Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2

Abstract

In chronic kidney disease (CKD), proteinuria results in severe tubulointerstitial lesions, which ultimately lead to end-stage renal disease. Here we identify 4-phenylbutyric acid (PBA), a chemical chaperone already used in humans, as a novel therapeutic strategy capable to counteract the toxic effect of proteinuria. Mechanistically, we show that albumin induces tubular unfolded protein response via cytosolic calcium rise, which leads to tubular apoptosis by Lipocalin 2 (LCN2) modulation through ATF4. Consistent with the key role of LCN2 in CKD progression, Lcn2 gene inactivation decreases ER stress-induced apoptosis, tubulointerstitial lesions and mortality in proteinuric mice. More importantly, the inhibition of this pathway by PBA protects kidneys from morphological and functional degradation in proteinuric mice. These results are relevant to human CKD, as LCN2 is increased in proteinuric patients. In conclusion, our study identifies a therapeutic strategy susceptible to improve the benefit of RAS inhibitors in proteinuria-induced CKD progression.

ARTICLE

Received 28 Apr 2015 | Accepted 30 Nov 2015 | Published 20 Jan 2015

Khalil El Karoui1,2, Amandine Viau1, Olivier Dellis3, Alessia Bagattin4, Clment Nguyen1, William Baron1, Martine Burtin1, Mlanie Broueilh1, Laurence Heidet5, Graldine Mollet5, Anne Druilhe1, Corinne Antignac5, Bertrand Knebelmann1, Grard Friedlander1,2, Frank Bienaim1,2, Morgan Gallazzini1,* & Fabiola Terzi1,*

In chronic kidney disease (CKD), proteinuria results in severe tubulointerstitial lesions, which ultimately lead to end-stage renal disease. Here we identify 4-phenylbutyric acid (PBA), a chemical chaperone already used in humans, as a novel therapeutic strategy capable to counteract the toxic effect of proteinuria. Mechanistically, we show that albumin induces tubular unfolded protein response via cytosolic calcium rise, which leads to tubular apoptosis by Lipocalin 2 (LCN2) modulation through ATF4. Consistent with the key role of LCN2 in CKD progression, Lcn2 gene inactivation decreases ER stress-induced apoptosis, tubulointerstitial lesions and mortality in proteinuric mice. More importantly, the inhibition of this pathway by PBA protects kidneys from morphological and functional degradation in proteinuric mice. These results are relevant to human CKD, as LCN2 is increased in proteinuric patients. In conclusion, our study identies a therapeutic strategy susceptible to improve the benet of RAS inhibitors in proteinuria-induced CKD progression.

DOI: 10.1038/ncomms10330 OPEN

Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2

1 Mechanisms and Therapeutic Strategies of Chronic Kidney Disease, INSERM U1151CNRS UMR 8253, Universit Paris Descartes, Institut Necker Enfants Malades, Dpartement ) Croissance et Signalisation *, Hpital Necker Enfants Malades, 149 Rue de Svres, Paris 75015, France. 2 Service dExplorations

Fonctionnelles, Assistance Publique-Hpitaux de Paris, Hpital Necker Enfants Malades, 149 Rue de Svres, Paris 75015, France. 3 UMR-S 757 INSERM, Universit Paris Sud 11, Rue des Adles, Orsay 91405, France. 4 INSERM U1016, CNRS UMR 8104, Universit Paris Descartes, Institut Cochin, Paris, France.

5 INSERM U1163, Universit Paris Descartes, Institut Imagine, Hpital Necker Enfants Malades, 149 Rue de Svres, Paris 75015, France. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to F.T. (email: mailto:[email protected]

Web End [email protected] ).

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Chronic kidney disease (CKD) is characterized by a progressive decline in renal function to end-stage renal disease that can occur, irrespective of the cause of

renal damage, when a critical number of functional nephrons has been lost. CKD is now a worldwide concern: 410% of the US residents suffer from this disease1 and similar rates are found in developing countries2. Understanding the pathophysiology of CKD progression is therefore a critical challenge for public health.

Most CKD are characterized by abnormalities of the glomerular ltration barrier, leading to increased glomerular permeability and abnormal ltration of macromolecules, such as albumin. Convergent evidences from clinical and experimental studies indicate that albuminuria and proteinuria are not simply markers of CKD progression, but active players in the evolution of the disease3. Mechanistically, it has been proposed that the proteins that escape into the glomerular ltrate have a toxic effect on tubular cells and that, once damaged, tubular cells lead to the development of interstitial brosis and inammation46. Remarkably, several clinical studies have shown that the decline of renal function correlates more closely with the tubulointerstitial lesions than with the glomerular damage7. Hence, over the last 20 years, researchers have focused their efforts on the discovery of the molecular links between proteinuria and the development of tubulointerstitial lesions. Several candidates have been identied, that is, endothelin-1, MCP-1, RANTES or complement components5,6,8. However, so far, this did not lead to the development of novel therapeutic strategies susceptible to slow down CKD progression in humans. The only available strategy to counteract the deleterious effect of proteinuria is the reninangiotensin system (RAS) inhibition, which reduces the leakage of proteins from the glomerular ltration barrier9. However, a residual proteinuria is observed in most patients under RAS blockade and the nephroprotective effect of RAS inhibitors may decline over time1012. Moreover, attempts to further increase RAS blockade in proteinuric patients have revealed a high rate of severe side effects, such as hypotension or life-threatening hyperkalemia13. Thus, there is an urgent need to identify novel therapeutic targets susceptible to add benet to RAS inhibition by preventing the toxic effect of residual proteinuria.

The endoplasmic reticulum (ER) has emerged as a signalling platform that responds to various cellular stresses by inducing a coordinated response, the unfolded protein response (UPR)14. During UPR, inositol-requiring enzyme 1a (IRE1a) promotes the phosphorylation of c-JUN and the specic splicing of UPR transcription factor X-box binding protein 1 (XBP1). Besides, protein kinase R-like kinase (PERK) phosphorylates eukaryotic translation-initiation factor 2a (eIF2a): this reduces general translation but promotes translation of activating transcription factor 4 (ATF4), which activates the CCAAT/enhancer-binding protein homologous protein (CHOP). If this adaptive response cannot overcome ER stress, it triggers apoptotic cell death. UPR and ER stress can be targeted by various therapeutic compounds either Food and Drug Administration approved or in preclinical studies15. Interestingly, a few studies showed that UPR is activated in tubular cells exposed to albumin1618, but the pathophysiological role of such activation remains unknown17,18.

Here we combined in vivo and in vitro studies to dissect a novel molecular pathway in which albumin leads, via calcium-dependent ER stress activation, to Lipocalin 2 (LCN2) over-expression, which in turn triggers tubular cell apoptosis and renal lesions. More importantly, we showed that inhibiting ER stress with 4-phenylbutyric acid (PBA) prevents proteinuria-induced renal lesions and LCN2 overexpression.

ResultsProteinuria leads to UPR activation in vitro and in vivo. To investigate whether ER stress is a common response to protein overload, we rst studied UPR activation in several complementary models of genetic and acquired glomerular diseases. We observed that both PERK and IRE1 pathways were activated in proteinuric WT1 /mut mice, which carry a mutation of Wt1 (ref. 19), a gene encoding an essential transcription factor of podocyte homeostasis (Fig. 1a) and Nphs2D/ mice, which display dramatic podocin downregulation on tamoxifen exposure20 (Supplementary Fig. 1a). The activation of these pathways was conrmed in mice receiving doxorubicin (doxo) or serum bovine albumin, two widely used models of acquired proteinuric nephropathy21 (Supplementary Fig. 1b,c). To ascertain that UPR was the direct consequence of proteinuria, we incubated renal tubular cells with albumin. Immunouorescence experiments using albuminuorescein isothiocyanate showed the progressive (from 1 h) accumulation of albumin in endocytotic vesicles of mouse inner medulla collecting duct (mIMCD-3) cells, followed by a later co-localization of albumin with lysosomal markers (LAMP2; Fig. 1b). Nevertheless, albumin exposure was associated with phosphorylation of PERK, eIF2a and c-JUN from 5 min of incubation, which was followed by the subsequent overexpression of ATF4 and CHOP, and XBP1 splicing (Fig. 1ce).These results demonstrate that the activation of PERK and IRE1 pathways occurs before albumin internalization.

Albumin-induced UPR activation is calcium dependent. We then investigated the mechanisms by which albumin induces UPR in tubular cells. Previous studies have demonstrated that albumin exposure leads to reactive oxygen species (ROS) production and intracellular calcium increase, two biological processes known to induce ER stress22,23. Interestingly, within 30 min of albumin exposure, whereas we failed to detect any increase of ROS (Fig. 1f), we observed a rapid increase of cytosolic calcium concentration ([Ca2 ]cyt; Fig. 1g).

Thapsigargin, a known inhibitor of the sarco/ER Ca2 -ATPase that leads to ER Ca2 release, and thereafter to a

Ca2 inux called store-operated calcium entry (SOCE), induced a very similar [Ca2 ]cyt rise (Fig. 1g). Addition of albumin after thapsigargin stimulated a slight second [Ca2 ]cyt increase, suggesting that albumin and thapsigargin share the same pathway to mobilize Ca2 . To conrm the role of the

SOCE in this mobilization, we repeated the same experiments in the presence of two SOCE-specic inhibitors gadolinium (Gd3 ) and SKF96365 (ref. 24). As shown in Fig. 1h, Gd3 and SKF96365 partially abolished the albumin-induced [Ca2 ]cyt rise, demonstrating that albumin induces the rise of [Ca2 ]cyt by triggering Ca2 release from the ER, which in turn activates a SOCE. We then used 1,2-bis(oaminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), a calcium-specic aminopolycarboxylic acid, to chelate the extracellular Ca2 . When BAPTA was added at the same time as albumin, chelation of extracellular Ca2 fully prevented the [Ca2 ]cyt rise (Supplementary Fig. 2a), demonstrating that in the absence of free Ca2 albumin can induce neither the

Ca2 release from the ER nor the subsequent SOCE. However, when we added BAPTA 5 min after albumin exposure, albumin triggered a rapid increase of [Ca2 ]cyt; however, as soon as

BAPTA was added, [Ca2 ]cyt started to slowly decrease (Supplementary Fig. 2a), demonstrating that albumin required extracellular Ca2 to sustain the rise of [Ca2 ]cyt. Depletion of

Ca2 from the ER compartment is known to induce ER stress and UPR activation. To demonstrate that albumin-induced

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Figure 1 | UPR is induced in tubular cells exposed to albumin in vivo and in vitro. (a) p-eIF2a, ATF4, CHOP and p-c-JUN evaluated in kidneys of WT1/mut mice (n 10) and their wild-type littermates (Control, n 6) 6 weeks after birth (representative panels scale bar, 100 mm; insert scale bar,

10 mm). (b) Internalization of extracellular albumin in mIMCD-3 cells (exposed to 1% albuminuorescein isothiocyanate (Alb-FITC) for 30 min and 1, 3 and 24 h) evaluated using anti-LAMP2 antibodies (red), a marker of the lysosomal compartment. Pictures show endocytosis within 30 min to 1 h of the albumin and its targeting to the lysosomal compartment after 24 h of treatment (n 3; scale bar, 20 mm). (c) pPERK, p-eIF2a and p-c-JUN expression during time-

course exposition of mIMCD-3 cells to 1% albumin (n 5). (d) ATF4 and CHOP expression in mIMCD-3 cells exposed to 1% albumin (Alb) for 24 h (upper

panel) and splicing of XBP1 evaluated by reverse transcriptasePCR (down) in mIMCD-3 cells exposed to 1% albumin for 3 h (lower panel; n 3).

(e) Quantications of ATF4 and CHOP protein abundance and mRNA of CHOP or spliced XBP1 in mIMCD-3 cells exposed to 1% albumin for 24 or 3 h, respectively (n 6 and n 5 for protein and mRNA quantications, respectively). (f) Measurement by dichlorodihydrouorescein diacetate (DCFh-DA) of

intracellular ROS generation in mIMCD-3 cells exposed for 30 min to 1% albumin or 100 mM H2O2 (n 3). (g) Measurement of intracellular calcium in

mIMCD-3 cells exposed to 1% albumin (black trace) or rst exposed to 1.0 mM thapsigargin (red trace, red arrow 1) and then to 1% albumin (red trace, red arrow 2; n 3). (h) Measurement of intracellular calcium in mIMCD-3 cells exposed to 1% albumin for indicated times in control conditions (black trace),

with gadolinium (Gd3 , green trace) or with SKF96365 (red trace) (n 3). (i) Representative western blotting (upper panel) and quantication (lower

panel) of pPERK evaluated in mIMCD-3 cells exposed to 1% albumin for 30 min in the presence or the absence of NAC, Cd2 or Gd3 (n 5). Data are

means.e.m. Statistical analysis: one way analysis of variance followed by TukeyKramer test; MannWhitney test when only two groups are compared; *Po0.05, **Po0.01, ***Po0.001 versus controls.

Ca2 release is responsible for the observed UPR activation, we treated tubular cells exposed to albumin with either N-acetylcystein (NAC), an inhibitor of ROS generation, or cadmium (Cd2 ) and Gd3 , two inhibitors of Ca2 transporters.

Consistent with the observation that ROS did not increase after 30 min of albumin exposure, NAC treatment affected neither PERK nor eIF2a phosphorylation (Fig. 1i and

Supplementary Fig. 2b). In contrast, Cd2 and Gd3 administration completely prevented UPR activation (Fig. 1i

and Supplementary Fig. 2b), indicating that intracellular Ca2 is the critical intermediate between albumin and UPR activation in this pathological setting.

Albumin-induced UPR leads to LCN2 overexpression. As all these results indicated that albumin leakage from damage glomeruli leads to UPR in tubular cells, we next aimed at identifying the potential targets of UPR activation during

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tubulointerstitial lesion development. Among the possible candidates, we focused on LCN2 (also known as NGAL, 24p3, siderocalin or uterocalin), a small, secreted iron-transporting protein, as (i) it has been recently reported that UPR activation is associated with LCN2 overexpression in cancer cells25 and (ii) we previously showed that LCN2 is critically involved in the

progressive deterioration of tubules following nephron reduction26. We observed that Lcn2 messenger RNA and protein expression progressively increased in proteinuric WT1 /mut mice as compared with control littermates (Fig. 2ac). Co-localization experiments revealed that LCN2 was expressed in tubules (mainly proximal tubules, Henles loops

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Figure 2 | Albumin-induced UPR leads to LCN2 overexpression through ATF4. LCN2 expression evaluated by (a) immunohistochemistry (scale bar, 100 mm), (b) quantitative reverse transcriptasePCR and (c) western blotting in kidneys in WT1 /mut mice at 3 (n 5), 4 (n 6) and 6 (n 7) weeks after

birth and their control littermates (n 5). LCN2 overexpression occurred before apparition of tubular lesions. (d,e) LCN2 protein (upper panel) and

mRNA (lower panel) abundance in (d) mIMCD-3 cells and (e) in mouse renal primary cultured cells exposed to 1% albumin (Alb) for 24 h (n 3).

(f) Representative western blotting (upper panel) and quantication (lower panel) of LCN2 protein abundance in mIMCD-3 cells exposed to 1% albumin for 24 h, treated with NAC, Cd2 or Gd3 (n 5). (g) LCN2 protein expression in mouse renal primary cultured cells exposed to 1% albumin or heat-

denatured (denatured) 1% albumin for 24 h (n 3). (h) CHOP and LCN2 protein, and (i) Lcn2 mRNA expression in mice injected with thapsigargin (Thap),

tunicamycin (Tuni) or the vehicle (Ctrl). (n 5 per group; scale bar, 100 mm; insert scale bar 10 mm). (j) LCN2 expression in mIMCD-3 cells exposed to

Thap (left panel) or Tuni (right panel) for 24 h (n 3). The different apparent size of LCN2 protein due to the inhibition of LCN2 glycosylation by Tuni is

worth noting. (k) LCN2 protein (upper panel) and mRNA abundance (lower panel) in mIMCD-3 cells transiently transfected with different constructs (control, ATF4, ATF6, CHOP, unspliced XBP1 (XBP1u) and spliced XBP1 (XBP1p)). (n 3). (l) Luciferase activity in mIMCD-3 cells co-transfected

with different transcription factors and a Lcn2 promoter luciferase reporter (n 3). (m) LCN2 protein and mRNA expression in wild-type ATF4 / and

ATF4 / MEFs exposed to 1% albumin or 0.5 mM thapsigargin (Th) for 24 h (n 3). (n) LCN2 mRNA expression in ATF4 / MEFs transfected with

empty vector (EV) or ATF4 construct and exposed to 1% albumin or 0.5 mM thapsigargin (Th) for 24 h (n 3). Statistical analysis: one way analysis of

variance followed by TukeyKramer test; MannWhitney test when only two groups are compared; *Po0.05, **Po0.01, ***Po0.001 versus controls.

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and few collecting ducts) but not in glomeruli (Supplementary Fig. 3ac). Consistent with this observation, LCN2 was not found to be increased in glomeruli from WT1 /mut mice as compared with wild-type littermates (Supplementary Fig. 3a,b).

These results were conrmed in the other experimental models of proteinuria (Supplementary Fig. 4ac). More importantly, we observed that these ndings were not restricted to mice: in patients with proteinuric nephropathies LCN2 immunoreactivity remarkably increased, but exclusively in tubular cells (Supplementary Fig. 5), suggesting that LCN2 overexpression is a common response to protein overload. In favour of this idea, we demonstrated that the expression of LCN2 dramatically increased when cultured tubular cells were directly exposed to albumin (Fig. 2d,e). Inhibition of calcium entry by Cd2 and Gd3 completely prevented LCN2 upregulation (Fig. 2f), suggesting that UPR is an essential step in albumin-induced LCN2 overexpression. On the other hand, heat denaturation experiments showed that LCN2 induction was similar, regardless of the albumin state (Fig. 2g), excluding the possibility that compounds bound to albumin account for the overexpression of LCN2, as reported for other biological outcomes27. Moreover, we showed that thapsigargin and tunicamycin, two compounds known to trigger ER stress, lead to a marked induction of Lcn2 gene expression both in vivo, in treated mice (Fig. 2h,i), and in vitro, in mIMCD-3 cells (Fig. 2j), demonstrating that ER stress per se induces LCN2 expression.

Albumin induces LCN2 overexpression through ATF4. We then investigated the molecular links between UPR and LCN2 expression. UPR induces several transcription factors such as ATF4, CHOP, XBP1 or ATF6 (ref. 14). To determine whether one of them is involved in LCN2 overexpression, we transfected mIMCD-3 cells with vectors encoding each of these factors and studied Lcn2 mRNA and protein expression. Interestingly, only ATF4 and CHOP were able to induce the expression of the endogenous Lcn2 gene (Fig. 2k). However, when we used a Lcn2-luciferase reporter vector, we observed that ATF4, but not CHOP, signicantly stimulated the Lcn2 promoter activity (Fig. 2l), indicating that the effect of CHOP on the endogenous Lcn2 promoter was either indirect or the result of a binding to a site located outside of the sequence used to drive the reporter gene. Consistent with the Lcn2-luciferase experiment, in silico analysis of the Lcn2 promoter conrmed the presence of a putative ATF4 binding site located at 1,091/ 1,070 upstream

of the transcription start site (TSS: Chr2, 32243259). To ascertain that ATF4 is required for UPR-induced LCN2 expression, we exposed Atf4-null mouse embryonic broblast (MEF) to either albumin or thapsigargin. Experiments showed that albumin exposure induced ER stress in MEF as in tubular cells (Supplementary Fig. 6a,b). We observed that in the absence of ATF4, UPR could no more stimulate LCN2 synthesis (Fig. 2m), despite the persistent overexpression of CHOP under ER stress (Supplementary Fig. 6a). In addition, rescue experiments using an ATF4 expression vector (Supplementary Fig. 6b,c) showed that ATF4 overexpression in Atf4-null MEF restored the albumin- and thapsigargin-induced LCN2 expression (Fig. 2n). Taken together, these results demonstrate that ATF4 is necessary for the efcient induction of Lcn2 gene expression during UPR.

LCN2 deciency protects tubular cells from proteinuria. To investigate whether LCN2 is the critical effector of this novel molecular pathway, we generated double transgenic mice in which the Wt1 mutant allele was introduced in mice lacking the Lcn2 gene26. As expected, WT1 /mutXLcn2 / mice developed

severe renal lesions 6 weeks after birth (Fig. 3a,b). However, the frequency and the severity of tubular lesions were signicantly reduced in proteinuric WT1 /mut mice lacking Lcn2 (Fig. 3a). In contrast, neither the severity of glomerular lesions (Fig. 3b) nor the level of proteinuria (Fig. 3c) was affected by Lcn2 gene inactivation. Consistent with the persistence of severe proteinuria, the induction of UPR was similar in tubular cells of WT1 /mut mice, regardless of the Lcn2 genotype (Supplementary Fig. 7a,b).

Remarkably, the improvement of kidney lesions was associated with a signicant delay of the overall mortality (median survival rate 96 versus 56 days) in WT1 /mutXLcn2 / mice as compared with WT1 /mutXLcn2 / littermates (Fig. 3d). Of note, LCN2 deciency did not prevent the development of mild interstitial brosis in WT1 /mutXLcn2 / mice at 6 weeks (Supplementary Fig. 7c).

LCN2 deciency reduces apoptosis via ROS modulation. We next wondered which were the cellular mechanisms involved in LCN2-induced tubular injury during proteinuria. Uncontrolled and prolonged ER stress leads to apoptosis28 and LCN2 has been shown to favour apoptosis in multiple cellular types29. As tubular cell apoptosis has been shown to play a role in CKD progression in proteinuric nephropathies30, we combined in vivo and in vitro experiments, to investigate whether LCN2 might regulate tubular cell apoptosis during proteinuria. In vivo, we observed that the number of TUNEL (TdT-mediated dUTP nick end labelling)-positive tubular cells was signicantly reduced in proteinuric WT1 /mut mice lacking Lcn2 as compared with

WT1 /mutXLcn2/ littermates (Fig. 3e). Similarly, the expression of active caspase 3, a well-known marker of apoptosis, was signicantly reduced in the kidneys of WT1 /mutXLcn2 / mice (Supplementary Fig. 8).

Consistently, in vitro, the number of apoptotic cells was signicantly decreased in Lcn2-silenced tubular cells when stressed with albumin (Fig. 3f and Supplementary Fig. 9). The mechanisms of the pro-apoptotic role of LCN2 are still a matter of debate29. As ROS generation has been involved in ER stress-induced apoptosis31, we investigated whether LCN2 acts in a ROS-dependent manner in stressed cells. Our results showed that LCN2 deciency signicantly reduced ROS content in tubular cells after 24 h of albumin exposure (Fig. 3g). Consistent with this observation, Lcn2-silencing prevented albumin-induced overexpression of heme oxygenase 1 (HO-1), a gene involved in the anti-oxidative response (Supplementary Fig. 10a). Notably, when NAC was added to albumin-treated cells, apoptosis was reduced at the same extent as those in cells lacking Lcn2 (Fig. 3h). As expected, we observed that NAC treatment prevented albumin-induced intracellular ROS accumulation (Supplementary Fig. 10b). As NAC did not inhibit LCN2 expression (Fig. 2f), we concluded that LCN2 induces apoptosis after prolonged albumin exposure by promoting ROS generation.

PBA delays CKD progression during proteinuria. Several therapeutic strategies have been developed to modulate pathological ER stress, such as chemical chaperones that improve ER function, possibly by attenuating protein misfolding and stabilizing protein conformation15. Among them, the PBA is already used with a good safety prole in humans32. To provide the rational bases for a novel pharmacological strategy susceptible to counteract the toxic effect of proteinuria during CKD progression, we treated WT1 /mut mice with PBA. As expected, PBA treatment resulted in a marked reduction of ER stress in kidneys of proteinuric WT1 /mut mice, as judged by the decrease of p-eIF2a, ATF4, CHOP and p-c-JUN (Supplementary

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Figure 3 | LCN2 deciency protects from proteinuria-induced tubular lesions and apoptosis. (a) Renal tubular (periodic acidSchiff (PAS); scale bar, 100 mm) and (b) glomerular (PAS; scale bar, 50 mm) morphology (upper panels) and quantications (lower panels) in control (n 6), WT1/mutXLcn2/

(n 10) and WT1/mutXLcn2 / (n 10) mice, 6 weeks after birth. (c) Quantication of urine albumin/creatinine ratio in control (n 6),

WT1/mutXLcn2/ (n 5) and WT1 /mutXLcn2 / (n 10) mice at the time of killing. (d) Survival curve of WT1/mutXLcn2/ (n 37) and

WT1/mutXLcn2 / (n 24) mice. (e) Tubular cell apoptosis evaluated using TUNEL (upper panel; scale bar, 100 mm) and quantication (lower panels)

in control, WT1/mutXLcn2/ and WT1/mutXLcn2 / mice (n 6 per group). (f) Apoptosis measurement by annexin V staining in scramble and Lcn2

sh-RNA expressing mIMCD-3 cells exposed to albumin for 24 h (n 3). (g) ROS abundance in scramble and LCN2 shRNA expressing mIMCD-3 cells

exposed to albumin for 24 h (n 4). (h) Apoptosis measurement by annexin V staining in Lcn2/ or Lcn2 / MEFs exposed to 10% albumin for 24 h in

the presence of either NAC or the vehicle (PBS) (n 3). Data are means.e.m. Statistical analysis: one way analysis of variance followed by TukeyKramer

test; *Po0.05, **Po0.01, ***Po0.001 versus control; #Po0.05, ###Po0.001 versus Lcn2/ ; log rank test (d) Po0.05.

Fig. 11a). Remarkably, PBA administration was associated with a dramatic improvement of kidney lesions and function: tubular lesions (Fig. 4a), interstitial brosis (Fig. 4c) and plasma creatinine (Fig. 4b) were reduced in WT1 /mut PBA-treated mice as compared with WT1 /mut vehicle-treated littermates.

Similarly, the number of tubular apoptotic cells was signicantly reduced in WT1 /mut mice receiving PBA as compared with the vehicle-treated animals (Fig. 4d). It is worth noting that as observed in WT1 /mut mice lacking LCN2, the reduction of tubulointerstitial lesions was independent of the severity of glomerular injury and proteinuria (Supplementary Fig. 11bd). Importantly, the benecial effect of PBA was associated with a dramatic reduction of LCN2 expression in both proteinuric WT1 /mut mice (Fig. 4e,f) and mIMCD-3 cells exposed to

albumin (Fig. 4g,h). The decrease of LCN2 synthesis was associated with reduced ATF4 expression (Fig. 4g), suggesting that LCN2 downregulation by PBA was directly related to UPR inhibition. Consistently, PBA also prevented thapsigargin-induced LCN2 expression (Supplementary Fig. 12).

To extend these observations to a model of acquired proteinuric nephropathy, we treated doxo-injected mice with PBA from either the day of doxo injection (preventive study) or 4 days later, when proteinuria was already established (intervention study). All doxo-injected mice developed severe proteinuria 2 weeks after injection, regardless of the PBA treatment (Fig. 5a). However, the severity of tubular lesions was signicantly reduced in mice receiving PBA as compared with the vehicle-treated mice, in both the preventive and

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Figure 4 | PBA protects from proteinuria-induced tubular lesion development and renal dysfunction by modulating LCN2 expression. (ae) WT1 /mut mice and wild-type littermates were treated with PBA or vehicle for 2 weeks. As no signicant difference between vehicle and PBA-treated WT1 / mice was observed, only one group (Control) is represented. (a) Renal tubular morphology (left panels, periodic acidSchiff (PAS) staining; scale bar, 100 mm) and quantication (right panel) in control (n 6), vehicle-treated WT1 /mut (n 6) and PBA-treated WT1/mut (n 11) mice. (b) Interstitial brosis (left panels,

Sirius red staining; scale bar, 100 mm) and quantication (right panel) in control (n 6), vehicle-treated WT1/mut (n 6) and PBA-treated WT1/mut (n 6)

mice. (c) Plasma creatinine levels in control (n 6), vehicle-treated WT1 /mut (n 5) and PBA-treated WT1 /mut (n 9) mice. (d) Tubular cell apoptosis

evaluated using TUNEL (left panel; scale bar, 100 mm) and quantication (right panel; n 6 per group). (e) LCN2 protein (scale bar, 100 mm; insert scale bar,

10 mm) and (f) mRNA expression in control (n 6), vehicle-treated WT1 /mut (n 6) and PBA-treated WT1/mut (n 10) mice. (g,h) mIMCD-3 cells were

exposed to 1% albumin for 24 h in the presence of either PBA or the vehicle (n 3). (g) Western blotting of ATF4 and LCN2 (left panel), and quantication

(right panel; n 3). (h) Lcn2 mRNA expression (n 3). (i) LCN2 immunohistochemistry in the renal biopsy of a proteinuric patient (before PBA

administration) and a control (scale bar, 100 mm). (j) Evolution of urinary LCN2 excretion (black line) and proteinuria (red line) before and during PBA treatment (arrow indicates the beginning of the treatment) in the proteinuric patient. Data are means.e.m. Statistical analysis: one way analysis of variance followed by TukeyKramer test; *Po0.05, **Po0.01, ***Po0.001 versus controls: #Po0.05, ##Po0.01 versus vehicle-treated WT1/mut mice or cells.

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Figure 5 | PBA decreases proteinuria-induced tubular lesions in doxo-treated mice. Doxo (12 mg kg 1) or saline (Control) injected mice were treated with PBA or vehicle (Vh) from day 0 (D0, preventive study) or day 4 (D4, intervention study) and killed 2 weeks after injection. As no signicant difference was observed between vehicle and PBA-treated mice after saline exposure, only one group (Control) is represented. (a) Quantication of urine albumin/ creatinine ratio at time of killing (n 9, n 10, n 10 and n 5 in control, Doxo-Vh, Doxo-PBA D0 and Doxo-PBA D4 group, respectively). (b) Renal

tubular morphology (periodic acidSchiff (PAS) staining; scale bar, 100 mm), glomerular morphology (PAS staining; scale bar, 50 mm), p-eIF2a, p-c-JUN and LCN2 expression (scale bar, 100 mm) and tubular cell apoptosis evaluated using TUNEL (scale bar, 100 mm; n 4 per group). (c) Lcn2 mRNA expression

(n 3, n 5, n 4 and n 5 in control, Doxo-Vh, Doxo-PBA D0 and Doxo-PBA D4 group, respectively). Data are means.e.m. Statistical analysis: one

way analysis of variance followed by TukeyKramer test; *Po0.05, **Po0.01 versus controls.

the intervention study (Fig. 5b). In contrast, the severity of glomerular lesions was unchanged by PBA, regardless of the treatment schedule. Consistently, ER stress-induced LCN2 expression and tubular apoptosis were reduced in mice receiving PBA (Fig. 5b,c), conrming the potential benet of this therapeutic approach in acquired proteinuric nephropathies.

PBA decreases urinary LCN2 in a proteinuric patient. To nally explore the potential relationship between PBA and LCN2 in humans, we took advantage of a patient followed in our nephrology department for a lysinuric protein intolerance (a urea cycle disorder due to a SLC7A7 gene mutation), which was associated with amyloidosis and proteinuria. PBA, which is currently used in patients with urea cycle disorders as an

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ammonium-scavenging agent32, was recommended because of persistent hyperammoniema. A renal biopsy performed before PBA treatment revealed that the amyloid deposits involved exclusively the glomeruli and not the interstitial or vascular compartments. Proteinuria was severe (2.6 g per day), despite the use of a RAS inhibitor. Immunohistochemistry revealed that LCN2 expression was remarkably increased in renal tubular cells (Fig. 4i). Urinary LCN2 excretion was also dramatically increased as compared with healthy controls. Remarkably, serial urine collections revealed that PBA treatment resulted in a progressive reduction of urinary LCN2 excretion, despite sustained levels of residual proteinuria (Fig. 4j).

DiscussionProteinuria is now recognized not simply as a marker but also as a major risk factor of CKD progression. Despite the fact that RAS inhibitors reduce the leakage of proteins in some patients, therapeutic strategies capable of counteracting the deleterious effect of residual proteinuria on the tubulointerstitial compartment are still lacking. By combining experimental models of proteinuric nephropathies with in vitro and in vivo genetic and pharmacological approaches, we uncovered a novel ER stress pathway. We demonstrated that proteinuria stimulates, via a calcium release-induced ER stress, the overexpression of LCN2, which in turn leads to tubular apoptosis and renal lesions (Fig. 6). More importantly, we showed that inhibition of this pathway by PBA, a pharmacological strategy already used in humans, delayed renal deterioration in proteinuric mice. Collectively, these data identify a novel therapeutic strategy and suggest a crucial role for albumin/ER stress/LCN2 pathway in modulating the progression of CKD.

Our study showed that ER calcium release is a crucial event in the signalling pathway leading to albumin-induced ER stress. Indeed, we observed that extracellular albumin rapidly induces an ER calcium release followed by a store-operated calcium entry that maintains the cytosolic calcium increase. Interestingly, a previous study showed that albumin induces ER stress and

[Ca2 ]cyt increase in cultured podocytes through TRPC6 (transient receptor potential cation channel, subfamily C, member 6) activation33. Whether TRPC6, which is expressed in tubular cells34, plays a role in our experimental model remains to be elucidated. Furthermore, our experiments using BAPTA revealed that albumin requires extracellular calcium to induce ER calcium release. Taking all these data together, it is tempting to speculate that albumin binds to a protein receptor in a calcium-dependent manner, to trigger the intracellular events responsible for ER calcium release. Interestingly, LRP2/Megalin, a well-known albumin receptor, binds its ligands in an extracellular calcium-dependent manner35,36. It is worth noting that LRP2 is expressed in mIMCD3 cells (Supplementary Fig. 13a,b).

In the present study, we showed that AFT4 is mandatory for the efcient induction of Lcn2 gene expression in albumin-overloaded tabular cells. In fact, albumin failed to induce Lcn2 in Atf4 / cells and overexpression of ATF4 completely rescued the phenotype. These data are consistent with the observation that ATF4 directly binds to the Lcn2 promoter31. A previous study has reported that CHOP is able to bind to the Lcn2 promoter as well and is able to induce LCN2 expression, at least, in human cancer cells37. As a recent work has shown that CHOP and ATF4 interact to induce target genes during UPR31, it is possible that ATF4 and CHOP cooperate to induce LCN2 expression in albumin-overloaded tubular cells.

We previously showed that LCN2 plays a crucial role in CKD progression during nephron reduction and cystogenesis, by mediating the mitogenic effect of epidermal growth factor receptor26. In the present study, we demonstrated that LCN2 acts rather by triggering apoptosis in protein-overloaded tubular cells. Interestingly, albumin overload did not lead to epidermal growth factor receptor phosphorylation (Supplementary Fig. 14), suggesting that LCN2 might act as a central integrator of multiple signalling pathways leading to CKD progression. We also observed that LCN2 triggers apoptosis through ROS generation. The mechanisms by which LCN2 promotes oxidative stress in this pathological setting are still unknown. It is possible that an increase in intracellular iron content plays a role, as LCN2 has been shown to transport iron and modulate mitochondrial apoptosis via Bcl-2-like protein 11 (BIM) expression38,39. In favour of this idea, LCN2 endocytosis has been shown to increase ROS generation through an intracellular accumulation of iron leading to apoptosis40. Alternatively, LCN2 might act by inhibiting the nuclear factor-erythroid 2 p45-related factor 2 antioxidant pathway, as LCN2 has been also shown to sequestrate catechol41, a small metabolite product that could activate nuclear factor-erythroid 2 p45-related factor 2 (ref. 42). Whether the secreted or the intracellular form of LCN2 is involved in ROS-induced tubular apoptosis remains to be elucidated.

The substantial increase of the survival rate in proteinuric WT1 /mut mice lacking LCN2 might appear bigger than expected, given the difference in renal function. However, the experimental design of the study could explain, at least in part, this discrepancy. In fact, kidney lesions and apoptosis were evaluated at 6 weeks, whereas the difference in mortality rate was discernable 4 weeks later. Considering the natural history of CKD progression, it is very likely to be that both kidney lesions and renal failure were much greater at this time point than at 6 weeks. In favour of this idea, it has been shown that this model progresses very rapidly towards the complete destruction of the kidney19. Nevertheless, as we studied mice bearing a germline Lcn2 inactivation, we cannot rule out the possibility that LCN2 deciency affected the lifespan of WT1 /mut mice via a systemic effect. It is known that CKD is associated with increased cardiovascular mortality43. On the other hand, LCN2 has been shown to modulate vascular and cardiomyocyte functions44.

Proteinuria

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Figure 6 | Proposed model of proteinuria-induced tubulointerstitial damage. This study shows that by inducing Ca2 -dependent ER stress, albumin activates ATF4 expression, which in turn stimulates LCN2 production. LCN2 then leads to increased apoptosis and tubulointerstitial damage.

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Whether LCN2 inhibition might reduce the cardiovascular lesions and mortality associated with CKD progression is an interesting question that deserves further investigation.

Urinary LCN2 excretion has been shown to be a valuable biomarker of CKD progression45, including in proteinuric nephropathies46. Despite the fact that proteinuria is now recognized as a major risk factor of CKD progression, the rate of progression varies among proteinuric patients47. By showing that LCN2 is a critical target of proteinuria, our data suggest that urinary LCN2 could be helpful to mark the proteinuric patients with the higher risk of CKD progression.

Chemical chaperones have recently received increasing interest as a novel therapeutic approach for the treatment not only of rare genetic diseases leading to misfolded proteins but also of more frequent diseases, that is, metabolic diseases or cancers, characterized by ER stress48. Besides, multiple new therapeutic compounds targeting specic UPR branches are currently under development15. In the present study, we showed that pharmacological modulation of ER stress is an efcient treatment for preserving renal function and morphology in proteinuric mice, at least in part, by decreasing LCN2 expression. The observation, however, that PBA administration, but not LCN2 deciency, decreased the severity of interstitial brosis in WT1 /mut mutant mice suggests that PBA might act by modulating additional actors of CKD progression. Notably, the benet was observed despite the persistence of severe proteinuria. More importantly, our clinical observation that PBA dramatically reduced LCN2 overexpression in a proteinuric patient suggests that this treatment might be valuable in humans. Although we are aware that these promising results must be conrmed, they provide new therapeutic perspectives for patients who display residual proteinuria despite RAS inhibition11 or develop adverse side effects on RAS inhibitors13.

In conclusion, by identifying a novel therapeutic strategy able to counteract the toxic effect of proteinuria on tubular cells, our study provides the rational for the development of an innovative ready-to-use multitarget therapy for proteinuric CKD patients, susceptible to inhibit not only proteinuria leakage but also its tubular toxicity. We expect that adding molecular chaperones will signicantly improve the benet of RAS inhibitors in delaying the progression of CKD, a major public health problem.

Methods

Animals. Mice were on FVB/N background unless otherwise specied. Mutant mice used for these studies were as follows: (i) mutant Wt1 knock-in mice (WT1 /mut mice) bearing a heterozygote Wt1 mutation19, (ii) mutant Nphs2 mice (Nphs2D/ mice), bearing a oxed Nphs2 exon 2 allele, a-null Nphs2 allele and a podocyte-expressed, tamoxifen-responsive Cre recombinase20, and (iii) Lcn2 /

mice26. Animals were fed ad libitum and housed at constant ambient temperature in a 12-h light cycle. Animal procedures were approved by the Departmental Director of Services Vtrinaires de la Prfecture de Police de Paris and by the ethical committee of the Paris Descartes University (approval number: A75-15-34).

For the WT1 /mut mice experiments, male and female mutant mice (n 10)

and their control littermates (n 6) were uninephrectomized at 4 weeks after birth

and killed 2 weeks later. For the time-course experiments, mutant WT1 /mut mice were killed at 3 (n 5), 4 (n 6) and 6 (n 7) weeks after birth. Data were

compared with a group of control littermates (WT1 / mice, n 5). PBA

(1 g kg 1 per day of absorbed PBA, Merck) or vehicle (water corrected for pH and Na concentration with HCl and NaCl) was given in drinking water after uninephrectomy (UNx) performed at 4 weeks and mice were killed 2 weeks later (n 6, n 6, n 6 and n 11 in vehicle-treated WT1 / , PBA-treated

WT1 / , vehicle-treated WT1 /mut and PBA-treated WT1 /mut group, respectively).

For WT1 /mutXLcn2 / mice experiments, male and female double mutants and their control littermates (WT1 / ) were uninephrectomized at 4 weeks after birth and killed 2 weeks later (n 6, n 10 and n 10 for WT1 / , WT1 /

mutXLcn2 / and WT1 /mutXLcn2 / groups, respectively). For survival experiments, male and female double mutant mice were followed up to 23 weeks after birth (n 37 and n 24 in WT1 /mutXLcn2 / and WT1 /mutXLcn2 /

mice, respectively).

For Nphs2 mice, experiments were performed on 6-week-old male and female triallellic Nphs2lox/ ,Cre mice and their control littermates (Nphs2lox/ ,Cre_ mice). Cre recombinase was induced by intraperitoneal administration of tamoxifen (33 mg kg 1 per day for 5 days; Sigma) and mice were killed 34 weeks after the last injection (n 6 for each genotype).

For the doxo experiments, Balb/c females were injected intravenously with doxo (10 or 12 mg kg 1; Sigma) or vehicle (saline) and killed either 14 or 25 days after injection (nZ5 per group). PBA or vehicle was given in drinking water as described above, either from the day of doxo injection (Day 0, preventive study) or from4 days after doxo injection (Day 4, intervention study), and mice were killed2 weeks later (n 6, n 10, n 10 and n 5 in control, vehicle-treated

doxo-injected, PBA-D0 doxo-injected and PBA-D4 doxo-injected group, respectively).

For BSA experiments, FVB/N females were uninephrectomized at 12 weeks and then intraperitoneally injected with BSA (25 mg g 1; Sigma) or vehicle (saline) for 4 weeks (n 4 per group).

For the thapsigargin and tunicamycin experiments, mice were injected with thapsigargin (0.5 mg g 1; Sigma), tunicamycin (1 mg g 1; Sigma) or vehicle (dimethyl sulfoxide) and killed 24 or 48 h later for thapsigargin and tunicamycin, respectively (n 5 per group).

For all the studies, urine and plasma samples were collected at the time of killing and the kidneys were removed for morphological, protein and mRNA studies.

Cell cultures. mIMCD-3 cells were cultured as previously described26. Cells were maintained in DMEM medium /HamF12 (1:1; Gibco, France) medium containing 10% fetal bovine serum (Sigma). Aft4 / mouse embryonic broblasts (MEFs; a kind gift of Dr David Ron) and Lcn2 / MEF (a kind gift from Dr Xiaoli Chen)

were maintained in DMEM (Gibco) medium containing non-essential amino-acid and 10% fetal bovine serum (Sigma). Subconuent cells were starved for 16 h and then stimulated for 24 h with 1% ltered fatty acid-free BSA (Roche) diluted in PBS. Tunicamycin (0.5 mg ml 1; Sigma) and thapsigargin (0.5 mM; Sigma) were used for 1624 h. For heat-denatured albumin experiments, fatty acid-free BSA was diluted in DMEM and heated for 5 min at 95 C. For treatment (Gd3, Cd2 ,

SKF96365, NAC and PBA) experiments, mIMCD-3 cells were rst starved for 6 h. For Gd3 (1 mM; Sigma), Cd2 (1 mM; Sigma), SKF96365 (1 mM; Sigma) and

BAPTA (10 mM; Sigma), the treatment was added at the same time than 1% BSA. BAPTA was added 5 min after 1% BSA incubation, in another set of experiments. For NAC (10 mM; Sigma) and PBA (2.5 mM; Merck), the treatment was added 30 min before 1% BSA incubation. Cells were lysed in RIPA buffer for protein extraction, whereas mRNA was extracted with the MachereyNagel mini-kit, according to the manufacturers protocol. All cell lines were mycoplasma free.

Primary cultures of renal cortical tubular cells were prepared as previously described49. Briey, kidney cortex tubules from 3- to 4-week-old mice were micro-dissected, dissociated by collagenase treatment, washed in Hanks solution by centrifugation and ltered to obtain an enriched preparation of dissociated tubular cells. Cells were then cultivated to reach subconuency. Cell cultures were exposed to 1% fatty acid-free BSA for 24 h and then cells were lysed in RIPA buffer for protein extraction.

Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions. The following plasmids were used: ATF4 (Addgene Inc., ID 21845), ATF6 (Addgene Inc., ID 11975), CHOP (Addgene Inc., ID 21898), XBP1u (Addgene Inc., ID 21832) and XBP1p (Addgene Inc., ID 21833). pcDNA3.1 vector was used as a control. After 36 h of transfection, cells were harvested for mRNA or protein preparation.

For stable cell lines, mIMCD-3 cells expressing short hairpin RNA (shRNA) against Lcn2 were obtained as follows. Double-stranded oligos encoding shRNA directed against Mus musculus Lcn2 were cloned into a lentiviral Tet-pLKO-puro plasmid (pLKO-Tet-on, Addgene 21915). Two double-stranded oligos were inserted between the Age1/EcoR1 restriction site of pLKO-Tet-on, to generate inducible RNA interference (RNAi) expression following doxycycline exposure (100 ng ml 1): scramble (TRCN0000072181) RNAi: sense: 50-CCGGACAACA

GCCACAACGTCTATACTCGAGTAT AGACGTTGTGGCTGT TGTTTTTT G-30, antisense: 50-AATTCAAAAAACAACAG CCACAACGTCTATACTCGA

GTATAGACGTT GTGGCTGTTGT-30; and Lcn2 (TRCN0000055328): sense: 50-CCGGTGCCACTCCATCTTTCCTGTTCTCGAGAACAGGAAAGATGGA GTGGCATTTTTG-30, antisense: 50-AATTCAAAAATGCCACTCCATCTTTC CTGTTCTCGAGAACAGGAAA GATGGAGTGGCA-30. The lentiviral particles were produced by co-transfection of HEK293T cells with three plasmids (pMD2G, psPAX2 and the shRNA vector) using Lipofectamine 2000 (Life Technologies). Cells were infected in the presence of 8 mg ml 1 polybrene overnight and were selected 2 days after viral transduction in puromycin (2 mg ml 1), to achieve 100%

positive cells. For mIMCD-3 stable cell experiments (expressing scramble or sh-Lcn2 RNAi), cells were exposed to medium containing 100 ng ml 1 doxycycline before treatment.

Transfections and Luciferase reporter gene assays. Luciferase gene reporter assays were carried out in 24-well plates. In brief, subconuent mIMCD-3 cells were co-transfected with pGL3 control vector or pGL324p3/Lcn2-Luc containing murine Lcn2 gene promoter (Addgene Inc., ID 25463) in combination with

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plasmids expressing protein of interest. pRL-SV40 Renilla control vector (Promega) was co-transfected in each condition. The luciferase activity was measured 36 h after transfection using a dual-luciferase reporter assay system (Promega) as recommended. Luciferase activity in each cellular lysates was normalized to Renilla luciferase activity. Results are presented as changes of transactivation of the murine LCN2 promoter constructs relative to the original activity of this promoter constructs in cells transfected with empty vector control plasmid.

Clinical samples. Renal biopsies were routinely performed in proteinuric(41 g per day) patients with diabetic nephropathy, membranous nephropathyor IgA nephropathy (n 3 per group). Renal biopsies from patients with resolute

kidney injury that did display neither signicant proteinuria nor histological

lesions were used as controls.

The patient treated with PBA was a 33-year-old man, who developed proteinuric CKD associated with lysinuric protein intolerance. Blood pressure was 116/84 mm Hg, measured glomerular ltration rate (iohexol clearance) was 51 ml min 1 per 1.73 m2, serum albumin levels were 38 g l 1 and proteinuria 2.6 g per day. The patient was treated with irbesartan, hydrochlorothiazide, citrullin and sodium benzoate, and successively PBA (3 g per day) was added. Morning spot urine samples were obtained before PBA and every week after the beginning of the treatment for 2 months. Urine samples were stocked at 80 C after

centrifugation. No other therapeutic modication was made once PBA was introduced. Informed consent was obtained from all patients. Our institutional review board approved the study (approval number: DC-2009-955).

Urine and plasma analysis. For mice samples, urinary albumin and creatinine levels were measured using an Olympus multiparametric autoanalyser (Instrumentation Laboratory). Plasma creatinine was measured using Konelab Analyzer (Thermo Fischer Scientic).

For human samples, urinary LCN2 concentration was evaluated by enzyme-linked immunosorbent assay (R&D Systems) according to the manufacturers instructions. Proteinuria and urinary creatinine was measured using a Hitachi 917 analyser (Roche Diagnostics).

Renal morphology. For morphology analysis, kidneys were xed in 4% paraformaldehyde, parafn embedded and 4 mm sections were stained with periodic acidSchiff or Sirius red. The degree of glomerular lesion was evaluated using a semiquantitative score methodology as previously described50. The degree of tubular lesions was evaluated using semiquantitative score methodology as previously described51, with minor modications in the evaluation of kidney lesions (0 normal, 1 involvement of o10% of the cortex, 2 involvement of

1025% of the cortex, 3 involvement of 2550% of the cortex, 4 involvement of

450% of the cortex). The degree of brosis was automatically quantied using Sirius red staining in eight representative elds (magnication 100) with a

Nikon digital camera Dx/m/1200 and NIS software (Nikon), and expressed as the ratio between the Sirius red-positive surface and the total section area, as previously described26.

Immunohistochemistry. For mouse samples, 4-mm sections of parafn-embedded kidneys were incubated with the following: a goat anti-mouse LCN2 antibody (R&D Systems, AF1857) at 1:100, a rabbit anti-mouse CHOP (Santa Cruz Biotechnology, SC-575) at 1:50, a rabbit anti-mouse ATF4 at 1:50 (Santa Cruz Biotechnology, SC-200), a rabbit anti-mouse p-c-JUN (Cell Signaling Technology, 2361) at 1:200, a rabbit anti-mouse p-eIF2a (Cell Signaling Technology, 3597) at 1:50, followed by a rabbit anti-goat biotinylated antibody (Dako, E0466) at 1:200, a donkey horseradish peroxidase (HRP)-conjugated anti-rabbit (GE Healthcare, NA934V) at 1:200 or a donkey biotinylated anti-rabbit antibody (GE Healthcare, RPN1004V) at 1:200. Biotinylated antibodies were detected using HRP-labelled streptavidin (Southern Biotech, 7100-05) at 1:2,000 and 3-3-diamino-benzidinetetrahydrochloride (Dako, K3468).

For human samples, 4 mm sections of parafn-embedded kidneys were incubated with a goat anti-human LCN2 antibody (R&D Systems, AF1757) at 1:100, followed by a HRP-labelled rabbit anti-goat antibody (Dako, P0449) and 3-3-diamino-benzidine-tetrahydrochloride revelation.

For co-localization experiments, lotus tetragonolobus lectin was detected using biotinylated-lotus tetragonolobus lectin (Vector, B1325) at 1:50, followed by HRP-labelled streptavidin at 1:2,000. For TammHorsfall staining, a goat anti-TammHorsfall antibody (AbDSerotec, 8595-0054) at 1:200 was used, followed by a biotinylated goat antibody (Dako) at 1:500 and HRP-labelled streptavidin at 1:2,000. For aquaporin (AQP2) 2 staining, sections were incubated with a rabbit anti-AQP2 antibody (Sigma, A7310) 1:400, followed by a donkey HRP-conjugated anti-rabbit antibody (GE Healthcare, NA934V) at 1:300.

Apoptosis was detected in 4 mm sections of parafn-embedded kidneys by TUNEL assay using the In Situ Cell Death Detection kit (Roche) according to the manufacturers protocol. The number of apoptotic cells was determined as the number of TUNEL-positive nuclei per tubule in eight representative elds (magnication 400).

Albumin uptake in mIMCD-3 cells. Cells exposed to 1% uorescein isothiocyanatealbumin (Sigma) for different time were exposed to rat anti-LAMP2 antibody (Abcam, Ab 13524). The secondary antibody was uorescent goat anti-rat antibody (Invitrogen). Images were acquired using a Leica TCS SP5 AOBS microscope with a 40/1.25 (differential interference contrast (DIC)) (Leica Microsystems). Resulting digital images were acquire with LAS AF software and analysed using ImageJ.

Cell death measurement. Cellular death was assessed based on propidium iodide (PI) staining (Sigma) and Annexin V/PI staining (Molecular Probes) according to the manufacturers protocols. Cell uorescence was measured by ow cytometry. PI-negative cells were dened as viable. Flow cytometry analysis was performed on a FACSCalibur (BD Biosciences) followed by analysis using FlowJo.

mRNA analysis. mRNAs were quantied in mouse kidneys and cultured cells by quantitative reverse transcriptasePCR using an ABI PRISM 7700 Sequence Detection system (Applied Biosystems). Primers were as follows: Lcn2 (fwd) 50-GGACCAGGGCTGTCGCTACT-30 and (rev) 50-GGTGGCCACTTGCACAT

TGT-30; CHOP (fwd) 50-GGAGGTCCTGTCCTCAGATGAA-30 and (rev) 50-GGACGCAGGGTCAAGAGTAGTG-30; spliced XBP1 (fwd) 50-GAGTCC GCAGCAGGTG-30 and (rev) 50-GTGTCAGAGTCCATGGGA-30; and HO-1: (fwd) 50-GTACACATCCAAGCCGAGAA-30 and (rev) 50-TGGTACAAGGA AGCCATCAC-30.

The splicing of XBP-1 mRNA was analysed by PCR using the following protocol: 94 C for 3 min, 29 cycles of 94 C for 30 s, 58 C for 30 s, 72 C for 30 s and 72 C for 3 min. We used the following primers: XBP-1 (fwd) 50-ACAC GCTTGGGAATGGACAC-30 and (rev) 50-CCATGGGAAGATGTTCTGGG-30.

RPL13, SDHA and HPRT were used as the normalization controls as previously described52.

Western blotting. Western blottings were performed as previously described26 using the following: a goat anti-mouse LCN2 antibody (R&D systems) at 1:1,000, a rabbit anti-mouse CHOP antibody (Santa Cruz Biotechnology) at 1:500, a rabbit anti-mouse p-c-JUN antibody (Cell Signaling Technology) at 1:1,000, a rabbit anti-mouse pPERK antibody (Cell Signaling Technology), a rabbit anti-mouse ATF4 antibody (Santa Cruz Biotechnology) at 1:500, a rabbit anti-mouse p-eIF2a antibody (Cell Signaling Technology) at 1:1,000 and a rabbit anti-mouse cleaved caspase 3 antibody (Cell Signaling Technology) at 1:1,000, followed by either a rabbit HRP-conjugated anti-goat antibody at 1:10,000 (Dako) or a donkey HRP-conjugated anti-rabbit antibody at 1:10,000 (Amersham). Mouse monoclonal anti-a-tubulin or anti-b-actin antibody (Sigma) was used as loading control. The uncropped versions of western blottings are shown in Supplementary Fig. 15.

ROS measurement. Two techniques were used to measure intracellular ROS content. mIMCD-3 cells were loaded with dichlorodihydrouorescein diacetate (10 mM; LifeTechnologies) and incubated for 30 min at 37 C. After incubation, cells were washed twice with PBS, lysed and acquired on a microplate reader (Tristar LB 941, Berthold). For analysis of intracellular ROS content, CellROX Deep Red Reagent was used according to the manufacturers instructions (LifeTechnologies) and analysed either by immunouorescence or ow cytometry. Flow cytometry analysis was performed on a FACSCalibur (BD Biosciences) followed by analysis using FlowJo.

Calcium spectrouorimetry. Intracellular Ca2 concentration variations were studied using spectrouorimetry. Recordings were made in a HEPES buffered saline medium (HBS) containing (in mM): 135 NaCl, 5.9 KCl, 2 CaCl2, 1.2 MgCl2,11.6 Hepes, 11.5 glucose pH 7.3 adjusted with NaOH. mIMCD-3 cells were grown on glass coverslips in Petri dishes. Before conuence, coverslips were washed three times with HBS medium and then loaded with 4 mM Fluo-4 AM (Molecular

Probes). After loading, coverslips were washed twice with Fluo-4 AM-free HBS medium and maintained in this medium. Just before recordings, a coverslip was introduced in a 1 cm width3 ml quartz cuvette, containing 2 ml of HBS medium and inserted in a spectrouorimeter (RF-1501 Shimadzu Corporation, Japan). Fluo-4 was excited at 494 nm and uorescence was measured at 516 nm. The baseline uorescence was recorded during 10 min before adding BSA. The [Ca2 ]cyt was expressed as a ratio value of the uorescence intensity divided by the baseline average uorescence intensity.

Data analysis and statistics. Data were expressed as meanss.e.m. Differences between the experimental groups were evaluated using analysis of variance followed by the TukeyKramer test. The log-rank test was used for survival analysis. When only two groups were compared, MannWhitney test was used. Assumptions for statistical analyses were met (that is, normal distribution and equal variance). Replicates used were biological replicates, which were measured by using different samples derived from distinct mice. The results are representative examples of more than three independent experiments. We estimated the sample size considering the variation and mean of the samples. No randomization or

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blinding was done. No animals were excluded from the analysis. The statistical analysis was performed using Graph Prism Software.

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Acknowledgements

We are grateful to Sophie Berissi, Nomie Gadessaud and to all the persons working in

the LEAT facility for technical assistance. We are grateful to David Ron (Institute of

Metabolic Science, University of Cambridge, Cambridge, UK) and Pierre Fafournoux

(UMR 1019 INRA, Universit Clermont 1, Clermont-Ferrand, France) for providing

Atf4-null MEF cells and to Xiaoli Chen (Department of Food Science and Nutrition,

University of Minnesota-Twin Cities, St Paul, MN, USA) for providing Lcn2-null MEF

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10330 ARTICLE

cells. We thank Aurlie Hummel, Aude Servais and Marion Rabant for patient data and

sample collection. We thank Marco Pontoglio and Marie-Claire Gubler for critical

advice. This work was supported by INSERM, Universit Paris Descartes, Assistance

Publique-Hpitaux de Paris (AP-HP), Fondation pour la Recherche Mdicale, Agence

Nationale Recherche, Subvention Genzyme/Socit de Nphrologie, Association

pour lInformation et la Recherche sur les maladies rnales Gntiques (AIRG), pRED

Roche Laboratories (Basel), Institut Roche de Recherche et Mdecine Translationnelle

(Paris).

Author contributions

K.E.K. and M.G. designed and performed the experiments, analysed the data and wrote

the paper. A.V., O.D. and A.B. also performed some experiments and analysed the data.

C.N. and M. Burtin performed the mouse experiments (breeding, surgery and treat

ments). W.B. performed some in vitro studies. M. Broueilh performed the enzyme-linked

immunosorbent assay experiments. L.H., G.M. and C.A. provided the mutant Wt1 and

Nphs2 mice and revised the manuscript. A.D., B.K., G.F. and F.B. were involved in data

analysis. F.T. and M.G. provided the conceptual framework and designed the study,

supervised the project and wrote the paper.

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How to cite this article: El Karoui, K. et al. Endoplasmic reticulum stress drives

proteinuria-induced kidney lesions via Lipocalin 2. Nat. Commun. 7:10330

doi: 10.1038/ncomms10330 (2016).

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