BASP1 Promotes Apoptosis in Diabetic Nephropathy

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Maria Dolores Sanchez-Nin˜o,* Ana Belen Sanz,† Corina Lorz,* Andrea Gnirke,‡ ʈ Maria Pia Rastaldi,§ Viji Nair, Jesus Egido,* Marta Ruiz-Ortega,* ʈ Matthias Kretzler, and Alberto Ortiz*

*Nefrologıá, Fundacioń Jimeńez Dıáz, Universidad Autonoma de Madrid and Instituto Reina Sofia de Investigaciones Nefrolo´gicas-IRSIN, Madrid, Spain; †Servicio de Nefrologia, Fundacion para la Investigacion Biomedica del Hospital Universitario La Paz, Madrid, Spain; ‡Xantos, Munich, Germany; §Fondazione D’Amico per la ʈ Ricerca sulle Malattie Renali & Fondazione IRCCS Policlinico, Milan, Italy; and Nephrology/Internal Medicine and Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan

ABSTRACT Apoptosis contributes to the development of diabetic nephropathy (DN), but the mechanisms that lead to diabetes-induced cell death are not fully understood. Here, we combined a functional genomics screen for cDNAs that induce apoptosis in vitro with transcriptional profiling of renal biopsies from patients with DN. Twelve of the 138 full-length cDNAs that induced cell death in human embryonic kidney cells matched upregulated mRNA transcripts in tissue from human DN. Confirmatory screens identified induction of BASP1 in tubular cross sections of human DN tissue. In vitro, apoptosis-inducing conditions such as serum deprivation, high concentrations of glucose, and proinflammatory cytokines increased BASP1 mRNA and protein in human tubular epithelial cells. In normal cells, BASP1 localized to the cytoplasm, but in apoptotic cells, it colocalized with actin in the periphery. Overexpression of BASP1 induced cell death with features of apoptosis; conversely, small interfering RNA (siRNA)-mediated knockdown of BASP1 protected tubular cells from apoptosis. Supporting possible involvement of BASP1 in renal disease other than DN, we also observed significant upregulation of renal BASP1 in spontaneously hypertensive rats and a trend toward increased tubulointerstitial BASP1 mRNA in human hypertensive nephropathy. In summary, a combined functional genomics approach identified BASP1 as a proapoptotic factor in DN and possibly also in hypertensive nephropathy.

J Am Soc Nephrol 21: 610–621, 2010. doi: 10.1681/ASN.2009020227

Diabetic end-organ damage contributes to at least lated by a host of checks and balances with a multi- 233,000 yearly deaths in the United States alone, tude of positive and negative regulators, many of with diabetic nephropathy (DN) being the most which are disease and organ specific.6 common cause of ESRD.1 The diabetic milieu is known to induce apoptosis in various end-organ Received February 28, 2009. Accepted December 8, 2009. 2,3 systems and is believed to contribute to the grad- Published online ahead of print. Publication date available at ual loss of renal function and mass in DN, because www.jasn.org. apoptosis has been detected in tubular epithelial, C.L.’s current affiliation is Ciemat, Madrid, Spain; A.G.’s current endothelial, and interstitial cells of renal biopsy affiliation is Fresenius Biotech GMBH, Munich, Germany. 4,5 samples from patients with DN. Correspondence: Dr. Alberto Ortiz, Dialysis Unit, Fundacio´n Cell death via apoptosis is an active response to Jime´nez Diaz, Avd. Reyes Cato´licos 2, 28040 Madrid, Spain. an altered microenvironment and is characterized Phone: 34-91-5504940; Fax: 734-764-3157; E-mail: [email protected]; or Dr. Matthias Kretzler, Division of Nephrology, University of by the activation of specific intracellular pathways. Michigan, 1570 MSRB II, 1150 W. Medical Center Drive, Ann The presence of injurious factors and/or the lack of Arbor, MI 48109-0676. Phone: 734-764-3157; Fax: 734-763-0982; survival factors may lead to engagement of the ap- E-mail: [email protected] optotic molecular machinery. Apoptosis is modu- Copyright ᮊ 2010 by the American Society of Nephrology

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BASP1 (also neuron-enriched acidic protein, having a molecular mass of 22 kD/cortical cytoskeleton-associated protein) is a 23-kDa myr- istoilated protein originally isolated from brain extracts10,11 that shares 70% homology in human and rat.12 It was initially described as a brain-specific protein,10,11 but later studies re- vealed that BASP1 is also expressed by human endothelium,13 developing mammary gland, kidney, testis, and lymphoid tissues.12–16 BASP1 is involved in cytoskeletal and lipid raft dynamics, as well as in the nuclear regulation of transcription. However, a role in apoptosis has not been previously reported. BASP1 contains an ef- fector domain (ED) that dynamically binds to the plasma membrane, to calmodulin, and to actin fibrils. Reversible phosphorylation of ED by protein kinase C modulates these interactions.12 In the plasma membrane BASP1 local- izes to lipid rafts and may influence the behavior and composition of the membrane.17 In addition, BASP1 promotes actin dynamics, includ- ing loss of stress fibers and bleb formation.18 Ad- ditionally, BASP1 is present in the developing nephron structures of the embryonic kidney co- inciding with the transcriptional regulator Wilm’s tumor gene (WT1). In the adult kidney the main site of BASP1 expression is the podo- cyte, where it behaves as a transcriptional cosup- Figure 1. Integrative functional genomics screen for apoptosis mediators in DN. pressor of WT1. Low levels of BASP1 expression 138 of 18000 unique cDNA clones containing full-length open reading frames are present in the cytoplasm of tubular cells in induced cell death upon overexpression in HEK293 cells. Genome-wide expres- 15 sion analysis of renal biopsies from DN and control patients identified 12 of the vivo and in cell lines. 138 unique clones to be upregulated in the tubulointerstitium with q Յ 0.05 BASP1 expression is increased in human (Table 1). Of these, only BASP1 was positive in confirmatory apoptosis screening DN tubulointerstitium and in tubules from tests and was chosen for further analysis. experimental DN. BASP1 is also expressed by cultured renal tubular cells, where it is regu- The combination of unbiased gene expression profiling lated by stimuli that promote cell death and has a role in with focused data mining has proven to be a powerful tool to apoptotic cell death. expand our knowledge of relevant pathways and players in human disease.7,8 We have recently used this approach to find specific changes in apoptosis-related genes in the renal tubu- RESULTS lointersitium transcriptome of the human diabetic kidney using gene ontology.6 A key limitation of this method is the need Functional Genomics cDNA Screening for Cell-Death- of a priori knowledge of the cell-death-associated function of Inducing Genes the regulated transcript. Genome-wide functional assays for In a previous search for genes with a potential tumor suppres- the identification of novel cell-death-associated molecules sor phenotype, a high throughput (HT) functional genomics have recently been developed.9 For this study this strategy was screen was established to identify novel cell-death-inducing adapted to identify apoptosis-inducing full-length reading genes.9 In brief, 596,832 independent clones from a human frames in human embryonic kidney cells as a primary screen- embryo cDNA expression library and 17,680 defined full- ing step. The in vitro assay was followed by analysis of the length cDNA expression plasmids were screened for cell death regulation of the corresponding mRNA transcripts in human induction after transient transfection into human embryonic DN. From the genes passing both filter steps, brain acid soluble kidney (HEK293) cells. Of these, 194 unique cDNAs induced protein 1/brain-abundant signal protein 1 (BASP1) mRNA cell death and DNA fragmentation: 138 contained full-length and protein was induced in the tubulointerstitial compartment open reading frames (Figure 1). Thirty-nine genes were known of human DN. inducers or associated with apoptosis.

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The list of full-length cDNAs whose overexpression in- rRNA ratio was induced 2.4 times in DN compared with duced cell death in the HT functional screen was compared controls [mean Ϯ SD; control 0.5 Ϯ 0.25, DN 1.18 Ϯ 0.6, with genome-wide expression profiles from tubulointerstitial P ϭ 0.02]. BASP1 expression in nondiabetic renal diseases is compartments of DN biopsies.6,7 Twelve genes were upregu- summarized in Supplemental Table 1). Immunofluores- lated in DN (q Ͻ 0.05) and induced cell death in the HT func- cence confirmed an increase of tubular expression of BASP1 tional screen as assessed by ␤-galactosidase/chlorophenolred- in human DN compared with minimal detectable levels in ␤-D-galactopyranoside and DNA fragmentation assays in control kidneys (Supplemental Figure 1), as well as co-stain- HEK293 cells (Table 1). Of these genes, only BASP1 overex- ing for BASP1 and active caspase 3 in human DN (Figure 2B). pression yielded positive results in all three subsequent confir- The percentage of positive tubulointerstitial area was signifi- matory screens for caspase activation, externalization of phos- cantly higher in DN (18.25 Ϯ 2.49, versus MCD 7.2 Ϯ 3.11% phatidylserin, and loss of mitochondrial potential (Figure 1). and control kidney 0%, P Ͻ 0.05) and this was associated with an increased apoptosis rate (active caspase 3 staining DN BASP1 Is an Apoptosis Inducer and Is Upregulated in 5.25 Ϯ 2.96% of cells, versus MCD 0.6 Ϯ 0.89 versus control the Tubulointerstitium of DN kidney 0.33 Ϯ 0.57%, P Ͻ 0.05). In addition, total kidney Overexpression of BASP1-induced death as assessed by BASP1 protein was increased in two animal models of chronic ␤-galactosidase/chlorophenolred-␤-D-galactopyranoside kidney disease: diabetes induced by streptozotocin and hyper- and DNA fragmentation assays in HEK293 cells. BASP1 was tensive nephropathy of spontaneously hypertensive rats (Fig- also positive in screening tests for caspase activation, exter- ure 3, A and B). Immunohistochemistry localized increased nalization of phosphatidylserine, and of loss of mitochon- BASP1 expression to tubular epithelia in these models (Figure drial potential. BASP1 mRNA was increased 1.6-fold in the 3C). Lectin binding experiments identified proximal tubules as renal tubulointerstitium of DN patients when compared the main source of tubular BASP1 in DN (Supplemental Figure with controls (Figure 2A). No significant changes were ob- 2). As previously reported in rodent models of DN, an in- served in the glomerular compartment (1.09-fold change, creased tubular apoptosis rate was observed in diabetic kidneys data not shown). BASP1 mRNA expression correlated with (Supplemental Figure 3A).4,19–21 Apoptotic cells tended to be 24-hour proteinuria (R2 ϭ 0.40, P ϭ 0.04) in DN patients grouped in certain tubular epithelia where colocalization of but not in minimal change disease (MCD) patients (R2 ϭ active caspase 3 and BASP1 expression was noted (Supplemen- 0.14, P ϭ 0.61). In an independent cohort of patients with tal Figure 3B). DN, BASP1 mRNA was evaluated by quantitative real-time reverse-transcriptase PCR (RT-PCR). The BASP1-to-18S BASP1 Regulation in Cultured Human Tubular Epithelial Cells Table 1. Open reading frames capable of induction of cell Cultured human proximal tubular epithelial cells constitu- death when overexpressed and with concomitant tively express BASP1 mRNA and protein (Figure 4). Depri- upregulation in the tubulointerstitial compartment in vation of the survival factors from serum increased BASP1 human DN biopsies (q Յ 0.05)a mRNA (Figure 4A) and protein (Figure 4, B and C) expres- GeneSymbol FC LogFC q sion. Confocal fluorescence localized the protein to the perinuclear area (Figure 4D). This is consistent with the EMP3 1.75 0.81 0.00 known localization of BASP1 in human umbilical vascular BASP1 1.62 0.70 0.00 13,15 PLP2 1.61 0.69 0.00 endothelial cells and COS-1 cells. The increased protein ARL6IP5 1.41 0.50 0.00 expression found in serum-deprived cells was mainly local- PTPLAD1 1.39 0.48 0.00 ized to the perinuclear area. A peripheral location near the GNAS 1.38 0.47 0.00 cell membrane was more conspicuous in apoptotic cells, CYBA 1.33 0.41 0.00 where it colocalized with actin and ␣-tubulin in the cell PPIB 1.29 0.36 0.00 periphery (Figure 4E). BASP1 expression was also increased CLDN4 1.58 0.66 0.03 by a high glucose concentration and by proinflammatory TMEM59 1.22 0.29 0.03 cytokines (Figure 5 A and B), but not by mannitol used as an ITGB2 1.84 0.88 0.04 osmotic control (Ͻ5% variation versus control). Depriva- SSR4 1.31 0.39 0.04 tion of survival factors and exposure to a high glucose envi- FC, fold change over control; EMP3, epithelial membrane protein 3; PLP2, proteolipid protein 2 (colonic epithelium-enriched); ARL6IP5, ADP- ronment and proinflammatory cytokines are known induc- ribosylation-like factor 6 interacting protein 5; PTPLAD1, protein tyrosine ers of apoptosis in tubular cells.22–24 phosphatase-like A domain containing 1; GNAS, guanine nucleotide binding protein, alpha stimulating complex locus; CYBA, cytochrome b-245, alpha polypeptide; PPIB, peptidylprolyl isomerase B (cyclophilin B); CLDN4, claudin BASP1 Overexpression Induces Apoptosis in Tubular 4; TMEM59, transmembrane protein 59; ITGB2, integrin, beta 2 (complement Epithelial Cells component 3 receptor 3 and 4 subunit); SSR4, signal sequence receptor, Once we had demonstrated that BASP1 expression is regu- delta. aData are presented as the fold-change increase in mRNA expression in DN lated by an adverse microenvironment in tubular epithelia as assessed by Affymetrix GeneChip. in vivo and in cell culture, we approached the consequences

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Figure 2. Increased BASP1 expression in human DN. (A) BASP1 mRNA was increased in the renal tubulointerstitium of DN patients (n ϭ 11) when compared with the controls (n ϭ 7) (*q ϭ 0.001). There was no difference in BASP1 expression between living donors and MCD biopsies, thus they were grouped as controls. The array data are expressed as normalized log-transformed expression units. The box extends from 25th percentile to the 75th percentile and the line gives the median at the 50th percentile. (B) Most cells in a tubule from a type II DN biopsy co-stain for BASP1 (green) and active caspase 3 (red) in DN tubulointerstitium. Note co-stained cells in the merged image (yellow, arrows). Original magnification 400ϫ. of BASP1 overexpression. As predicted by the screening as- DISCUSSION say, overexpression of BASP1 decreased cell survival in a Recent studies suggest a role for cell death in the progression of dose-dependent manner (Figure 6A). Inhibitors of specific human DN.4,25 Gene ontology identified 112 cell-death-re- caspases, such as caspase 3 [Z-Asp(OMe)-Glu(OMe)- lated genes that were significantly differentially regulated in the Val-DL-Asp(OMe)-fluoromethylketone (DEVD)], caspase 8 tubulointerstitium of renal biopsies from DN patients.24 To [Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoro- search for further apoptosis regulatory genes not previously methylketone (IETD)], and caspase 9 [Z-Leu-Glu(OMe)-His- identified as such, we took advantage of HT screening for genes Asp(OMe)-fluoromethylketone (LEHD)], as well as multiple for which overexpression induced cell death. BASP1 was in- caspases [Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD)] pre- creased in the tubulointerstitium of human DN samples, in- vented the decrease in cell viability associated with BASP1 over- duced apoptosis upon overexpression, and was thereby iden- expression (Figure 6B). In addition P5, a KU-70-derived Bax in- tified as a novel death-related molecule. Animal experiments hibitory peptide, also prevented cell death (Figure 6B). confirmed that BASP1 expression is increased in tubular cells Overexpression of BASP1 also increased the percentage of hypo- in progressive nephropathies and cell culture demonstrated diploid apoptotic cells (Figure 7A) and of apoptotic cells staining induction of apoptosis upon BASP1 overexpression and protection for caspase-generated cytokeratin fragments (Figure 7B), as from apoptosis when BASP1 expression was suppressed. well as caspase 3 activity (Figure 7C). Cotransfection with a BASP1 is widely expressed. Sites of expression include the de- green fluorescent protein (GFP) plasmid allowed quantifi- veloping and adult kidney15 and mammary gland.14 The localiza- cation of apoptotic cells with the typical nuclear morphol- tion of the protein is cell-type-specific. Although its nuclear ex- ogy among transfected cells and showed that BASP1 over- pression in podocytes was recently stressed, the protein was also expression promoted apoptosis that was prevented by found in the cytoplasm of tubular and interstitial cells in vivo.15 zVAD or P5 (Figure 8). Taken together, these data suggest BASP1 contains a nuclear localization sequence, and BASP1 is the involvement of caspases and mitochondria in the cell exclusively nuclear in some cell lines.26 BASP1 associates with death process activated by BASP1 overexpression. WT1 in the nucleus of cells that naturally express both proteins (e.g., podocytes).15 Despite its wide expression, the functions of Inhibition of BASP1 Expression Protects against BASP1 have only been characterized in neurons and podocytes Apoptosis while remaining poorly understood in other cell types. Available A siRNA approach was used to assess the requirement of endog- data suggest that BASP1 is a multifunctional protein that may enous BASP1 for cell death induction in human kidney epithelial behave as a transcription factor and may have lipid raft and cy- cells. Western blot confirmed efficient gene silencing (Figure 9A). toskeletal organizational properties15,18,27; either of these proper- Deprivation of survival factors is a classical inducer of apoptosis ties could contribute to apoptosis. BASP1 regulates transcription that also upregulates BASP1 expression in tubular cells. HK2 cells by acting as a cosuppressor for WT1 in glomerular epithelial cells. underwent apoptosis when deprived of serum that was prevented The subcellular localization of BASP1 in the course of tubular by targeting of BASP1 (Figure 9B). BASP1 targeting also protected epithelial cellapoptosis points to a relationship between BASP1 from high-glucose-induced apoptosis but not from cytokine-in- actions and actin and/or lipid raft organization. Lipid rafts and the duced death (Figure 9, B and C). Surviving cells looked healthy by actin cytoskeleton are key determinants of the cell susceptibility to contrast-phase microscopy (not shown). apoptosis and there is an intimate relationship between them.28–30

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with the apoptotic actin ring. BASP1 also colocalizes at this location with tubulin in a pattern similar to the tubulin cocoon of apoptotic cells.36 Taken together, these data point to a previously undescribed proapoptotic role for BASP1. The precise localization of BASP1 in the chain of events that leads to apoptosis is not clear. Pre- vention of BASP1-induced apoptosis by Bax an- tagonists and caspase inhibitors suggests that it has a role upstream of mitochondrial injury and caspase activation or that, at least, participates in the positive feedback loop, leading to further mitochondrial injury and caspase activation.35 Re- cent reports have implicated changes in the dynamics of the actin cytoskeleton in mitochondrial injury and subsequent cell death.32,34 Thus, actin binding proteins such as gelsolin and cofilin regulate apoptosis at the mitochondrial injury and caspase activation levels.37–39 BASP1/cortical cytoskeleton-associated protein 23, growth-associated protein 43 (GAP43), and myristoylated alanine-rich C-kinase substrate (MARCKS) define a novel class of functionally related proteins (termed “GMC proteins”) that share the ED domain and certain properties (e.g., acyla- tion-mediated targeting to the cell membrane and association with the cortical cytoskeleton).18 BASP1, GAP43 and MARCKS bind to and can regulate key actin binding proteins such as gelsolin and cofilin.40,41 Although a role for BASP1 in apoptosis is novel, it has been suggested for other GMC proteins, although the molecular pathways are not well understood. GAP43 and a novel xenopus MARCKS-related protein induce apoptosis.42–44 Figure 3. BASP1 expression in experimental DN and hypertensive nephropathy. The ED domain is essential for the proapoptotic (A) Representative image of Western blot of whole kidney extracts (SHR, sponta- function of the Xenopus MARCKS-like protein.44 neously hypertensive rats). (B) Quantification of Western blot results. *P Ͻ 0.05 Phosphorylation of the ED in adducin precedes Ϯ versus Wistar control rats. Mean SD of 5 rats. (C) Immunohistochemistry localized caspase activation, actin cytoskeleton changes, increased BASP1 expression to tubular cells. Detail: Note an apoptotic BASP1- and apoptosis in tubular epithelia.45–47 stained cell in the tubular lumen. Original magnification 200ϫ and 1000ϫ. In summary, we have identified BASP1 as a pro- Cytoskeletal remodeling is a hallmark of apoptosis. Concomi- tein for which expression is increased in tubular cells in the course tantly with rounding of the apoptotic cell, actin rearranges into of tubulointerstitial injury in DN and in cultured cells stressed by a peripheral cortical ring.31,32 Appropriate remodeling of a proapoptotic microenvironment. BASP1 may contribute to the the actin cytoskeleton is required for cell survival, because apoptotic loss of tubular cells induced by an inadequate supply of disassembly and excessive crosslinking adversely affect sur- survival factors or by high glucose concentrations in DN. BASP1 vival.33,34 In tubular epithelia BASP1 overexpression in- expression may be increased in other nephropathies, suggesting a duces apoptosis and endogenous BASP1 expression is in- potential role in kidney disease of different etiologies. creased by stimuli that promote apoptosis. Conversely, BASP1 downregulation protects from serum deprivation CONCISE METHODS and high-glucose-induced apoptosis. Lack of protection from cytokine-induced cell death suggests that BASP1 may Functional Genomics cDNA Screening for Cell-Death- be critical for some, but not all, forms of apoptotic cell death Inducing Genes and supports a role in DN.35 In addition, in apoptotic cells The HT functional genomics approach has been previously detailed.9 BASP1 redistributes to the cell cortex where it colocalizes In brief, a cDNA expression library generated from human embryo

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tissue (Scinet, Braunschweig, Germany) and a full- length cDNA expression plasmid collection have been propagated in E. Coli SURE. In a robot cascade, individual colonies were picked and automatically trans- ferred for cDNA plasmid preparation in a 96-well for- mat followed by transient transfection into HEK293 cells. The cells were grown in DMEM medium/5% FCS in 96-well plates and transfected by calcium phosphate coprecipitation. Cotransfection with CRE-lacZ reporter plasmid encoding for ␤-galactosidase enabled assessment of growth and viability 48 hours later by stimulation with forskolin and measuring of ␤-galactosidase activity (chlorophenolred-␤-D-galactopyranoside, Roche). In total 596,832 independent clones from the human embryo cDNA library and all 17,680 defined full-length cDNA expression plasmids were screened. Positive clones inducing cell death by overexpression were verified after transient transfection by DNA-fragmentation ELISA (CDDϩ, Roche) and were bioinformatically annotated after sequencing. Flow cy- tometric determinations of active caspases in HeLa cells (CaspaTag assay with carboxyfluorescein and propidium iodide, Intergen, Oxford, United Kingdom), apoptotic membrane alterations (Annexin V, externalization of phosphatidylserine), and measurements of loss of mitochondrial potential with the fluorochrome DiOC6 were performed on selected genes.

Human Renal Biopsy Samples for Oligonucleotide-Array-Based Gene Expression Profiling Human renal biopsies were collected in a multicenter study, the European Renal cDNA Bank (ERCB; see Acknowlegdments). The biopsies were performed according to the local ethical committees Figure 4. Serum deprivation increases BASP1 expression in human proximal tu- after informed consent was obtained from the pa- bular epithelial HK2 cells. (A) Real-time RT-PCR analyses of BASP1 mRNA expression in cells cultured in the presence or absence of 10% FBS. Mean Ϯ SD of three tients and the samples processed according to the independent experiments. *P Ͻ 0.05 versus 10% FBS for 24 hours. (B) Quantifica- ERCB protocol.8 For oligonucleotide-array-based tion of Western blot results. Mean Ϯ SD of three independent experiments. *P Ͻ gene expression profiling, a total of 18 kidney biop- 0.05 versus 10% FBS for 24 hours. **P Ͻ 0.01 versus 10% FBS for 48 and 72 hours. sies from individual patients were included: 3 pre- (C) Representative Western blot of BASP1. Cells were cultured in the presence or transplantation kidney biopsies from living donors absence of 10% FBS for 72 hours. (D) Confocal microscopy confirmed increased [n ϭ 3, age 39 Ϯ 7 years, female:male ratio (F:M) 1:2, BASP1 expression in cells deprived of the survival factors from serum for 24 hours, serum creatinine Ͻ1 mg/dl, proteinuria absent], 4 although in the presence and absence of serum BASP1 was mainly cytoplasmic and biopsies with histologic diagnosis of MCD (n ϭ 4, perinuclear in live cells. By contrast, in apoptotic cells found among serum-deprived age 25 Ϯ 4.1 years, F:M 1:3, serum creatinine 1.0 Ϯ cells BASP1 displayed a peripheral distribution consistent with its presence in the 0.14 mg/dl, proteinuria 4.9 Ϯ 2.3 g/24 h), and 11 cell cortex. BASP1, green; propidium iodide, red. Original magnification 320ϫ. (E) biopsies with histologic diagnosis of DN (n ϭ 11, age Colocalization of BASP1 was further explored in cells undergoing apoptosis upon Ϯ Ϯ serum deprivation. BASP1 localized to the cell periphery of apoptotic cells with 58.3 3.2 years, F:M 3:8, diabetes duration 9.8 3.3 Ϯ pyknotic nuclei, where it appeared to colocalize with tubulin (arrows, upper panel, years, hemoglobin A1C levels 7.1% 0.3%, serum cre- conventional fluorescence microscopy). Confocal microscopy confirmed colocaliza- atinine 1.98 Ϯ 0.37 mg/dl, proteinuria 2.95 Ϯ 0.8 g/24 tion with tubulin (middle panel, confocal microcopy) and actin (arrow, lower panel, h). Detailed clinical and histologic patient characteris- confocal microscopy) in the periphery of apoptotic cells. In the two lower panels the tics have been recently published.48 image is focused on the apoptotic cells. Nevertheless, the different pattern of actin For validation quantitative, real-time RT-PCR filament distribution between the apoptotic cell (arrow) and the living cells is clearly analysis of biopsies from progressive DN samples (n ϭ observed in the lower panel. 7, age 57.83 Ϯ 3.15 years, F:M 2:5, serum creatinine

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Figure 5. BASP1 expression is increased by apoptosis-inducing stimuli. Cells exposed to high glucose concentration or proinflammatory cytokines (100 ng/ml TWEAK, 30 ng/ml TNF␣, and INF␥ 30 Figure 6. Overexpression of BASP1 induces caspase-dependent U/ml) for 48 hours. (A) Representative Western blot. (B) Quantifica- death in human tubular epithelial cells. (A) Dose-response curve: tion of Western blot results. Mean Ϯ SD of three independent Cells were co-transfected with a ␤-galactosidase expression plas- experiments *P Ͻ 0.05 versus control, **P Ͻ 0.01 versus control. mid and different amounts of BASP1 expression vector. ␤-galactosidase activity was quantified at 24 hours. Mean Ϯ SD of three independent experiments. *P Ͻ 0.05 versus control. (B) Protective 2.48 Ϯ 0.79 mg/dl, proteinuria 3.68 Ϯ 0.97 g/24 h), pretransplan- effect of caspase inhibition and Bax antagonism. Cells were trans- ϭ Ϯ tation kidney biopsies from living donors (n 6, age 63.3 8.44 fected with BASP1 expression vector and treated with 200 ␮M Ϯ years, F:M 1:5, serum creatinine 1.02 0.09 mg/dl, proteinuria DEVD, LEDH, P5, or zVAD. Cell survival was assessed at 24 hours. absent) using ABI assay on demand for BASP1 and 18S rRNA and Mean Ϯ SD of three independent experiments. *P Ͻ 0.05 versus the relative standard curve method for BASP1 quantification were control, #P Ͻ 0.005 versus BASP1 alone. performed as described.48 Cortical tissue segments were manually microdissected in glo- Probes, Invitrogen, S. Giuliano Milanese, Italy). Double-staining merular and tubulointerstitial compartments.7 Total RNA was iso- sections were sequentially incubated with the primary antibodies lated from the microdissected tubulointerstitial tissue. HG-U133A (rabbit polyclonal anti-BASP1, Abcam; mouse anti-active caspase 3, Affymetrics microarray chips displayed 22,283 probe sets. Image eBioscience Ltd., Hatfield, United Kingdom), followed by proper flu- files were initially obtained through Affymetrix GeneChip soft- orescence-labeled secondary antibodies (Alexa Fluor 488 goat anti- ware (MAS5). Subsequently, robust multichip analysis was applied rabbit IgG, Alexa Fluor 546 goat anti-mouse IgG, Invitrogen). Speci- using RMAexpress (http://statwww.berkeley.edu/users/bolstad/ ficity of antibody labeling was demonstrated by the lack of staining after RMAExpress) for the preprocessing of data, and significance anal- substituting proper control immunoglobulins (rabbit primary antibody ϳ ysis of microarrays (http://www-stat.stanford.edu/ tibs/SAM/) isotype control, Zymed, Invitrogen) for the primary antibody. Slides were was used to identify differentially expressed genes.7 mounted with Fluorsave aqueous mounting medium (Calbiochem, VWR International, Milan, Italy) and observed under a Zeiss Axioscope Immunofluorescence in Human Samples 40FL microscope equipped with an AxioCam MRc5 digital videocamera Stainings were performed by indirect immunofluorescence. and immunofluorescence apparatus (Carl Zeiss SpA). A total of 16 biop- Briefly, 5-␮m thick acetone-fixed frozen sections from human sies were studied: DN samples (n ϭ 8, serum creatinine 1.55 Ϯ 0.33 control and diabetic kidneys were incubated with the primary an- mg/dl, proteinuria 1.82 Ϯ 0.77 g/24 h), MCD samples (n ϭ 5, serum tibody (rabbit polyclonal anti-BASP1, AbCAM, Cambridge, creatinine 0.84 Ϯ 0.11 mg/dl, proteinuria 5.72 Ϯ 1.66 g/24 h), and con- United Kingdom), followed by fluorescence-labeled secondary an- trol kidney biopsy samples (n ϭ 3, serum creatinine 0.80 Ϯ 0.10 mg/dl, tibody (Alexa Fluor 546 goat anti-rabbit IgG (HϩL) (Molecular proteinuria 0.02 Ϯ 0.01 g/24 h).

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transferase-mediated dUTP nick-end labeling (In Situ Cell Death Detection Kit; Promega) as described previously. 51

Cell Lines and Reagents HK-2 human proximal tubular epithelial cells (ATCC, Rockville, MD) were grown on RPMI 1640 (Life Technologies, Grand Island, NY) with 10% heat-inactivated FBS, 2 mM glu- tamine, 100 U/ml penicillin, 100 ␮g/ml strepto- mycin, 5 ␮g/ml insulin, 5 ␮g/ml transferrin, 5 Figure 7. Cell death induced by BASP1 has features of apoptosis. (A) BASP1 induces ng/ml sodium selenite, and 5 ng/ml hydrocorti- apoptosis in tubular cells. Quantification of hypodiploid cells by flow cytometry of DNA sone in 5% carbon dioxide at 37°C. For experi- content. Note the increase in the hypodiploid, apoptotic population in cultured cells ments cells were cultured in serum-free media ␮ Ϯ transfected with 5 g BASP1 for 24 hours. Mean SD for three independent exper- 24 hours previous to the addition of the stimuli Ͻ iments. *P 0.05 versus control. (B) BASP1 overexpression increases the percentage and throughout the experiment. of apoptotic cells stained with caspase-specific cytokeratin fragments (cytodeath), as The pancaspase inhibitor zVAD (Bachem, assessed by flow cytometry. Mean Ϯ SD for three independent experiments. *P Ͻ 0.05 Bubendorf, Switzerland), the caspase 3 inhibitor versus control. (C) Caspase 3 activity is increased in tubular cells overexpressing BASP1. Mean Ϯ SD for three independent experiments #P Ͻ 0.005 versus control. DEVD, and the caspase 9 inhibitor LEHD (Calbio- Cells were transfected with 5 ␮g BASP1 for 20 hours. chem, San Diego, CA) were dissolved in DMSO and used at 200 ␮M.23 Final concentration of DMSO in culture was 0.05% and it did not influ- Animal Model ence apoptosis. Bax inhibitor peptide P5 (Tocris, Ellisville, MO) was used Diabetes was induced by a single intraperitoneal injection of streptozo- at 200 ␮M.52,53 tocin (Sigma, St. Louis, MO; 50 mg/kg in 0.01 M citrate buffer, pH 4.5)19 in ten 10-week-old Wistar Kyoto rats (Criffa, Barcelona, Spain). Ten con- Transient Transfection trol rats received the vehicle. Rats were sacrificed 7 months after induc- For transient transfection, cells were plated at a density of 8 ϫ 104 tion of diabetes. Additionally, ten 10-week-old spontaneously hyperten- cells/plate in six-well plates (Costar, Cambridge, MA) with RPMI sive rats were followed for 7 months. Insulin (1 to 4 IU subcutaneously, 1640 (10% FBS) 24 hours before transfection. Cells were trans- Insulatard NPH, Novo Nordisk, Denmark) was administered weekly to fected with 5 ␮g of BASP1 containing plasmid (unless otherwise prevent death from 7 days after administration of streptozotocin, having indicated) or empty vector and 1 ␮g of reporter vector (pCMV␤) checked that all of the animals had blood glucose levels Ͼ400 mg/dl. or GFP (kindly donated by Miguel Angel del Pozo, CNIC, Madrid, Systolic blood pressure was measured monthly in conscious, restrained Spain) using FuGENE 6 (Roche, Indianapolis, IN) according to the rats by a tail-cuff sphygmomanometer (NARCO, Biosystems, CO). The manufacturer’s protocol. The efficacy of transfection was visual- average of three separate measurements was calculated at each time ized by confocal microscopy or galactosidase staining. Real-time point. Albuminuria was assessed by ELISA every 24 hours (Celltrend, RT-PCR analyses of BASP1 mRNA in cells transfected with BASP1 Luckenwalde, Germany). Table 2 presents animal model data. All studies confirmed a 300-fold BASP1 overexpression. BASP1 was inserted were performed in accordance with the European Union normative. into modified pcDNA3 and sequenced to confirm its identity.

Immunostaining of Rat Tissue Samples Cell Death and Apoptosis Immunohistochemistry was carried out in 5-␮m thick paraffin-em- Cells were cultured to subconfluence in 12-well plates and transfected bedded rat tissue sections.49 The primary antibody was a rabbit poly- with BASP1 plasmid (5 ␮g) for 24 hours. Apoptosis was assessed by clonal anti-BASP1 (1:200). Sections were counterstained with Caraz- functional and morphologic studies. Flow cytometry of DNA content zi’s hematoxylin. Negative controls included incubation with a was used to quantitate apoptosis. Adherent cells were pooled with nonspecific Ig of the same isotype as the primary antibody. Some spontaneously detached cells; stained in 100 ␮g/ml propidium iodide, sections were subsequently incubated with the proximal tubule 0.05% NP-40, and 10 ␮g/ml RNAse A in PBS; and incubated at 4°C for marker, fluorescein-conjugated Tetranogolobus lotus (diluted 1:33) Ͼ1 hour. This assay permeabilizes the cells. Permeabilization allows (Sigma) or the collecting tubule marker, fluorescein-conjugated Doli- entry of propidium iodide in all cells, dead and alive. A lower DNA chos biflorum (diluted 1:100) (Sigma) lectins. These sections were content (hypodiploid cells) due to nuclear fragmentation character- mounted in 90% glycerol/PBS and photographed immediately.50 izes apoptotic cells; thus, this assay is not based on the known ability of Immunofluorescence was used to co-stain with anti-BASP1 anti- propidium iodide to enter dead cells. The percentage of apoptotic 54 body (1:100) and anti-active caspase 3 (1:100, Promega). Secondary cells with decreased DNA content (A0) was counted. Several pro- antibodies were anti-goat Alexa Fluor 488 and anti-rabbit Alexa Fluor apoptotic stimuli were studied, such as deprivation of growth factors 633 (1:300, Invitrogen). from serum, exposure to high (450 mg/dl) glucose concentration, Apoptotic cells were stained using the terminal deoxynucleotidyl- or exposure to a lethal cytokine cocktail (30 ng/ml TNF␣, 100

J Am Soc Nephrol 21: 610–621, 2010 BASP1 in Diabetic Nephropathy 617 BASIC RESEARCH www.jasn.org

Nagoya, Japan) was measured following the manufacturer’s instructions.23 In brief, cell extracts (100 ␮g of protein) were incubated in half-area 96-well plates at 37°C with 200 ␮M DEVD-pNA peptide in a total volume of 100 ␮l. The pNA light emission was quantified using a spectrophotometer plate reader at 405 nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase activity.

Survival Assays To provide an objective assessment of cell loss, the ␤-Galactosidase Enzyme Assay System (Promega) was used to assay ␤-galactosidase activity in lysates prepared from cells transfected with ␤-galactosidase reporter vector and BASP1 vector or empty vector. Cell lysates were prepared 24 hours after transfection and the absorbance was read at 420 nm with a Mi- croplate Absorbance Reader Anthos 2020 (Anthos Labtec Instruments, Wals, Austria). Absorbance for the control vector (empty vector) was considered to be 100% and percent survival refers only to the transfected population.55 To observe the morphology of ␤-galactosidase- transfected cells, 24 hours after transfection cells were fixed with 2% paraformaldehyde/0.2% glutar- aldehyde in PBS (5 minutes, 4°C), washed with PBS twice, and stained (1 mg/ml X-gal, 5 mM potassium

ferricyanide, 2 mM MgCl2, 0.02% NP-40, and 0.01% SDS) at room temperature until cells turn blue. Western Blot Figure 8. BASP1 overexpression promotes human tubular epithelial cell apo- Western blots were performed as described previ- ptosis. (A) Tubular cells were cotransfected with GFP plasmid and BASP1 or ously.23 Membranes were incubated overnight at 4°C empty vector for 24 hours. Note the presence of apoptotic nuclei (shrunk, with anti-BASP1 antibody (1:2000, AbCAM) or bright) among cells transfected with BASP1 (arrows). (B) Quantification of apo- mouse anti-tubulin monoclonal antibody (1:5000, ptotic cells expressed as percentage of cells with apoptotic nuclei among green Sigma, St. Louis, MO) followed by incubation with cells. Mean Ϯ SD for three independent experiments. #P Ͻ 0.005 versus control, **P Ͻ 0.01 versus BASP1, ##P Ͻ 0.004 versus BASP1. horseradish-peroxidase-conjugated secondary antibody (1:2000, Amersham, Aylesbury, United King- dom). Blots were developed with the enhanced ng/ml TWEAK, and 30 U/ml IFN␥) for 24 hours. In addition, flow chemiluminescence method following the manufacturer’s instruc- cytometry was used to quantify the appearance of a specific epitope tions (Amersham). generated by caspase cleavage of cytokeratin 18 and identified by the M30 cytodeath antibody (Roche Biochemicals, Mannheim, Confocal Microscopy Germany). Cells plated onto Labtek slides were fixed in 4% paraformaldehyde To assess the typical nuclear changes seen in apoptosis, cells and permeabilized in 0.2% Triton X-100 in PBS for 10 minutes each. were cultured in chamber slides (Labtek, Nunc, Naperville, IL), After washing in PBS cells were incubated overnight at 4°C with rabbit transfected with GFP and BASP1 plasmid, fixed with methanol: polyclonal anti-BASP1 antibody (1:100) (AbCAM), followed by acetone (1:1), and stained with 4Ј,6-diamidino-2-phenylindole 1-hour incubation with FITC secondary antibody (1:200, Sigma). Cell (Sigma) or FITC-M30 cytodeath stain (1:250 Roche) and BASP1. nuclei were counterstained with propidium iodide. After washing, Thus, the typical condensed, shrunk, and fragmented nuclei of cells were mounted in 70% glycerol in PBS and analyzed with a DM- apoptotic cells were identified by a laser scanning confocal micro- IRB confocal microscope (Leica DM, Bannockburn, IL).49 F-actin was scope (Leika). stained with 800 ␮M TRITC-phalloidin (Sigma) in the dark for 30 Caspase 3 activity was assessed in cells transfected with BASP1 minutes at room temperature and anti-␣-tubulin antibodies (1:200, plasmid (5 ␮g) or vehicle for 24 hours. Caspase 3 activity (MBL, Sigma) followed by a 1-hour incubation with TRITC secondary anti-

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Transfection of siRNA Cells were grown in six-well plates (Costar, Cambridge, MA) and transfected with a mixture composed of 10 nmol/ml siRNA (Am- bion, Applied Biosystems, Foster City, CA), Opti-MEM I Reduced Serum Medium, and siPORT Amine Transfection Agent (Ambion, Applied Biosystems). After 18 hours cells were washed and cultured for 48 hours in complete medium and serum-depleted for 24 to 72 hours. These time points were selected from a time course of BASP1 protein expression in response to siRNA. A negative control scrambled siRNA provided by the manufacturer did not re- duce BASP1 protein.

Statistical Analysis Results are expressed as mean Ϯ SD of at least three independent experiments. Significance at the P Ͻ 0.05 level was assessed by non- parametric Mann–Whitney test for two groups of data and Kruskal– Wallis or one-way analysis (ANOVA) with a Bonferroni post hoc cor- rection for analysis of differences between three or more groups by SPSS software (SPSS Inc., Chicago, IL). Correlation of BASP1 tubular expression to proteinuria in DN (n ϭ 11) was estimated using Pearson correlation method by GraphPad Prism version 3.00 (GraphPad Soft- ware, San Diego CA).

ACKNOWLEDGMENTS

We thank all members of the ERCB and their patients for their sup- port and cooperation. C.L. was funded by Instituto de Salud Carlos III Figure 9. Inhibition of BASP1 expression by siRNA prevents apoptosis in human tubular epithelial cells. (A) BASP1 siRNA de- (ISCIII), A.B.S. by ISCIII, and M.D.S.N. by Ministerio de Ciencia y creased the expression of BASP1 protein. (B) Cells transfected Tecnología-MCYT. This study was supported by the following grants: with BASP1 siRNA were protected from serum-deprivation and EU Framework V Program “Progressive Renal Disease” QLRT-2001- high glucose-induced apoptosis but not from cytokine-induced 01215, Comunidad de Madrid (08.2/0030.1/2003, S-BIO-0283–2006) apoptosis. Cell death was assessed by flow cytometry of DNA EU project DIALOK: LSHB-CT-2007-036644, MCYT SAF 2003/884, content (hypodiploid cells) after culture in the absence of FBS FIS 06/0046 y PI081564, FIS PS09/00447 ISCIII-RETICS: RedinREN (control, 5 mM glucose) and in the presence of high glucose (25 06/0016, and Programa de Intensificacio´n de la Actividad Investiga- ␣ ␥ Ϯ mM) or TWEAK/TNF /IFN for 48 hours. Mean SD of three dora in the Sistema Nacional de Salud of the ISCIII and the Agencia Ͻ independent experiments. *P 0.05 versus 0% FBS control, “Pedro Laín Entralgo.” We are indebted to Dr. Almut Nitsche and Dr. **P Ͻ 0.01 versus high glucose. Bodo Brunner (Sanofi-Aventis Deutschland GmbH) for DNA chip hybridizations and delivery of the data set of hybridization results. body (1:200, Sigma). Apoptosis was characterized by morphologic Mar Gonzalez García-Parren˜o helped with confocal microscopy and and functional criteria. Nuclei of formalin-fixed cells were stained Susana Carrasco with histologic studies. with 4Ј,6-diamidino-2-phenylindole (Vector Laboratories, Inc., Bur- Members of ERCB-Kroener Fresenius Bank at the time of this lingame, CA) to observe the typical morphologic changes. study: J.P. Rougier, P. Ronco, Paris; M. P. Rastaldi, G. D’Amico, Mi-

Table 2. Animal modela Weight SBP Glycemia Albuminuria Animal Gain (g) (mmHg) (mg/dl) (␮g/24 h) WKY 222 Ϯ 15 138.1 Ϯ 6.7 87 Ϯ 3 390 Ϯ 70 WKY-D 102 Ϯ 32c 136.2 Ϯ 7.6 422 Ϯ 4c 1071 Ϯ 247b SHR 160 Ϯ 11b 205.7 Ϯ 2d 90 Ϯ 6 1800 Ϯ 464c WKY, Wistar-Kyoto rats; WKY-D, diabetic WKY rats; SHR, spontaneously hypertensive rats; SBP, systolic blood pressure. aData at latest follow-up (month 7), except for glycemia, which represents mean values throughout the study. Mean Ϯ SD. bP Ͻ 0.02. cP Ͻ 0.002 versus WKY. dP Ͻ 0.05 versus WKY.

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