BASIC RESEARCH www.jasn.org BASP1 Promotes Apoptosis in Diabetic Nephropathy 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* *Nefrologı´a, Fundacio´n Jime´nez Dı´az, 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 spontane- ously hypertensive rats and a trend toward increased tubulointerstitial BASP1 mRNA in human hyper- tensive 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 610 ISSN : 1046-6673/2104-610 J Am Soc Nephrol 21: 610–621, 2010 www.jasn.org BASIC RESEARCH BASP1 (also neuron-enriched acidic protein, having a molecular mass of 22 kD/cortical cy- toskeleton-associated protein) is a 23-kDa myr- istoilated protein originally isolated from brain extracts10,11 that shares 70% homology in hu- man 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 dynam- ics, as well as in the nuclear regulation of tran- scription. 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 interac- tions.12 In the plasma membrane BASP1 local- izes to lipid rafts and may influence the behavior and composition of the membrane.17 In addi- tion, 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 us- ing 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. J Am Soc Nephrol 21: 610–621, 2010 BASP1 in Diabetic Nephropathy 611 BASIC RESEARCH www.jasn.org 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.