Albumin suppresses vascular endothelial growth factor via alteration of hypoxia-inducible factor/hypoxia-responsive element pathway

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Biochemical and Biophysical Research Communications 367 (2008) 305–310 www.elsevier.com/locate/ybbrc

Albumin suppresses vascular endothelial growth factor via alteration of hypoxia-inducible factor/hypoxia-responsive element pathway Pisut Katavetin a,b, Reiko Inagi a, Toshio Miyata c, Tetsuhiro Tanaka a, Ryoji Sassa d, Julie R. Ingelfinger e, Toshiro Fujita a, Masaomi Nangaku a,* a

Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan b Division of Nephrology, Department of Medicine, Chulalongkorn University, Bangkok, Thailand c Molecular and Cellular Nephrology, Institute of Medical Sciences and Department of Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan d Okazaki Kita Clinic, Aichi, Japan e Division of Pediatric Nephrology, Massachusetts General Hospital, Boston, MA, USA Received 7 December 2007 Available online 26 December 2007

Abstract Reduction of vascular endothelial growth factor (VEGF) expression plays a crucial role in chronic kidney disease (CKD). In order to clarify a cause of VEGF suppression in CKD, we examined an interaction between proteinuria and VEGF. Rat proximal tubular cells were subjected to hypoxia with or without albumin to mimic proteinuric conditions, and VEGF expression was assessed by real-time quantitative PCR and enzyme-linked immunosorbent assays. Albumin significantly reduced VEGF expression under hypoxia. Luciferase activity controlled by hypoxia-responsive element (HRE) was suppressed by albumin, demonstrating suppression of the hypoxia-inducible factor (HIF)/HRE pathway. Studies utilizing a proteasome inhibitor and a prolyl hydroxylase inhibitor showed that mechanisms of HIF/HRE pathway suppression by albumin load did not involve degradation of HIF protein levels. Further, albumin did not change HIF mRNA levels. Our data, for the first time, suggest a clear ‘link’ between proteinuria and hypoxia, the two principal pathogenic factors for CKD progression. Ó 2007 Elsevier Inc. All rights reserved. Keywords: VEGF; Albumin; Hypoxia; Proximal tubule; HIF; Proteinuria

Chronic kidney disease (CKD) is currently a major public health problem worldwide. Extensive evidences suggest that progression of CKD is largely due to final common pathways that are unrelated to the activity of the initial kidney disease [1–3]. Loss of peritubular capillaries with subsequent cellular hypoxia has been regarded as a major cause of tubulointerstitial injury which is the most important pathological changes leading to CKD progression [1,3,4]. Although several pathogenic mechanisms potentially contribute to the loss of capillaries, reduction of vascular endothelial growth factor (VEGF) is likely to be the most critical factor [5,6]. *

Corresponding author. Fax: +81 3 5800 8806. E-mail address: [email protected] (M. Nangaku).

0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.086

VEGF is a potent stimulator of angiogenesis and also a survival factor for endothelial cells. Expression of VEGF is primarily regulated by local oxygen tension through the hypoxia-inducible factor (HIF)/hypoxia-responsive element (HRE) pathway [7–9]. HIF is a transcription factor composed of two subunits, an oxygen-sensitive HIF-a subunit and a constitutively expressed HIF-b subunit. HIF-a protein is continuously synthesized, but it is rapidly degraded in the oxygen-dependent mechanisms [10,11]. Activity of the HIF/HRE pathway under normoxia is substantially suppressed by prolyl hydroxylation and subsequent proteasomal degradation of HIF-a protein [12,13]. Decreasing oxygen concentrations during hypoxia inhibits hydroxylation reaction and eventually rescues the HIF-a protein from proteasomal degradation. Consequently, the

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rescued HIF-a protein heterodimerizes with HIF-b, enters the nucleus, binds to the HRE in the regulatory region of the genes, and activates transcription of various HIFdependent genes including VEGF. Peritubular capillaries are likely to be regulated by renal tubular cells. VEGF produced by hypoxic proximal tubular cells can augment angiogenesis in vitro [14]. In the kidney, the loss of peritubular capillary density correlates well with reduced tubular VEGF expression in the remnant kidney model [5]. Recent studies using kidney biopsies from patients with diabetic nephropathy also demonstrated a correlation between reduced VEGF expression and peritubular capillary loss [15]. However, causes of reduced VEGF expression in CKD remain to be elucidated. Proteinuria is another principal risk factor for CKD progression [16–18]. The mechanisms of renal injuries from proteinuria have been proposed and are likely to be multifactorial [19]. We hypothesized that proteinuria may cause CKD progression by detrimentally altering renal tubular VEGF expression. In the present study, we performed studies in a proximal tubular cell line to clarify the effect of albumin on hypoxia-induced VEGF expression. Methods Cell culture and experimental design. Immortalized rat proximal tubular cells (IRPTC) [20] were cultured in normal glucose (5.5 mM) Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan) buffered with 25 mM HEPES (Sigma–Aldrich, St. Louis, MO, USA) at pH 7.4, supplemented with 5% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 50 U/ml penicillin, 50 lg/ml streptomycin, and 0.01 mM non-essential amino acids. The cells were grown at 37 °C under a humidified atmosphere of 5% CO2/95% air. IRPTC were synchronized for 24 h in serum-free DMEM at 80–90% confluence before the experiments. Albumin-containing culture medium (40 mg/ml) was prepared by addition of fatty acid free and endotoxin free bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA) into the serum free DMEM. Hypoxia was induced by incubation at 1% O2/5% CO2 balanced with nitrogen in a multigas incubator, APM-30D (ASTEC, Fukuoka, Japan). All experiments were performed in the serum free media to avoid the confounding effects of serum. To investigate the effect of albumin on cellular responses to hypoxia, IRPTC were incubated for 24 h in: (1) normal medium under normoxia as a control group; (2) albumin-containing medium (40 mg/ml) under normoxia; (3) normal medium under hypoxia (1% O2); (4) albumin-containing medium (40 mg/ml) under hypoxia. Experiments in HRE-luciferase stable-transfected IRPTC. The HREluciferase stable-transfected immortalized rat proximal tubular cells (HRELUC IRPTC) were developed as previously reported [21]. This transgene construct has a high sensitivity and high specificity of the HIF/HRE pathway activation [22]. The HRE-luciferase stable-transfected IRPTC (HRELUC IRPTC) were cultured for 24 h under the conditions described above. To further explore a mechanism that albumin down-regulated HIF/ HRE pathway, HRE-LUC IRPTC were also cultured under hypoxia for 24 h in culture medium containing 5 lM Z-Leu-Leu-al (MG132; Sigma– Aldrich), culture medium containing 40 mg/ml albumin, and 5 lM MG132, culture medium containing 500 lM ethyl 3,4-dihydroxybenzoate (EDHB; Sigma–Aldrich), or culture medium containing 40 mg/ml albumin, and 500 lM EDHB. Total RNA isolation and real-time RT-PCR. Total RNA was extracted with ISOGEN (Nippon gene, Tokyo, Japan). Reverse-transcription was carried out by ImProm-IITM Reverse Transcription System (Promega, Madison, WI, USA) using oligo (dT)15 primer and 1 lg of isolated RNA.

One-twentieth (v/v) of cDNA product was subjected to real-time PCR (iCycler iQ; Bio-Rad Laboratories, Hercules, CA) utilizing iQ SYBR Green Supermix (Bio-Rad) with the specific primers as follows: vascular endothelial growth factor (VEGF): 5 0 -TTACTGCTGTACCTCCAC-3 0 , 5 0 -ACAGGACGGCTTGAAGATA-3 0 ; heme oxygenase-1 (HO-1): 5 0 -TC TATCGTGCTCGCATGAAC-3 0 , 5 0 -CAGCTCCTCAAACAGCTCAA3 0 ; glucose transporter-1 (GLUT-1): 5 0 -CAGTTCGGCTATAACACCG GTGTC-3 0 , 5 0 -ATAGCGGTGGTTCCATGTTT-3 0 ; hypoxia-inducible factor-1 a (HIF-1 a): 5 0 -GCACAGTTACAGGATTCCAGCAGA-3 0 , 5 0 CCTTAGCAGTGGTCATTTCTTGAG-3 0 ; and b-actin: 5 0 -CTTTCTAC AATGAGCTGCGTG-3 0 , 5 0 -TCATGAGGTAGTCTGTCAGG-3 0 . After an initial hold of 15 min at 95 °C, the cDNA samples were cycled 40 times at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The mRNA levels of genes were normalized by b-actin. Measurement of VEGF protein. After 24 h, culture medium was collected, followed by immediate centrifugation for 10 min at 10,000 rpm. VEGF levels of cell culture supernatant were measured by quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using rat VEGF DuoSet (R&D Systems, Inc., Minneapolis, MN, USA). Reporter assay. The luciferase (LUC) activity of the HRE-LUC IRPTC cell lysate was determined using PicaGene firefly luciferase assay kit (Toyo Ink, Tokyo, Japan). The bioluminescent of the reaction was analyzed by Lumat 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). Measurement of prostaglandin E2. IRPTC were cultured in the media with or without albumin for 24 h as described above (n = 3 for each), and lipids were extracted from cells as described previously [23]. Measurements of prostaglandin E2 (PGE2) were performed utilizing a PGE2 EIA kit (Cayman Chemical, Ann Arbor, MI, USA). Statistical analysis. Data were expressed in means ± standard error of the mean. Unpaired t-tests were performed to compare the mean between two groups. One-way ANOVA with post hoc analysis by the least significant difference (LSD) method were used to determine the differences of mean between more than two groups. The significance level was set at 5% for all tests.

Results Albumin suppressed VEGF expression under hypoxia VEGF mRNA expression in IRPTC was estimated by quantitative real-time PCR analysis (Fig. 1A). VEGF mRNA expression under hypoxia was significantly increased (3.06 ± 0.57-fold versus the control group). In order to study effects of proteinuria on tubular cells, we added albumin in the culture media. The hypoxic response of VEGF mRNA expression was significantly blunted by albumin (1.51 ± 0.18-fold in the hypoxia and albumin group versus 3.06 ± 0.57-fold in the hypoxia group; p < 0.05). Albumin did not affect the VEGF mRNA expression in IRPTC cultured under normoxia (1.02 ± 0.38-fold versus the control group). We also measured VEGF protein in cell culture supernatants (Fig. 1B). Hypoxia increased the level of VEGF protein (from 107.0 ± 3.0 to 260.0 ± 13.0 pg/105 cells; p < 0.05). This hypoxia-induced VEGF protein expression was significantly blunted by albumin (176.6 ± 7.5 pg/105 cells in the hypoxia and albumin group versus 260.0 ± 13.0 pg/105 cells in the hypoxia group; p < 0.05). However, albumin did not alter the VEGF protein expression under normoxia (96.6 ± 8.6 pg/105 cells in the albumin group versus 107.0 ± 3.0 pg/105 cells in the control group).

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Fig. 2. Albumin suppressed the HIF/HRE pathway activation under hypoxia. The relative amount of luciferase activity was normalized by total protein and expressed as an arbitrary unit in which the hypoxia group value equaled to one. The relative amount of the HO-1 and GLUT1 mRNA was normalized by b-actin and expressed as an arbitrary unit in which the hypoxia group value equaled to one. Hypoxia, hypoxia without BSA; H + Alb, hypoxia with 40 mg/ml BSA; *p < 0.05 compared with the hypoxia group.

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Fig. 1. Albumin blunted the VEGF response to hypoxia (1% O2 for 24 h). (A) Quantitative real-time RT-PCR analysis of VEGF gene expression. The relative amount of VEGF mRNA was normalized by b-actin and expressed as an arbitrary unit in which the control group value equaled to one. (B) VEGF protein levels in cell culture supernatant. Concentrations of VEGF protein were measure by ELISA, normalized by number of cells and expressed as picogram per 105 cells. Control, normoxia without BSA glucose; Albumin, normoxia with 40 mg/ml BSA; Hypoxia, hypoxia without BSA; H + Alb, hypoxia with 40 mg/ml BSA; *p < 0.05 compared with the control group; #p < 0.05 compared with the hypoxia group.

inhibitor, EDHB (Fig. 3). Interestingly, MG132 as well as EDHB did not inhibit the suppressive effect of albumin (0.77 ± 0.05 versus 2.33 ± 0.12 and 0.87 ± 0.04 versus 2.19 ± 0.07, hypoxia plus albumin versus hypoxia alone, in the presence of MG132 and EDHB, respectively, p < 0.05 both), suggesting that albumin is unlikely to suppress HIF/HRE pathway activation by increasing degradation of HIF-a protein. We then explored an alternative mechanism of HIF suppression, decreasing mRNA expression. However, hypoxic HIF-1 a mRNA expression was 3

Albumin suppressed HIF/HRE pathway activation under hypoxia Since VEGF is primarily regulated by the HIF/HRE pathway, we further explored the effect of albumin on activation of the HIF/HRE pathway using HRE-LUC IRPTC (Fig. 2). LUC activity in HRE-LUC IRPTC served as a reporter of HIF/HRE pathway activation. Albumin significantly reduced LUC activity under hypoxia (0.62 ± 0.06-fold versus the hypoxic control, p < 0.05). Hypoxic up-regulation of other downstream HIF/HRE dependent genes, HO-1 and GLUT-1, was also blunted by albumin (0.70 ± 0.09 and 0.80 ± 0.07-folds versus the hypoxic control, p < 0.05 both). Next, we explored a mechanism that albumin suppresses the HIF/HRE pathway. Because the HIF/HRE pathway is basically down-regulated by prolyl hydroxylation and subsequent proteasomal degradation of HIF-a protein, we first examined the involvement of these mechanisms using a proteasome inhibitor, MG132, and a prolyl hydroxylase

Relative LUC activity

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Fig. 3. Proteasome inhibitor and prolyl hydroxylase inhibitor did not abolish HIF/HRE pathway suppression by albumin. The relative amount of luciferase activity was normalized by total protein and expressed as an arbitrary unit in which the hypoxia group value equaled to one. Control, HRE-LUC IRPTC were cultured in normal media; MG132, HRE-LUC IRPTC were cultured in media containing 5 lM Z-Leu-Leu-al (MG132); EDHB, HRE-LUC IRPTC were cultured in media containing 500 lM ethyl 3,4-dihydroxybenzoate (EDHB); Hypoxia, hypoxia without BSA; H + Alb, hypoxia with 40 mg/ml BSA; *p < 0.05, Hypoxia versus H + Alb.

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Relative HIF mRNA expression

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Hypoxia

H+Alb

Fig. 4. Albumin did not suppress the HIF mRNA expression under hypoxia. The relative amount of the HIF-1 a mRNA was normalized by bactin and expressed as an arbitrary unit in which the hypoxia group value equaled to one. Hypoxia, hypoxia without BSA; H + Alb, hypoxia with 40 mg/ml BSA.

not significantly decreased by albumin under hypoxic conditions (Fig. 4). We also measured levels of PGE2, which is known to be involved in the nuclear translocation of HIF1, within the cell. We did not observe significant changes of PGE2 in cells treated with albumin when compared with control cells (91 ± 39 versus 100 ± 15 of % control, p = 0.84; representative results from two independent sets of experiments).

Discussion The present studies demonstrated that albumin suppresses the hypoxic VEGF response in cultured proximal tubular cells, at both mRNA and protein levels. We used fatty acid free and endotoxin free albumin, alleviating the concern that a non-proteinaceous compound attached to albumin in the manufacturing process might mediate the observed effect in our studies. Previous papers reported various protein concentrations in proximal tubules in normal animals by means of micropuncture techniques [24– 26], and we employed the albumin concentrations based on previous studies of the model of albumin overload in this study [27–31]. VEGF is a principal growth factor for endothelial cells. In the kidney, VEGF expression in renal tubular epithelium is likely to play a substantial role in the maintenance of peritubular capillaries [5,11]. The hypoxia-induced increase in VEGF levels in proximal tubules could be viewed as a feedback mechanism that helps to ensure adequate cellular oxygenation by maintaining peritubular capillary flow. If this feedback loop is altered, the integrity of the peritubular vasculature becomes impaired, leading to loss of functional peritubular capillaries. Albumin loading, which was shown to suppress the hypoxia-induced VEGF response in the present study, is therefore likely to lead to loss of peritubular capillaries, tubulointerstitial hypoxia and, eventually, to progression of CKD in proteinuric

patients. Our findings not only provide a plausible cause of the reduced VEGF expression in CKD, albumin overloading, but also suggest a possible mechanism of renal injuries caused by proteinuria, reduction of VEGF with subsequent hypoxia. In addition, proteinuria has been demonstrated to activate the local renin-angiotensin system in the kidneys, which can also lead to vasoconstriction with subsequent hypoxia [32]. These results reconcile the two putative pathogenic factors of CKD progression, proteinuria and hypoxia. Recent studies demonstrated a correlation of an inverse correlation between renal VEGF expression and degree of proteinuria in diabetic nephropathy [32]. While our findings suggest that proteinuria reduces VEGF expression, other studies suggested that neutralization of VEGF might, in turn, cause proteinuria [33,34]. It is clear that these two reciprocal mechanisms can create a vicious cycle of renal destruction, perpetually increasing proteinuria and decreasing renal VEGF expression with subsequent capillary loss. The blunted hypoxic response by albumin was also evident in other hypoxia-inducible genes expressed in cultured proximal tubular cells such as HO-1 and GLUT-1. These genes, as well as VEGF, are mainly regulated by the HIF/HRE pathway. Moreover, the hypoxic HIF/HRE pathway activation, measured by luciferase activity in IRPTC transfected with HRE-LUC, was suppressed by albumin. Taken together, these findings suggest that albumin suppressed VEGF expression by altering the HIF/ HRE pathway. The proximal tubule can reabsorb albumin via a clathrin-dependent receptor-mediated endocytic pathway (reviewed in [35]). Exposure of tubular cells to albumin activates a wide array of diverse intracellular signaling pathways. Accumulating evidence suggests that albumin may induce cellular responses by membrane receptor-mediated signaling [36], or by endocytosis-associated mechanisms [28,37]. We examined possible mechanisms of HIF suppression by albumin load. The HIF/HRE pathway is physiologically controlled by continuous synthesis and oxygendependent degradation of HIF-a protein by prolyl hydroxylation and subsequent proteasomal digestion. When cells were treated with the proteasome inhibitor, MG132, and the prolyl hydroxylase inhibitor, EDHB, induction of HRE-LUC activity by hypoxia was enhanced, probably due to more complete shutdown of the degradation pathway. However, the proteasome inhibitor and the prolyl hydroxylase inhibitor did not inhibit the suppressive effect of albumin on hypoxic HIF/HRE pathway activation. These data suggested that albumin does not suppress HIF/HRE pathway activation by enhancing HIF-a protein degradation. On the other hand, hypoxic HIF-1 a mRNA expression was not significantly changed by albumin, indicating that albumin does not suppress HIF-1 a synthesis at the level of mRNA expression. PGE2 is known to be involved in the nuclear translocation of HIF-1 [38], and

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deprivation of PGE2 by fatty acid free albumin may have led to suppression of the HIF/HRE pathway. However, our measurements of PGE2 in the cells showed that sequestration of PGE2 did not contribute to suppression of the HIF/HRE pathway under our experimental conditions. Other possible mechanisms of HIF regulation include suppression of HIF-1 a protein translation, reduction of HIF transport into the nucleus, modulation of the HIF transcriptional activity, or combination of these mechanisms. The exact mechanism that albumin suppresses HIF/HRE pathway activation remains to be elucidated in future studies. In summary, we have provided evidence that albumin blunts the hypoxia-induced VEGF response in IRPTC. These effects are likely to be mediated by suppression of the HIF/HRE pathway, suggesting a potential ‘link’ between proteinuria and hypoxia, the two principal pathogenic factors for CKD progression. Acknowledgments This work was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (19390228) to M.N. and (19590939) to R.I. We are grateful to Prof. Kriang Tungsanga (Chulalongkorn University, Bangkok, Thailand) and Dr. Tomoko Takano (McGill University, Quebec, Canada) for their supports. References [1] K.A. Nath, Tubulointerstitial changes as a major determinant in the progression of renal damage, Am. J. Kidney Dis. 20 (1992) 1–17. [2] B.M. Brenner, Remission of renal disease: recounting the challenge, acquiring the goal, J. Clin. Invest. 110 (2002) 1753–1758. [3] M. Nangaku, Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure, J. Am. Soc. Nephrol. 17 (2006) 17–25. [4] K.U. Eckardt, W.M. Bernhardt, A. Weidemann, C. Warnecke, C. Rosenberger, M.S. Wiesener, C. Willam, Role of hypoxia in the pathogenesis of renal disease, Kidney Int. Suppl. (2005) S46– S51. [5] D.H. Kang, A.H. Joly, S.W. Oh, C. Hugo, D. Kerjaschki, K.L. Gordon, M. Mazzali, J.A. Jefferson, J. Hughes, K.M. Madsen, G.F. Schreiner, R.J. Johnson, Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular endothelial growth factor and thrombospondin-1, J. Am. Soc. Nephrol. 12 (2001) 1434–1447. [6] M. Matsumoto, T. Tanaka, T. Yamamoto, E. Noiri, T. Miyata, R. Inagi, T. Fujita, M. Nangaku, Hypoperfusion of peritubular capillaries induces chronic hypoxia before progression of tubulointerstitial injury in a progressive model of rat glomerulonephritis, J. Am. Soc. Nephrol. 15 (2004) 1574–1581. [7] A.P. Levy, N.S. Levy, S. Wegner, M.A. Goldberg, Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia, J. Biol. Chem. 270 (1995) 13333–13340. [8] Y. Liu, S.R. Cox, T. Morita, S. Kourembanas, Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5 0 enhancer, Circ. Res. 77 (1995) 638–643. [9] J.A. Forsythe, B.H. Jiang, N.V. Iyer, F. Agani, S.W. Leung, R.D. Koos, G.L. Semenza, Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1, Mol. Cell. Biol. 16 (1996) 4604–4613. [10] C.W. Pugh, P.J. Ratcliffe, Regulation of angiogenesis by hypoxia: role of the HIF system, Nat. Med. 9 (2003) 677–684.

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