2^–/– mice, 2 months after surgery. G, glomerulus. Original magnification, ×600. Because no differences were detected between wild-type and mutant control mice, results for only 1 group are shown. (C) Cell apoptosis quantification in scrambled shRNA and Lcn2 shRNA mIMCD-3 cells, 24 hours after EGF treatment. Data are mean ± SEM; n = 3 and 4 for in vitro and in vivo experiments, respectively. ANOVA followed by Tukey-Kramer test: **P < 0.01, ***P < 0.001, control versus Nx mice; ^##P < 0.01, ^###P < 0.001, Nx Lcn2^+/+ versus Nx Lcn2^–/– mice.

Unbiased profiling analyses offer a powerful approach to uncover critical mediators and dissect novel molecular networks of complex biological processes such as CKD progression. By combining experimental models of CKD in mice from different genetic backgrounds with microarray analyses, we have established a pivotal role for Lcn2 in regulating the progression of CKD and cyst formation. Furthermore, we have defined an important pathophysiological mechanism by which Lcn2 mediates the mitogenic effect of EGFR, consistent with its role in cell proliferation in cystogenesis. Inhibition of this pathway by Lcn2 gene inactivation or by the expression of a dominant negative EGFR isoform prevented lesion development in the transgenic mice. Conversely, overexpression of Lcn2 significantly correlated with hyperproliferation and CKD progression in both mice and humans. We have further identified Hif-1α as a crucial intermediate between EGFR and Lcn2 upregulation. Collectively, these results elucidate what we believe to be a novel molecular pathway of CKD progression and show that Lcn2 acts as a growth-promoting factor whose overexpression identifies patients with rapid CKD progression.

Lcn2, like all members of the lipocalin superfamily, binds hydrophobic ligands; the ligand is thought to define the function of the protein. Lcn2 binds enterochelin (20), parabactin (20), and carboxymycobactin (30), which are siderophores produced by bacteria for the purpose of binding iron. The siderophore-chelating property of Lcn2 renders it a bacteriostatic agent (20). Consistent with this finding, Lcn2 mutant mice have a profound defect in the defense against E. coli (19, 31) and Mycobacterium tuberculosis (32). Nonetheless, Lcn2 expression dramatically increases in several aseptic pathological conditions such as cancers (33), inflammatory diseases (34), and acute kidney injury (24), suggesting that Lcn2 may have other functions. To date, study of its noninfectious activities has focused on its effects on cell proliferation and/or apoptosis (24), but proof of these in a physiological setting in vivo has been lacking. Even in the case of acute kidney injury, a disease that is related to CKD, it remains unclear whether Lcn2 is a critical mediator of tubular changes (19). Hence, our work is the first clear demonstration to our knowledge that in vivo Lcn2 has a critical function in a pathological condition other than infection, namely serving as a growth regulator that mediates CKD progression. Our findings in mice and humans may be generally applicable to many forms of CKD, because Lcn2 is also expressed in obstructive uropathy (16), diabetic nephropathy (16), and in damaged kidneys of patients with IgA nephropathy (17) or HIV-associated nephropathy (18).

Our study shows that Lcn2, which is induced by EGFR, controls aberrant growth of renal tubules and cysts. Notably, we demonstrated that Lcn2 gene inactivation inhibited proliferation of tubular cells, which led to a marked decrease in cyst formation in mice. Consistently, we identified cystic tubular epithelia as the major source of Lcn2 production. These data suggest that Lcn2 might act as a tubulogenic factor that controls cell growth. This is supported by additional evidence: first, Lcn2 induced tubular development in in vitro assays in the rat (35); second, Lcn2 infusion favored tubular regeneration after ischemic injury in mice (36); third, high LCN2 levels were associated with a higher rate of cystic growth in humans (37). This property was not limited to mammalian cells: Lpr-1, a newly identified lipocalin family member, controlled unicellular tube development in the excretory system of Caenorhabditis elegans (38). Our data also suggest that Lcn2 may modulate tubular shape by controlling both cell proliferation and apoptosis. In fact, the beneficial effect of Lcn2 gene inactivation in mutant mice was accompanied by a decrease in tubular apoptosis, consistent with a previous observation in proximal tubule lacking Pkd1, a cystic disease–associated gene (39). However, this effect may be indirect, since Lcn2 silencing in vitro did not affect the number of apoptotic tubular cells. Whether the growth-promoting effect of Lcn2 is mediated by the binding of Lcn2 to a unique receptor, thus inducing a signaling cascade, or alternatively by iron mobilization, as suggested by the DFO experiments, remains to be elucidated.

In the present study we observed that Lcn2 gene inactivation protected from glomerulosclerosis and interstitial fibrosis after nephron reduction, despite the fact that Lcn2 was expressed only by tubules. The mechanism for this observation remains unknown. It may result from the perfusion and filtration of serum Lcn2 that we found by immunostaining in the tubules. Alternatively, since injuries to tubular cells, i.e., proteinuria, result in the expression of tubular cytokines and growth factors that ultimately lead to mesangial cell proliferation and matrix synthesis (40), it is tempting to hypothesize a crosstalk between tubular and surrounding renal cells. Studies in transgenic mice strongly support this idea. For example, it has been observed that mice that overexpressed VEGF selectively in tubules developed interstitial fibrosis and glomerular disease (41). And we have previously shown that the overexpression of a dominant negative isoform of EGFR in proximal tubules prevented the development of glomerular and interstitial lesions after nephron reduction (26). On the other hand, it has been shown that interstitial scarring resulted in the loss of microvessels, which, in turn, impacted the adjacent unaffected glomeruli (42). It is worthy of note that the synthesis of paracrine mediators may increase in proliferating tubular cells (43). Hence, we speculate that by inhibiting tubular cell proliferation, Lcn2 might protect glomeruli and interstitium from lesions development.

Activation of EGFR has been implicated in the evolution of CKD. Overexpression of an active EGFR form, the c-erb-B2 receptor, induces tubular hyperplasia and the development of renal cysts in transgenic mice (44). Conversely, expression of a dominant negative EGFR isoform inhibits cell proliferation, leading to reduced tubular dilations after nephron reduction (26). Other genetic and pharmacological approaches have confirmed the key role of EGFR and cell proliferation in polycystic kidney diseases (45, 46), and overexpression and mislocalization of EGFR were observed in cystic epithelia of jck mice (15). On the other hand, we have previously established that EGFR acts as a central integrator of angiotensin II pathway, a potent mediator of CKD (47). While the exact molecular networks that mediate the deleterious effect of EGFR during CKD have not been yet elucidated, our data point to Lcn2 as the crucial transcriptional target of EGFR during cyst formation and glomerulosclerosis. It is worthy of note that a very recent study showed that Lcn2 is also required for c-erb-B2 receptor signaling in breast cancer (48). In addition, our data show that Hif-1α is a critical intermediate between EGFR and Lcn2, consistent with the finding that Lcn2 is upregulated in most pathological conditions characterized by hypoxia, such as ischemia or cancers (24, 33). Whether Hif-1α is more extensively involved in the control of Lcn2 gene expression requires further investigations.

Clinical studies have suggested that urinary Lcn2 excretion might mark patients with the most severe clinical course (49), but whether Lcn2 is simply a marker of tubular damage or a key mediator of the deterioration process has been unknown. Our data now show a direct relationship between Lcn2 expression and disease progression and provide the first demonstration to our knowledge that Lcn2 is instrumental in CKD. CKD is a progressive disease, and there are many possible medical interventions over its course if the disease is recognized and treated in a timely manner. Current biomarkers of CKD progression, i.e., creatinine and albuminuria, have their limitations in terms of achieving this goal (50). An ideal biomarker should reflect tissue pathology, act as a critical component of disease, and be easily detectable by noninvasive approaches. By showing that Lcn2 unites these characteristics, we have provided strong evidence for the use of this molecule as a candidate biomarker of CKD progression.

In conclusion, we have uncovered what we believe to be a novel function of Lcn2 and highlighted its crucial role in the pathogenesis of progressive CKD. This is the first in vivo demonstration to our knowledge that Lcn2 acts as a growth regulator by mediating the mitogenic effect of EGFR signaling. Moreover, we have identified Lcn2 as one of the key effectors of renal damage and cystogenesis and one of the most promising biomarkers of CKD progression, worthy of study in large patient cohorts. We suspect that our findings will be critical in other pathological conditions that are also characterized by aberrant growth, such as cancers that demonstrate both EGFR activation and intensive Lcn2 expression (51, 52).

View Supplemental data

We are grateful to Sophie Berissi and Nicolas Cagnard for technical assistance. We are grateful to Genopole d’Evry, Laurent Beck, and Thierry Gilbert for microarrays, pSuppressor Retro vector, and in situ hybridization protocol, respectively. We thank Françoise Leviel for serum creatinine measurement by HPLC and David De Brauwere and the Biochemistry department of Necker Hospital for biological assays on patients’ urine. We thank Marco Pontoglio and Corinne Antignac for critical advice and Annette Bonnell for text revision. This work was supported by INSERM, Université Paris Descartes, AP-HP, Subvention AMGEM/Societé de Néphrologie, AURA (Paris), Agence Nationale Recherche (ANR-06-PHYSIO-029-01 grant), Fondation de la Recherche Médicale (DEQ20061107968 grant), AIRG, and Fondation Groupama. J. Barasch is supported by grants from the NIH and the March of Dimes.

Address correspondence to: Fabiola Terzi, INSERM U845, Team: Mechanisms and Therapeutic Strategies in Chronic Nephropathies, Hôpital Necker Enfants Malades, Tour Lavoisier, 6ème étage, 149 Rue de Sèvres, 75015 Paris, France. Phone: 33.1.44495245; Fax: 33.1.44490290; E-mail: fabiola.terzi@inserm.fr.

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

Reference information: J Clin Invest. 2010;120(11):4065–4076. doi:10.1172/JCI42004.

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