Tubular damage is a major cause of renal failure, and both ischemic and toxic tubular injuries are very frequent causes of acute and chronic renal failure.1 The course of any renal tubular injury typically starts with loss of specialized function (e.g., proximal tubular cells lose the brush border and their ability to reabsorb low-molecular-weight proteins) and ends with cell death through necrosis, apoptosis, or necroptosis.2 Repair occurs through proliferation of the surviving epithelial cells and their redifferentiation into specialized tubular cells.3
Most patients with transplanted kidneys are treated with calcineurin inhibitors, which cause vascular and tubular damage.4 These drugs, mainly cyclosporin A (CsA) and tacrolimus, can initiate tubular lesions,5,6 but the exact mechanism is currently unknown. Calcineurin is a serine phosphatase involved in the dephosphorylation of various proteins and, most importantly, a family of transcription factors—the nuclear factors of activated T cells (NFATs). There are five of these; NFATs 1–4 are regulated by calcineurin, and NFAT5 is regulated by osmotic stress.7 Calcineurin, by dephosphorylating NFAT, allows it to enter the nucleus and begin its transcriptional activity. NFAT target genes involved in the inflammatory response have been extensively studied in lymphocytes, where NFAT acts as a transcriptional activator but also as a repressor for some genes.8In vivo, CsA induces renal vasoconstriction and tubular lesions.9 The spontaneous development of a renal phenotype reminiscent of cyclosporin toxicity in mice deficient for calcineurin A α indicates that the decrease in calcineurin activity is sufficient to trigger renal damage.10,11 Genetic deficiency of either cyclophilin or NFATc1 (i.e., the two substrates known to be inhibited by cyclosporin) is not sufficient to initiate tubular damage, but may increase it by impeding the tubular repair process.12–14
The mechanisms by which cyclosporin alters tubular function or repair are still poorly understood. Its vasoconstrictive properties on afferent arterioles might be involved in vivo, by causing subintrant ischemia downstream.4 A direct effect within the epithelium is also plausible since, like many xenobiotics, calcineurin inhibitors are metabolized by tubular epithelial cells. Endoplasmic reticulum (ER) stress is one hallmark of in vitro cyclosporin toxicity.15–17 It is a complex phenomenon with antagonistic consequences.18 The term refers both to the accumulation of misfolded proteins in the ER and to the molecular events triggered in response to this accumulation. The accumulation of misfolded proteins recruits the chaperone protein BiP, thus in turn activating double-stranded RNA activated protein kinase-like ER kinase (PERK), a kinase that phosphorylates the transcription factor EIF2α. EIF2α phosphorylation is the common end signal of kinases activated by amino-acid limitation (general control nonrepressible 2 [GCN2]), viral infection (double-strand RNA-activated kinase), heme limitation (heme-regulated inhibitor kinase) and protein misfolding (PERK).19 The biologic role of EIF2α phosphorylation is to decrease protein translation in general, except for that of adaptive proteins, including ATF4, a master transcription factor. ATF4 induces the expression of adaptive genes. If sustained, however, ATF4-induced upregulation of protein synthesis eventually causes ATP depletion and cell death.20 It is thus essential to control the ER stress pathway. Cyclosporin toxicity is known to be associated with impaired energy status and can be alleviated by ATP.21 Together, these data strengthen the hypothesis that ER stress plays a role in cyclosporin-induced renal injury.
Our group has extensively studied the phenotypic changes observed in tubular epithelial cells from human kidney recipients exposed to cyclosporin and has repeatedly shown, in particular, that immunohistochemical staining of two mesenchymal markers, vimentin and β-catenin, in tubular epithelial cells detects cyclosporin tubular toxicity at an early, reversible stage.5,22 We have also shown that the expression of these mesenchymal markers in tubular epithelial cells of kidney grafts in patients treated with cyclosporin is associated with initial ischemic injury and is predictive of further development of kidney fibrosis.23,24 To further investigate the mechanisms of CsA-induced tubular injury, we characterized the in vivo tubular transcriptomic changes that occur at an early stage of cyclosporin toxicity. Our findings point to a new response gene encoding nuclear protein, transcriptional regulator 1 (Nupr1), which has a central role in the ER stress pathway. We show here that Nupr1 is induced in several animal models of acute tubular damage and confers resistance to renal tubular damage in these animals presenting with cyclosporin toxicity.
Cyclosporin Induces Tubular Transcriptomic Changes That Are Related to Protein Synthesis and Stress
We used laser microdissection coupled to an mRNA microarray to perform an in vivo comparative transcriptomic analysis of rat renal tubules exposed to either vehicle or nephrotoxic doses of cyclosporin (Figure 1, dataset one). Using a 1.5-fold threshold (with P<0.05 by t test), we found that 179 genes were downregulated and 286 genes were upregulated by cyclosporin (dataset one). The gene expression pattern confirmed that this transcriptomic analysis was limited to the tubular epithelium, since the expression of endothelial (Cd31) and podocyte-specific (Nephrin) genes was very low (three and two, respectively, on a logarithmic scale ranging from two to 14). Markers specific to the proximal tubule (Npt2a and Lrp2) were very strongly expressed (12 on the same logarithmic scale), whereas Aqp2, a marker of collecting ducts, was expressed only moderately (seven on the logarithmic scale). None of these cell-specific markers differed between the cyclosporin- and vehicle-treated groups. In contrast, we found that known markers of tubular damage—Havcr1 (also known as Kim1) and osteopontin (Opn)—were induced in renal tubules exposed to cyclosporin (three to seven for Kim1 and five to nine for Opn on the logarithmic scale). Figure 1 presents these expression data.
The pathway analysis using the Pathway Analysis using Logistic Regression (LRpath) identified significant changes in the expression of genes involved in five Kyoto Encyclopedia of Genes and Genomes pathways: ribosome, protein processing in the ER, protein export, peroxisome, and terpenoid synthesis (Figure 1, Supplemental Table 1). Notably, searching the Transcription Factor Binding Sites database (Biobase; Qiagen, Waltham, MA) for transcription factors associated with the observed transcriptomic changes did not identify NFAT or any other transcription factor. The first three pathways (ribosome, protein processing in the ER, and protein export), all involved in protein synthesis, showed overexpression in the CsA group, whereas the latter two (peroxisome and terpenoid synthesis) were underexpressed. This transcriptional signature of cyclosporin in vivo in the tubular epithelium is strongly reminiscent of the ATF4-dependent gene expression induced by tunicamycin (an ER stress inducer) in vitro.20 Accordingly, we observed that nephrotoxic doses of cyclosporin consistently enhanced the level of ATF4 in renal tubular cells, as shown by immunohistochemical analysis of kidney sections from CsA-treated mice. Moreover, ATF4 was induced by CsA in vitro in cultured mouse tubular epithelial cells, as shown by Western blot (Figure 2). Interestingly, Nupr1, an ATF4-responsive gene,25,26 was the gene most highly overexpressed (86 times) under cyclosporin in this microarray analysis (Supplemental Table 1). To our knowledge, the role of Nupr1 in acute tubular damage has not previously been studied in vivo.
Nupr1 Confers Resistance to Epithelial Injury
To gain insight into the role of Nupr1 in CsA nephrotoxicity, we translated our experimental model to mice. C57bl6/J wild-type (WT) mice showed evidence of epithelial toxicity with renal failure after 2 weeks of cyclosporin at doses between 50 and 100 mg/kg per day subcutaneously. A dose of 100 mg/kg per day induced AKI, weight loss, tubular damage, apoptosis, and proliferation together with dose-dependent Nupr1 induction by CsA and ER stress (Supplemental Figures 1–3). We used Nupr1−/− mice to investigate the role of this gene in cyclosporin-induced renal injury. Although these Nupr1-deficient mice showed no spontaneous renal phenotype, the renal tubular lesions they developed when challenged with cyclosporin were substantially worse than those observed in WT mice. They had more tubular vacuolization and dilation than their WT littermates (median semiquantitative score 2 interquartile range [0.88–2.5] versus 0.5 [0.5–1]; P=0.01 for vacuolization and 1.75 [0.5–2] versus 0 [0–0.5]; P=0.003 for dilation, Figure 3, A, B, G, and F). Accordingly, Kim-1 mRNA expression was significantly higher in the cortex of Nupr1−/− compared with WT mice; a trend toward higher plasma urea levels in the Nupr1−/− mice was also observed, but did not reach statistical significance (Supplemental Figure 4).
The Mechanism by Which Nupr1 Protects the Renal Epithelium Involves Cell Survival but Not Decreased Cyclosporin Exposure, Vascular Reactivity, or Enhanced Tubular Repair
Measurements of serum cyclosporin trough levels showed that the groups did not differ for cyclosporin exposure (Supplemental Figure 5A). Knockout mice did not differ from the WT mice in their expression of the Il27 gene, a close neighbor of the Nupr1 gene on the mouse genome and a potential regulator of cyclosporin-induced inflammation (Supplemental Figure 5B).27 Measurements of aortic BP and renal blood flow in Nupr1−/− and WT mice after 5 days of cyclosporin (100 mg/kg per day subcutaneously) or 5 days of vehicle showed no hemodynamic effect of Nupr1 (Supplemental Figure 5, C and D).
After cyclosporin treatment, apoptosis, assessed by terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling, was more frequent in renal tubular cells from Nupr1−/− (18.5 [9.8–33.3] nuclei/field) than in WT mice (4 [1.75–6.5]; P=0.01) (Figure 3, E, F, and J). This observation suggests that Nupr1 protects tubular cells from death. Tubular proliferation assessed by Ki67 staining was again more frequent in the renal tubular cells of the Nupr1−/− (89.5 [55.5–117.3] nuclei/field) than WT animals (43.5 [33.3–56.5]; P=0.03) (Figure 3, C, D, and I). Both tubular apoptosis and proliferative repair increased in the absence of Nupr1.
Nupr1 Limits Protein Synthesis, Proliferation, and Cell Death, and Preserves the Energy Status of Epithelial Proximal Tubular Cells during In Vitro Cyclosporin Toxicity
We used primary cultures of proximal epithelial tubular cells from Nupr1−/− and WT mice to investigate the effect of Nupr1 on apoptosis under CsA treatment. In WT cells, we measured ATF4 protein, Nupr1, Atf4, Chop, and Bip mRNA expression before and after 1, 2, 3, 6, and 24 hours of cyclosporin exposure. All these parameters were induced already after 1 hour, with ATF4 protein induction being the strongest at this early time point, except Bip which was induced only after 3 hours (Figure 2C). Nupr1−/− cells had a higher level of ATF4 expression after CsA treatment than WT cells (Supplemental Figure 6). Using a pulse of homopropargylglycine (a methionine analogue) to evaluate protein synthesis by immunofluorescence and flow cytometry, we showed that cyclosporin decreased protein synthesis in WT cells but did not do so in Nupr1−/− cells (Figure 4).
We measured cell proliferation using three different techniques: a simple cell count 48 hours after seeding various numbers of cells, continuous monitoring of cell density with xCELLigence technology, and 5-bromo-2′-deoxyuridine incorporation (Figure 5, Figure 6, A and B, Supplemental Figure 7). All techniques showed that Nupr1−/− proximal tubular cells proliferated faster than WT cells. Treatment with CsA virtually abolished proliferation in WT cells, but only decreased proliferation in knockout cells (Figure 6, A and B). Although proliferation was maintained, assessment by xCELLigence of CsA-treated Nupr1−/− cells showed a decrease in cell density, suggesting cell death.
Similar to CsA renal toxicity in vivo, 5 µg/ml CsA induced cell death, with more dead cells in Nupr1−/− cells compared with WT cells (Figure 6C).
We investigated the effect of Nupr1 on the energy status of proximal tubular epithelial cells by measuring ATP and ADP in Nupr1−/− and WT cells incubated for 24 hours with or without 1 µg/ml CsA (Figure 6D). ATP/ADP ratio was lower in Nupr1−/− mice compared with WT mice. Moreover, the ATP/ADP ratio decreased further still after cyclosporin was added. The effect of Nupr1 on the ATP/ADP ratio was explained mainly by a fall in ATP, although ADP increased slightly as well (Supplemental Figure 8).
Rescuing Nupr1 deficiency by transfecting a flag-Nupr1 transgene into Nupr1−/− proximal tubular cells did inhibit proliferation (less cells in S phase, and more cells in G2/M) in cells expressing the transgene, thus confirming the instrumental role of Nupr1 in proliferation and apoptosis (Figure 7, Supplemental Figure 9).
Nupr1 Induction is Relevant to Other Models of Kidney Injury in Mice and in Human Kidney Transplantation
To determine if Nupr1 was specifically upregulated after CsA treatment, we measured Nupr1 mRNA in kidney cortices of mice in control conditions and after ischemia-reperfusion injury, unilateral ureteral obstruction, and angiotensin II-induced hypertension. As shown in Supplemental Figure 10, we found that Nupr1 was upregulated in each of these different experimental models. This finding suggests that Nupr1 may exert its protective effects in many forms of kidney injury.
We studied the expression of Nupr1 in cultured human renal epithelial cells. Increasing the cyclosporin concentration dose-dependently induced Nupr1 mRNA expression in human proximal tubular cells (Figure 8A). We also performed immunohistochemical analysis for NUPR1 on normal kidney (stable renal allografts) and on injured kidney (preimplantation biopsies from donors with acute tubular necrosis and allografts undergoing acute tubular necrosis or allograft dysfunction). Using multiple stainings for NUPR1, megalin, and nuclei, we found that NUPR1 expression was absent in nuclei from renal proximal tubules in normal kidneys. Conversely, in injured kidneys, NUPR1 was strongly expressed in many nuclei from renal proximal tubules. (Figure 8, B–F).
Kidney resistance to injury varies greatly,28 as exemplified by cyclosporin toxicity; it is widely prescribed to patients in standardized conditions and results in variable toxicity, depending on the dose, on additional insults, and on the characteristics of the patient.4 Several genes involved in xenobiotic detoxification have been suggested as risk factors for calcineurin inhibitor nephrotoxicity.29 Nonetheless, cyclosporin nephrotoxicity remains poorly understood, especially its in vivo effect on renal tubular epithelium. Our investigation provides the first description of the genome-wide transcriptional effect of this drug there. Our primary hypothesis was that calcineurin inhibition would cause toxic effects by inactivating NFAT, a pleiotropic transcription factor. NFAT-induced genes (e.g.,Il2) are expected to be downregulated by cyclosporin through calcineurin inhibition.8 Other genes have been shown to be repressed by NFAT (e.g.,Cdk4) and might thus be enhanced under cyclosporin treatment.8,30–32 Interestingly, one of the genotypes at risk of cyclosporin toxicity is a low-activity variant of Cyp2c9,29 the gene most downregulated by cyclosporin in our data (Figure 1). However, the Cyp2c9 promoter contains no NFAT response element,33 and neither the NFAT nor the calcineurin pathways were modified by cyclosporin on a transcriptional level in our data. These seemingly contradictory results can be interpreted in two ways: (1) the calcineurin and NFAT pathways in the tubular epithelium (our data) are very different from these pathways in the lymphocytes (previously published data7,8,31), thus, bioinformatics tools like LRpath do not link our data to calcineurin and NFAT; and (2) the pattern of gene expression we observed is not directly caused by the inhibition of the calcineurin and NFAT pathways. Other substrates of calcineurin might be involved, or a secondary transcriptomic response to injury. This pattern is reminiscent of the genes induced by ATF4, a master transcription factor that orchestrates the expression of stress genes, including Nupr1 and other stress-related genes that were overexpressed in our analysis, like Asparagine synthetase and several aminoacyl-transfer RNA-synthetases. ATF4 induces a transcriptional program that upregulates the energy-expending mechanisms of protein synthesis potentially leading to cell death.20 Decreased ATP levels are instrumental in cell death, whether due to diminished ATP production (decreased fatty acid oxidation) or to enhanced ATP consumption (increased protein synthesis).20,34 Here, after administration of toxic doses of cyclosporin in vitro and in vivo, renal tubular cells expressed ATF4-dependent pathways associated with decreased expression of peroxisome genes and increased rates of protein synthesis, energy depletion, and cell death.
Nupr1, essentially unstudied in renal diseases, is a chromatin-binding protein induced by various epithelial stresses and has important properties: it is antiapoptotic and anti-inflammatory, and it regulates cell proliferation.35 Its expression is instrumental in endothelin-mediated mesangial hypertrophy.36 Because Nupr1 is an early stress response gene induced by ATF4 in the renal tubular epithelium and protects tubular cells from cell death,25 we hypothesized that it might protect against apoptosis by preserving the cell energy status.
We found that Nupr1 was strongly expressed during cyclosporin-induced renal epithelial lesions in vivo and in vitro and that it exerted a protective role on cell survival and energy status. Nupr1 also contributed to inhibiting protein synthesis and slowing the proliferation of proximal tubular cells. Ischemia-reperfusion injury causes p53-dependent G2/M arrest of renal tubular cells and subsequent renal fibrosis.37Nupr1 is a positive regulator of p53.38 Because we found a consistent increase in Nupr1 levels in all the kidney injury models we tested (including ischemia-reperfusion injury), Nupr1 induction appears to be a generic response to any epithelial injury and is not cyclosporin-specific. Whether Nupr1 induction is also protective in these other models remains to be tested. The mechanism by which the ATF4-NUPR1 pathway might influence evolution toward cell death or survival is outlined in Figure 9. Overall, Nupr1 inhibits energetically costly processes (proliferation and protein synthesis) and preserves ATP cellular concentrations. ADP/ATP ratio is associated with apoptosis, and may be instrumental in this model. However, the molecular targets of Nupr1 that lead to cell protection are still unknown. Further studies should therefore: (1) investigate the precise mechanisms that regulate energy consumption, protein synthesis, and proliferation downstream of Nupr1; (2) test strategies aimed at producing a time-limited expression of Nupr1-related pathways in AKI; and (3) determine whether Nupr1 variants are involved in susceptibility to kidney injury in humans.
For a full description of the methods please see Supplemental Material.
We used a model of in vivo renal toxicity created by daily injections of cyclosporin for 2 weeks.39 J.L.I. created the Nupr1−/− mouse strain.38 Blood and kidneys were harvested under general anesthesia. For creatinine clearance measurements, animals were placed overnight in metabolic cages for urine collection, and blood was collected from the retro-orbital sinus under isoflurane anesthesia.
Morphologic Studies and Laser Microdissection
Ki67 and terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end quantification were performed with the particle analysis function of ImageJ 1.45o software. For each sample, 200 cortical tubules were harvested by laser capture microdissection (Supplemental Figure 11).
RT-PCR and mRNA Microarray
Microdissected tubules were studied with the GeneChip Rat Genome 230 2.0 microarrays (Affymetrix, Santa Clara, CA). The pathway analysis was performed with LRpath web-based software.40 The quantitative RT-PCR expression results relative to the housekeeping gene were obtained by the ∆Cp method (see Supplemental Table 2 for primers sequences).
In Vitro Studies
Proximal epithelial cells were isolated from fresh kidneys of Nupr1−/− and WT mice.41,42
Statistical analysis used JMP Pro 9.0.2 software. Data are presented by their median values and interquartile ranges, and a nonparametric Wilcoxon test was used to assess statistical significance, except where otherwise indicated. A P value <0.05 was defined as significant.
Animals were handled in a licensed facility (approval number B-75–20–01) in compliance with French legislation and Institut National de la Santé et de la Recherche Médicale standards. Biopsies from renal-transplanted patients receiving cyclosporin treatment were used for NUPR1 immunohistochemistry. The biopsies were indicated for state-of-the-art routine care, and all patients provided written consent for use of the leftover material for research purposes. The study performed on human biopsies was approved by our institutional review board (Comité de Protection des Personnes Ile de France V).
P.G. designed and performed the experiments and drafted the manuscript. M.W., A.B., and C.M. participated in the in vivo experiments. D.L. and S.V. participated in the in vitro experiments. S.P. performed the hemodynamics study. N.B. participated in the in vivo studies and RT-qPCRs. Y.-C.X.-D., C.J., and D.B. participated in the histologic evaluation of the kidneys (mice and humans). A.H. participated in the interpretation of the results and reviewed the manuscript. J.I. created the Nupr1−/− mice and reviewed the manuscript. E.R. participated in the study design and in writing the manuscript.
Project funding: Assistance Publique–Hôpitaux de Paris, Agence de Biomédecine. Equipment funding: Domaine d'Intérêt Majeur Cardiovasculaire-Obésité-Diabète (CODDIM). Scholarships: Assistance Publique–Hôpitaux de Paris (to P.G. and A.B.), Société Francophone de Transplantation (to P.G.), Société de Néphrologie (to P.G.), Société Française d'Anesthésie-Réanimation (to A.B.), and Contrat d’Interface hospitalier Institut National de la Santé et de la Recherche Médicale - Assistance Publique—Hôpitaux de Paris (to A.H.)
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