Erythrophagocytosis of Lead-Exposed Erythrocytes by Renal Tubular Cells: Possible Role in Lead-Induced Nephrotoxicity

Background: Nephrotoxicity associated with lead poisoning has been frequently reported in epidemiological studies, but the underlying mechanisms have not been fully described. Objectives: We examined the role of erythrocytes, one of the major lead reservoirs, in lead-associated nephrotoxicity. Methods and results: Co-incubation of lead-exposed human erythrocytes with HK-2 human renal proximal tubular cells resulted in renal tubular cytotoxicity, suggesting a role of erythrocytes in lead-induced nephrotoxicity. Morphological and flow cytometric analyses revealed that HK-2 cells actively phagocytized lead-exposed erythrocytes, which was associated with phosphatidylserine (PS) externalization on the erythrocyte membrane and generation of PS-bearing microvesicles. Increased oxidative stress and up-regulation of nephrotoxic biomarkers, such as NGAL, were observed in HK-2 cells undergoing erythrophagocytosis. Moreover, TGF-β, a marker of fibrosis, was also significantly up-regulated. We examined the significance of erythrophagocytosis in lead-induced nephrotoxicity in rats exposed to lead via drinking water for 12 weeks. We observed iron deposition and generation of oxidative stress in renal tissues of lead-exposed rats, as well as the histopathological alterations such as tubulointerstitial lesions, fibrosis, and up-regulation of KIM-1, NGAL, and TGF-β. Conclusions: Our data strongly suggest that erythrophagocytosis and subsequent iron deposition in renal tubular cells could significantly enhance nephrotoxicity following lead exposure, providing insight on lead-associated kidney damages. Citation: Kwon SY, Bae ON, Noh JY, Kim K, Kang S, Shin YJ, Lim KM, Chung JH. 2015. Erythrophagocytosis of lead-exposed erythrocytes by renal tubular cells: possible role in lead-induced nephrotoxicity. Environ Health Perspect 123:120–127; http://dx.doi.org/10.1289/ehp.1408094


Introduction
Although environmental lead contamination has declined significantly since the 1970s, lead exposure is still observed in children and industrial workers, and even in the general population (Hernberg 2000). The average adult blood lead level (BLL) is 1-2 μg/dL, and the U.S. Centers for Disease Control and Prevention (CDC) defines lead poisoning as a BLL > 10 μg/dL (0.5 μM) (CDC 1997). Epidemiological and toxicological studies have reported lead-induced toxicity in the nervous, cardiovascular, and renal systems [Agency for Toxic Substances and Disease Registry (ATSDR) 2007]. The association between lead exposure and nephro toxicity has been well-established, even in a population with BLLs as low as 5 μg/dL (Ekong et al. 2006). Damage in kidney function is associated with albuminuria, reduced glomeru lar filtration rate, and decreased creati nine clearance in lead-exposed populations (Fadrowski et al. 2010;Navas-Acien et al. 2009). Histopathologically, renal impair ment associated with lead poisoning is charac terized by proximal tubular nephropathy, glomeru lar sclerosis, and fibrosis in peritubular and interstitial lesions (Cramér et al. 1974;Diamond 2005;Goyer 1989; Loghman- Adham 1997).
Oxidative stress has been suggested to be the most convincing mechanism underlying lead-associated nephrotoxicity (Daggett et al. 1998;Wang et al. 2009). Pro-oxidant and anti oxidant balance, along with decreased glutathione and increased lipid peroxidation, occurs in the kidney following lead exposure in animal models (Daggett et al. 1998;Liu et al. 2012b;Patra et al. 2001;Wang et al. 2010). There have been several attempts to determine how lead increases oxidative stress in the kidney (Stacchiotti et al. 2009;Wang et al. 2009Wang et al. , 2011, but the exact mechanism(s) has not been clearly elucidated.
There is increasing evidence (Madsen et al. 1982;Mimura et al. 2008;Sheerin et al. 1999;Trump et al. 1969) that the kidney may play a role in the clearance of erythrocytes. Infiltration of erythro cytes has been observed in proximal tubules and tubular lumen of renal biopsies from patients with acute glomerulo nephritis and hematuria (Trump et al. 1969) as well as in those from patients with acute renal failure (Mimura et al. 2008). Iron deposition in the kidney was also found in patients with various renal diseases (Wang et al. 2001), suggesting that the retention of iron-rich erythro cytes in the kidney may play a role in the pathogenesis of kidney diseases. Proximal tubular epithelial cells are capable of phagocytizing and degrading erythro cytes (Madsen et al. 1982;Sheerin et al. 1999), a phenomenon known as erythro phagocytosis. Erythrophagocytosis, which is primarily carried out by macrophages in the spleen and liver (Knutson and Wessling-Resnick 2003;Otogawa et al. 2007), occurs when aged or damaged erythro cytes are phagocytized and cleared from systemic circulation. This process is mediated by externalized phosphatidyl serine (PS) on the outer membrane (Kobayashi et al. 2007;Mercer and Helenius 2008) and by PS-bearing microvesicles (MVs) (Knutson and Wessling-Resnick 2003;Otogawa et al. 2007). Proximal tubule cells have been reported to actively phagocytize erythro cytes in renal injury (Madsen et al. 1982;Sheerin et al. 1999), but the toxicological significance of this process in the etiology of heavy metal-associated renal diseases remains to be established.
More than 99% of blood lead accumulates in erythrocytes, suggesting that erythrocytes may be a major target of systemic lead poisoning (Hernández-Avila et al. 1998;Schütz et al. 1996). We recently demonstrated that lead significantly increased PS externalization in erythro cytes and enhanced erythro phago cytosis by macrophages in the spleen (Jang et al. 2011). Fowler et al. (1980 observed iron deposition in renal proximal tubule cells, along with lead-associated histopathological lesions in the kidney, following adminis tration of lead via drinking water to rats. In the present study we examined the role of erythro cytes in lead-induced nephro toxicity in vitro in a co-culture system as well as in vivo in rats. On the basis of available evidence, we hypothesized that lead-induced PS externalization in erythro cytes promotes erythrophagocytosis by renal tubular cells, contributing to lead-associated kidney damage. Background: Nephrotoxicity associated with lead poisoning has been frequently reported in epidemiological studies, but the underlying mechanisms have not been fully described. oBjectives: We examined the role of erythrocytes, one of the major lead reservoirs, in leadassociated nephrotoxicity. Methods and results: Co-incubation of lead-exposed human erythrocytes with HK-2 human renal proximal tubular cells resulted in renal tubular cytotoxicity, suggesting a role of erythro cytes in lead-induced nephrotoxicity. Morphological and flow cytometric analyses revealed that HK-2 cells actively phagocytized lead-exposed erythro cytes, which was associated with phosphatidylserine (PS) externalization on the erythro cyte membrane and generation of PS-bearing microvesicles. Increased oxidative stress and up-regulation of nephrotoxic biomarkers, such as NGAL, were observed in HK-2 cells undergoing erythrophagocytosis. Moreover, TGF-β, a marker of fibrosis, was also significantly up-regulated. We examined the significance of erythro phago cytosis in leadinduced nephrotoxicity in rats exposed to lead via drinking water for 12 weeks. We observed iron deposition and generation of oxidative stress in renal tissues of lead-exposed rats, as well as the histopathological alterations such as tubulo interstitial lesions, fibrosis, and up-regulation of KIM-1, NGAL, and TGF-β. conclusions: Our data strongly suggest that erythrophagocytosis and subsequent iron deposition in renal tubular cells could significantly enhance nephrotoxicity following lead exposure, providing insight on lead-associated kidney damages. citation: Kwon SY, Bae ON, Noh JY, Kim K, Kang S, Shin YJ, Lim KM, Chung JH. 2015. Erythrophagocytosis of lead-exposed erythro cytes by renal tubular cells: possible role in lead-induced nephrotoxicity. Environ Health Perspect 123:120-127; http://dx.doi. org/10.1289/ehp.1408094

In Vitro Experiments
Preparation of erythro cytes. After obtaining approval from the Ethics Committee of Health Service Center at Seoul National University, we obtained human blood from healthy Korean male donors who provided informed consent (n = 20; 20-29 years of age). Blood was collected using a vacutainer containing acid citrate dextrose via a 21-gauge needle (Becton Dickinson, Franklin Lakes, NJ, USA) on the day of the experiment. Platelet-rich plasma and buffy coat were removed by aspiration after centrifugation at 300 × g for 15 min. Packed erythro cytes were washed three times with sterilized phosphatebuffered saline (PBS: 1 mM KH 2 PO 4 , 154 mM NaCl, 3 mM Na 2 HPO 4 , pH 7.4) and once with filter-sterilized Ringer's solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 32 mM HEPES, 5 mM glucose, pH 7.4). Washed erythro cytes were resuspended in Ringer's solution to a cell concentration of 5 × 10 7 cells/mL. CaCl 2 (final concentration, 1 mM) was added to erythrocytes prior to use. The erythro cytes were used immediately after isolation without storage.
Cell culture. We used renal proximal tubular cells, the cell population most susceptible to xenobiotic-induced toxicity in the kidney. Human proximal tubular epithelial cells [HK-2; ATCC (American Type Culture Collection), Manassas, VA, USA] were maintained in keratino cyte serum-free media supplemented with recombinant epidermal growth factor, bovine pituitary extract (BPE), and 1% penicillin/streptomycin at 37°C under a 5% CO 2 atmosphere (Ryan et al. 1994).
Cell viability measurement. Cell viability was measured using the MTT assay as previously described (Thiébault et al. 2007) with slight modification. To evaluate the effect of Pb 2+ on cell viability, we incubated HK-2 cells (2 × 10 5 cells/well in 6-well plates) with either vehicle (1% distilled water) or Pb 2+ (10 or 20 μM) for 24 hr. Cells were then incubated with MTT (0.5 mg/mL) for 2 hr and washed. The converted formazan was dissolved in 100% DMSO, and the absorbance at 570 nm was measured in a SpectraMax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
In experiments to examine the contribution of erythrocytes, erythrocytes were incubated with vehicle or Pb 2+ (10 or 20 μM) for 1 hr and then removed by centrifugation. HK-2 cells were co-incubated with Pb 2+ -treated erythro cytes (1 × 10 7 cells/well) for 24 hr. After co-incubation, erythrocytes were removed and MTT was added as described above.
Morphological examination. Erythrocytes were treated with either vehicle or Pb 2+ (20 μM) for 1 hr and washed. HK-2 cells were then co-incubated with the vehicle-or Pb 2+ -treated erythro cytes for 3 hr. Morphology was examined in order to measure the interaction between erythro cytes and HK-2 cells using phase-contrast microscopy (Olympus IX70; Olympus Corporation, Tokyo, Japan). For scanning electron microscopic observation (Jang et al. 2011;Noh et al. 2010), HK-2 cells co-incubated with vehicle-or Pb 2+ -treated erythro cytes were fixed with 2% glutaraldehyde for 1 hr at 4°C. Cells were washed three times with PBS and treated with 1% osmium tetroxide for 30 min at room temperature. After washing with PBS, the samples were dehydrated serially in 50%, 70%, 90%, and 100% ethanol. After drying and coating with gold, the images were obtained on scanning electron microscope (JEOL, Tokyo, Japan).
Measurement of in vitro erythro phago cytosis. After treatment with vehicle or Pb 2+ (10 or 20 μM) for 1 hr, erythro cytes were treated with 10 μM CFDA for 30 min. HK-2 cells were then co-incubated with vehicleor Pb 2+ -treated erythro cytes for 3 hr. After co-incubation, HK-2 cells were harvested and washed several times to remove remnant erythro cytes. Anti-CD13-PE was used to identify HK-2 cells. Samples were analyzed on a FACScalibur flow cytometer equipped with an argon ion laser emitting at 488 nm (Becton Dickinson). Cells were identified based on their forward and side scatter (FSC and SSC, respectively) characteristics. The fluorescence signals from FL1 and FL2 were also analyzed. We collected and analyzed data from 10,000 events positive for PE (FL1) using CellQuest Pro software (Becton Dickinson). Cells with double positive signals for PE (FL1) and CFDA (FL2) were counted as HK-2 cells with erythrophagocytosis.
To investigate the role of externalized PS in erythrophago cytosis, we used purified annexin V (0.5 μM) that binds to the externalized PS and interrupts PS-mediated phenomena. Erythrocytes were incubated with purified annexin V for 2 min and then exposed to lead for 1 hr. Erythrophagocytosis was measured as described above.
Analysis of PS exposure and MV genera tion. Erythrocytes were treated with vehicle or Pb 2+ (10 or 20 μM) for 1 hr at 37°C, and then incubated with annexin V-FITC, anti-glycophorin A antibody, and CaCl 2 (2.5 mM) for 30 min at room temperature in the dark to detect PS externalization. PE-labeled anti-glycophorin A was used to identify erythro cytes and erythrocyte-derived MVs. Isotype controls for measurement of non specific annexin V binding were stained with annexin V-FITC in the presence of EDTA (2.5 mM) instead of CaCl 2 . Samples were analyzed on the flow cytometer as described above.

Measurement of reactive oxygen species (ROS) in vitro.
Erythrocytes treated with vehicle or Pb 2+ (10 or 20 μM) for 1 hr were then loaded with 10 μM of calcein red-orange for 30 min. The HK-2 cells were loaded with H 2 DCFDA for 30 min and then co-incubated with vehicle-or Pb 2+ -treated erythro cytes for 4 hr. After co-incubation, HK-2 cells were harvested and washed several times to remove excess erythrocytes. Anti-CD13-perCP-Cy5.5 was used to identify HK-2 cells. Samples were analyzed by flow cytometry. As described above, HK-2 cells with erythro phagocytosis were defined as cells with double positive signals for perCP-Cy5.5 (FL3) and calcein red-orange (FL1). ROS generation was analyzed using the dichloro fluorescein signal from the cells with erythrophagocytosis.
In vitro quantitative realtime polymerase chain reaction (PCR). After co-incubation of HK-2 cells with vehicle-or Pb 2+ -treated erythro cytes for 4 hr, total mRNA was isolated from HK-2 cells using the Easy-Blue Total RNA Extraction Kit (Intron Biotechnology, Seongnam, Korea). Isolated mRNA was quantified using a Nanodrop spectro photometer (Thermo Scientific, Wilmington, DE, USA), and cDNA was synthesized from isolated RNA using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). We used quantitative real-time PCR (qRT-PCR) to determine the mRNA levels for NGAL (neutrophil gelatinase-associated lipocalin) and KIM-1 (kidney injury molecule-1), both or which are representative bio markers of nephro toxicity, and TGF-β (transforming growth factor β), an important mediator of renal fibrosis. volume 123 | number 2 | February 2015 • Environmental Health Perspectives qRT-PCR was conducted using 2× SYBR green reaction buffer mixed with 0.5 μg cDNA and forward/reverse primers. Quantification of gene copies was carried out on a CFX96™ Real-Time PCR Detection System using iQ™ SYBR Green supermix (both from Bio-Rad). PCR cycles consisted of an initial step at 95°C for 3 min followed by 45 cycles at 95°C for 10 sec, 55°C for 30 sec, and 72°C for 10 sec. Relative mRNA expressions were calculated by the comparative CT method (2 -ΔΔCt ) and normalized to the endogenous 18S control. The specific primer sequences were as follows:

In Vivo Experiments
Animal treatment. All animal protocols were approved by the Ethics Committee of the Animal Service Centre at Seoul National University, and the animals were treated humanely and with regard for alleviation of suffering. Male Sprague-Dawley rats (Samtako Co., Korea) weighing 200-250 g were used in all experiments. Before the experiments, animals were acclimated for 1 week in the laboratory animal facility, maintained at constant temperature (22 ± 2°C) and humidity (55 ± 5%) with a 12-hr light/dark cycle. Three rats were housed per cage (width, 260 mm; depth, 420 mm; height, 180 mm). Food (Purina Mills, Seongnam, Korea) and water were provided ad libitum. Lead was not assessed in food or untreated water, but BLLs in untreated rats were below the detection limit (0.5 ppb). We conducted our in vivo experiments in two independent trials, with rats randomly assigned to treatment groups. In the first trial, rats (n = 4/treatment group) were treated with 0 ppm or 1,000 ppm Pb 2+ in drinking water for 12 weeks. In the second trial, rats were treated with Pb 2+ at 0, 250, or 1,000 ppm (n = 5, 6, and 5, respectively) in drinking water for 12 weeks, and total mRNA was isolated from rat kidneys. The purity of one mRNA sample from a 250-ppm lead-treated rat was not good enough for qRT-PCR, thus leaving 5 rats/treatment group. We observed no significant difference in body weight between the groups. The rats were euthanized at various times during the day, in a random sequence, by exsanguination from the abdominal aorta under ether anesthesia. Blood, spleen, and kidney samples were collected and processed for bio chemical analysis. Each isolated kidney was blotted carefully, weighed, and immediately fixed or frozen for further histological examination, isolation of mRNA, or quantification of lead level. Spleens were fixed in 10% formalin for histological examination. Whole blood was used for isolation of serum or was immediately frozen for quantification of lead level. Serum, prepared by centrifugation of blood, was used to measure blood urea nitrogen (BUN) by the enzymatic-kinetics method (Neodin Vetlab, Seoul, Korea). Lead levels in frozen blood and kidney were analyzed by inductively coupled plasma mass spectrometry by the National Center for Inter-University Research Facilities, Seoul National University (Seoul, Korea). qRTPCR analysis of kidney. Total mRNA was prepared from kidney samples isolated from rats treated with Pb 2+ (0, 250, or 1,000 ppm). Frozen kidney tissue was homogenized in TRIzol reagent (Life Technologies, Carlsbad, CA), and mRNA was further isolated with chloroform and isopropanol. mRNA conversion to cDNA and qRT-PCR were conducted as described above. Relative mRNA expression of LCN2 (NGAL), KIM1, and TGFB1 were calculated by the comparative CT method (2 -ΔΔCt ), normalized to the endogenous 18S control. The specific primer sequences were as follows: Histological examination. To assess histological damage, iron accumulation, ROS generation, and collagen accumulation, we examined sections of kidney tissues using   Statistical analysis. Means ± SEs were calculated for all treatment groups. Data were subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple range test or Student's t-test to determine the statistical significance. In all cases, a p-value of < 0.05 was considered significant.

Pb 2+ induced erythrophagocytosis by renal tubular epithelial cells.
To examine the role of erythro cytes in lead-associated kidney injury, human erythro cytes treated with Pb 2+ (0, 10, or 20 μM) for 1 hr were co-incubated with HK-2 cells, as described in "Materials and Methods." As shown in Figure 1A, co-incubation of Pb 2+ -treated erythro cytes with HK-2 cells for 24 hr significantly reduced HK-2 cell viability, whereas Pb 2+ treatment alone (with no erythro cytes added) failed to affect HK-2 viability, supporting a central role of erythrocytes in Pb 2+ -induced HK-2 cytotoxicity. In microscopic examination, we observed adherence of Pb 2+ -treated erythro cytes to HK-2, whereas untreated discocytic erythro cytes did not bind to HK-2 cells ( Figure 1B). Images from scanning electron microscopy further confirmed the interaction between HK-2 cells and Pb 2+ -treated spherocytic erythro cytes, along with roughening of the HK-2 membrane ( Figure 1C). Next, we used flow cytometric analysis to investigate whether Pb 2+ could enhance erythro phagocytosis by HK-2 cells. The extent of phagocytosis, as determined by the number of HK-2 cells positive for the erythro cyte marker, was significantly increased by Pb 2+ exposure (p < 0.01; Figure 1D).

Role of Pb 2+ induced erythrophago cytosis in renal tubular damage.
Previous studies have shown that erythrophagocytosis by macrophages is mediated by PS on the outer membrane of erythro cytes (Jang et al. 2011;Noh et al. 2010). To clarify whether Pb 2+induced PS externalization may contribute to erythro phago cytosis by renal tubular cells, we examined PS externalization in erythrocytes after Pb 2+ treatment. The binding of annexin V, a marker for exposed PS, was increased in Pb 2+ -treated erythro cytes (Figure 2A), and the generation of PS-bearing MVs was also enhanced by Pb 2+ ( Figure 2B). Notably, when the exposed PS was neutralized by the added annexin V, Pb 2+ -induced erythro phago cytosis was significantly inhibited, suggesting that PS externalized on erythro cytes plays a key role in erythrophagocytosis by renal tubular cells ( Figure 2C). Next, we evaluated the potential role of erythro phago cytosis in renal tubular damage. Considering that erythro cytes contain a high amount of iron that can accumulate and induce excessive oxidative stress (Zager et al. 2002), we measured the generation of ROS in HK-2 cells. Co-incubation of Pb 2+ -treated erythro cytes increased ROS generation in HK-2 cells, indicating that Pb 2+ -induced erythrophago cytosis resulted in oxidative stress ( Figure 2D). We also evaluated tubular damage by measuring mRNA levels of the representative nephrotoxicity biomarkers NGAL and KIM-1 ( Figure 2E). NGAL mRNA expression in HK-2 cells was significantly increased by Pb 2+ -enhanced erythro phago cytosis, and there was a trend of increased KIM-1 expression although statistical significance was not achieved (p = 0.11).
In vivo erythrophagocytosis in the kidney. To evaluate the relevancy of our findings, we investigated the contribution of erythrophagocytosis in Pb 2+ -associated nephro toxicity in vivo. After exposure of rats to Pb 2+ (0, 250, or 1,000 ppm) in drinking water for 12 weeks kidney, spleen, and blood were isolated for biochemical and histological analysis. BLLs were 2.11 ± 0.54 μM for the 250-ppm Pb 2+ group and 3.53 ± 0.84 μM for the 1,000-ppm Pb 2+ group; BLLs of control rats were below the detection limit. The lead levels in kidney samples were 0.08 ± 0.05 μg/g, 12.29 ± 4.63 μg/g, and 26.67 ± 3.67 μg/g for the 0-, 250-, and 1,000-ppm Pb 2+ -treated rats, respectively. In Pb 2+ -treated rats, we observed iron accumulation in the spleen ( Figure 3A), which was in agreement with our previous report (Jang et al. 2011), and in the kidney ( Figure 3B). In addition, ROS generation was observed in kidney tissue from Pb 2+ -treated rats ( Figure 4A).
Evaluation of renal damage associated with Pb 2+ treatment. Along with iron accumulation and ROS generation in the kidney, we sought to determine whether Pb 2+ treatment induced kidney damage. Histopathological examination revealed Pb 2+induced morphological alterations, such as tubulo interstitial lesions (charac terized by basophilic regenerating tubules with altered morphology of epithelial cells) and interstitial lymphotic cell infiltration ( Figure 4B). Conventional nephro toxicity markers, such as relative kidney weight and serum BUN, were increased in kidneys from Pb 2+ -treated rats, but values were not statistically significant ( Figure 4C,D). In contrast, mRNA levels of the nephrotoxic biomarkers (KIM-1 and NGAL) were significantly increased in kidneys from Pb 2+ -treated rats ( Figure 4E), supporting Pb 2+ -induced kidney damage.
Chronic exposure to Pb 2+ has been associated with renal fibrosis (Cramér et al. 1974), which could ultimately disrupt normal kidney function. To evaluate the role of erythrophagocytosis in Pb 2+ -associated renal fibrosis, we examined collagen accumulation and the induction of TGF-β, an important mediator for renal fibrosis. We observed increased collagen deposition and increased TGFB1 mRNA expression in the kidneys of rats treated with Pb 2+ (Figure 5A,B). Notably, the expression of TGF-β was also significantly increased in HK-2 cells co-incubated with Pb 2+ -treated erythro cytes ( Figure 5C), demonstrating that Pb 2+ -associated erythrophago cytosis may play important roles in Pb 2+ -associated renal fibrosis.

Discussion
In the present study, we found that erythrocytes may play an important role in the potentiation of lead (Pb 2+ )-induced nephro toxicity through PS-mediated erythrophagocytosis.
Although treatment with Pb 2+ alone (up to 20 μM) failed to induce statistically significant cytotoxicity in HK-2 renal tubular cells, co-incubation of HK-2 cells with Pb 2+ -treated erythro cytes (at a concentration as low as 10 μM) significantly decreased cell viability. Increased oxidative stress and up-regulation of nephro toxic biomarkers, such as NGAL, were also observed in HK-2 cells co-incubated with Pb 2+ -treated erythro cytes. Moreover, TGF-β, a marker of fibrosis, was significantly up-regulated. In addition, the role of erythrocytes in lead-induced nephro toxicity was also observed in vivo in rats treated with Pb 2+ via drinking water for 12 weeks, as shown by increased iron deposition and generation of oxidative stress in renal tissues, as well as increased fibrosis and expression of KIM-1, NGAL, and TGF-β.
Lead contamination is a significant environ mental threat to public health. Among the lead-associated adverse health effects, nephro toxicity, represented by proximal tubular nephropathy and interstitial fibrosis, has been frequently reported in epidemiological studies (Cramér et al. 1974;Navas-Acien et al. 2009). High BLL (70-80 μg/dL) is known to be an established risk factor for chronic renal damage (Ekong et al. 2006;Fadrowski et al. 2010). Despite robust epidemiological evidence, the mechanisms under lying lead-induced nephro toxicity have not been fully understood. In vitro studies employing renal cells revealed that kidney cells were somewhat resistant to lead-induced cyto toxicity-as indicated by our resultsand that higher concentrations of lead than the reported human BLLs were required to manifest oxidative stress and cytotoxicity (Stacchiotti et al. 2009). In our study, we observed that erythro phago cytosis by kidney cells may underlie lead-induced nephro toxicity and cyt otoxicity, providing an important clue to explain the discrepancy. Although we cannot completely exclude the possibility of direct effects of lead on the kidney after chronic exposure in vivo, our data strongly suggest that erythro phago cytosis and subsequent iron deposition could contribute to lead-associated kidney damage. Experiments are needed to determine whether blocking or neutralizing the externalization of PS or the attenuation of iron-mediated ROS generation could reverse lead-associated nephrotoxicity.
Heavy metals that are absorbed into the bloodstream are continuously exposed to and accumulated into erythro cytes. In the case of lead, 99% of blood lead is associated with erythro cytes (Hernández-Avila et al. 1998;Schütz et al. 1996). Lead induced disruption of membrane-lipid asymmetry and subsequent PS externalization in erythro cytes, which is mediated by alteration of aminophospholipid translocase activities (Jang et al. 2011;Kempe et al. 2005) and activation of transbilayer lipid movement (Shettihalli and Gummadi 2013). Externalization of PS by lead treatment can induce erythro phago cytosis by inter acting with scavenger receptors on macrophages in the spleen (Willekens et al. 2005). PS-externalized erythro cytes can be also engulfed by other tissue, which can eventually cause certain patho genic effects (Fens et al. 2012;Otogawa et al. 2007). Otogawa et al. (2007) reported that a high-fat diet induced PS externalization on erythro cytes and erythro phagocytosis by Kupffer cells in the liver, resulting in inflammation and hepatic fibrosis. Fens et al. (2012) demonstrated that activated endothelial cells were capable of erythrophagocytosis, leading to endothelial cytotoxicity. Ichimura et al. (2008) showed that KIM-1 in kidney tubular epithelial cells specifically recognized PS, and was responsible for phagocytosis. Loss of phospholipid asymmetry in erythro cytes was also observed in uremia (Kong et al. 2001) and chronic renal failure (Bonomini et al. 1999(Bonomini et al. , 2001. Although erythro phagocytosis by renal tubular cells has been confirmed in several studies (Madsen et al. 1982;Mimura et al. 2008), its patho physiological significance has not been elucidated. The results of the present study indicate that lead-induced erythrophagocytosis was associated with increased oxidative stress and histological changes in renal cells. Additional research is needed to examine the potential role that erythro phagocytosis may play in the patho genesis of kidney disease.
In addition to lead accumulated in erythro cytes being transported to the kidney, the pathological role of erythro phagocytosis may stem from iron abundance in erythro cytes. Erythrocytes contain about 70% of the body's iron content in the form of hemoglobin, and abnormal uptake of damaged erythro cytes by intact tissues can result in iron accumulation and subsequent cellular damage. Iron over load stimulates ROS generation by an oxidation-reduction reaction (Fraga and Oteiza 2002;Valko et al. 2005). Deposition of large amounts of free iron is known to cause critical damage in the liver, heart, and other organs (Rasmussen et al. 2001). In the kidney, ROS-mediated lipid peroxidation (Zager et al. 2002) and renal fibrosis (Kovtunovych et al. 2010) can be induced by iron accumulation, ultimately leading to tubular cyto toxicity (Zager et al. 2004). The present study suggests that erythro phagocytosis may explain the potential source of iron accumulation and ROS generation in the kidneys following lead exposure.
According to the CDC (1997), the average BLL in adults is 1-2 μg/dL (~ 0.05-0.1 μM), and BLLs > 10 μg/dL (0.48 μM) are defined as lead poisoning. Lead exposure-associated nephro toxicity has been observed at BLLs as low as 5 μg/dL (Ekong et al. 2006), and BLL is a known risk factor for nephropathy (Muntner et al. 2003). During 2002-2011, the Adult Blood Lead Epidemiology and Surveillance program identified 11,536 adults in the United States with very high BLLs (≥ 40 μg/dL), and 19% of these adults had these very high BLLs during ≥ 2 calendar years (CDC 2013), showing that lead exposures continue to occur at unacceptable levels. In rat models, histologi cal and functional damages in kidney became evident at BLLs of 36-72 μg/dL (Liu et al. 2012a;Mahaffey et al. 1980). To achieve high BLLs for the experiments on the mechanism under lying lead-associated nephro toxicity, we treated rats with 1,000 ppm Pb 2+ in drinking water; in lead-treated rats, the mean BLL was 73.5 ± 17.4 μg/dL and the mean lead levels in kidney tissue was 26.67 ± 3.67 μg/g. Although the achieved BLL was epidemiologically reasonable, the lead level in drinking water may appear rather high; however, considering that 80% of environmental lead exposure is from other sources, such as food or inhalation, we consider the adoption of these high lead levels in drinking water necessary in our experimental system because drinking water was the only source of lead exposure. In our in vitro experiments, we observed that co-incubation of HK-2 cells with Pb 2+ -treated erythro cytes (concentrations up to 20 μM for 1 hr) resulted in erythrophagocytosis and subsequent cytotoxicity. Although we could not extend the duration of Pb 2+ exposure beyond 1 hr in our experimental setting due to technical limitations in maintaining erythro cyte integrity for 24-hr co-incubation, we believe that prolonged exposure to lower concentrations of Pb 2+ could affect renal tubular viability through PS-exposure-mediated erythrophagocytosis. In a previous study (Jang et al. 2011), we demonstrated that the level of PS externalization obtained using Pb 2+ 20 μM for 1 hr was similar to that obtained using Pb 2+ 0.5 μM for 24 hr, suggesting that erythrophagocytosis can be induced at a much lower concentration of Pb 2+ when exposed chronically, as observed in a real-life scenario.
We observed that erythrocyte uptake in the kidney induced iron deposition, ROS generation, and renal fibrosis. Fibrosis is a major determinant of progressive renal damage leading to end-stage renal failure (Eddy 1996), and it has been frequently observed in a lead-exposed population (ATSDR 2007). In the present study, we found typical charac teris tics of tubulo interstitial fibrosis, such as tubular loss and accumulation of collagen, the most abundant of the extra cellular matrix (ECM) proteins in kidneys from lead-exposed rats. ECM is primarily produced by myo fibroblasts (LeBleu et al. 2013); however, the active role of tubular epithelial cells in fibrosis has also been reported. In pathological states such as diabetes and hypertension, the potent profibrotic cytokine TGF-β is produced by tubular cells (Isaka et al. 2000), and stimulates renal fibroblasts to produce ECM (Vallon and Thomson 2012;Zhao et al. 2008). TGF-β also induces fibrogenic trans differentiation of tubular epithelial cells to harbor ECM-producing character (Li et al. 2002). TGF-β expression in tubular cells could be induced by excessive oxidative stress (Vallon and Thomson 2012;Zhao et al. 2008). In the present study, we observed ROS generation and TGF-β up-regulation in the kidney of lead-exposed rats in vivo and tubular cells undergoing erythrophagocytosis in vitro, suggesting the potential contribution of leadinduced erythrophagocytosis to renal fibrosis. These findings are in agreement with a previous study that showed the up-regulation of NGAL and KIM-1 in tubular cells to be key renal injury biomarkers (Lock 2010).

Conclusion
The results of the present study suggest that lead exposure can lead to the externalization of PS and generation of MVs in erythrocytes, which appears to be associated with increased erythrophagocytosis by renal tubular cells. Our data support the hypothesis that erythro phagocytosis seems to be associated with increased ROS generation, induction of nephrotoxicity biomarkers, TGF-β up-regulation, and decreased cell viability of renal tubular cells. Our in vivo experiments confirmed that chronic exposure to lead increased iron deposition in the kidney. The role that iron plays in lead-mediated oxidative stress and renal fibrosis warrants further research. We believe that our study gives a new insight into the mechanisms of lead exposure-associated nephrotoxicity.