Normal Levels of Urinary CC16 Protein. Comments on Beamer et al. Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media. Int. J. Environ. Res. Public Health 2016, 13, 521/Reply

Abstract

A proximal tubular dysfunction results in the diminished resorption of CC16 and increased levels in the urine. Since the early 1990s, the determination of urinary CC16 has sporadically been used as a marker of tubular renal proximal dysfunction [4]. Later, it was confirmed that chronic arsenic administration in experimental animals originates ultrastructural alterations that consist in swollen mitochondria and increased numbers of dense autophagic lysosome-like bodies in the proximal tubule cells [7]. [...]a subcytotoxic dose of arsenite induces necrotic changes in the cytoplasmic structure and mitochondrial morphology in the human proximal tubular cell line [8]. Under physiological conditions, the quasi-totality of this protein that is filtered is reabsorbed in the proximal tubule in such a way that the detectable levels in urine are very low. [...]in our first paper on the subject, the value of the urinary CC16-to-creatinine ratio was zero in 16 of the 31 control children (51.6%) [12].

In 1937, Max Clara described a new type of cell in the human lung, which was later determined to be an exocrine secretory cell type containing granules composed of proteins [1]. In 1988, in an exploratory study on proteins obtained from lung washes, Singh et al. identified a protein that is expressed in both rodents and humans, which was subsequently named Clara cell 16-kDa protein (CC16) [2]. It constitutes a major protein secreted by Clara cells in the respiratory tract and is secreted to the epithelial lining fluid of the lung and transported up along the bronchiolar tree [3].

Given its small size, with a molecular weight of 15.8 kDa, CC16 is rapidly filtered by the glomerulus, reabsorbed almost entirely, and catabolized in the renal proximal tubule cells. This mechanism is similar to that of ß2-microglobulin, cystatin C, and other low-molecular-weight proteins.

A proximal tubular dysfunction results in the diminished resorption of CC16 and increased levels in the urine. Since the early 1990s, the determination of urinary CC16 has sporadically been used as a marker of tubular renal proximal dysfunction [4].

Recently, Beamer et al. have published in this journal an interesting work whose objective was to determine if urinary CC16 levels in children are associated with arsenic concentrations in environmental media collected from their homes [5]. Their hypothesis was based on the fact that arsenic exposure has been associated with decreased CC16 levels in adults. The authors concluded that lower levels of CC16 in children's urine may be associated with exposure to arsenic via multiple routes.

These results have two controversial aspects. The first one is that the kidney is a known target organ of arsenic toxicity and is critical for both in vivo arsenic biotransformation and elimination. For more than a century, it has been well known that intoxication by arsenic produces vascular or tubular histological lesions for which the term "the arsenic kidney" was created [6]. Later, it was confirmed that chronic arsenic administration in experimental animals originates ultrastructural alterations that consist in swollen mitochondria and increased numbers of dense autophagic lysosome-like bodies in the proximal tubule cells [7]. Moreover, a subcytotoxic dose of arsenite induces necrotic changes in the cytoplasmic structure and mitochondrial morphology in the human proximal tubular cell line [8]. Recently, Tokumoto et al. described that inorganic arsenic may induce p53-dependent apoptotic pathways through the downregulation of gene expression of the Ube2d family in proximal tubular cells [9]. With the intention of examining the association between arsenic exposure and renal dysfunction, Chen et al. studied 1043 subjects from a population in Taiwan. The authors observed an abnormal urinary elimination of ß2-microglobulin when urinary levels with an arsenic-to-creatinine ratio were above 35 pg/g [10]. In a Mexican study, an association between urinary arsenic levels and a1-microglobulin urinary excretion, another low-molecular-weight protein that is a marker of early tubular proximal renal injury, was observed [11]. Consequently, the patients studied by Beamer et al. [5] should have elevated or at least normal CC16 levels, making difficult to accept that they had reduced levels upon exposure to arsenic.

The second controversial point refers precisely to the reduced urinary levels to which the paper of Beamer et al. refers [5]. Under physiological conditions, the quasi-totality of this protein that is filtered is reabsorbed in the proximal tubule in such a way that the detectable levels in urine are very low.

Therefore, in our first paper on the subject, the value of the urinary CC16-to-creatinine ratio was zero in 16 of the 31 control children (51.6%) [12]. In another recent study by our group [13], the average urinary CC16-to-creatinine ratio in control children (1.12 ± 0.59 pg/g) (n = 28) was even lower than that in the first study (1.22 ± 1.52 pg/g).

In summary, we believe that Beamer et al. [5] should reconsider the discussion of the results to which your paper refers given that having low urinary CC16 levels is normal.

Conflicts of Interest: The authors declare no conflict of interest.

Reference

1. Kuhn, C.; Callaway, L.A.; Askin, F.B. The formation of granules in the bronchiolar Clara cells of rats. II. Enzyme cytochemistry. J. Ultrastruct. Res. 1975, 53, 66-76.

2. Singh, G.; Singh, J.; Katyal, S.L.; Brown, W.E.; Kramps, J.A.; Paradis, I.L.; Dauber, J.H.; Macpherson, T.A.; Squeglia, N. Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein. J. Histochem. Cytochem. 1988, 36, 73-80.

3. Bernard, A.; Roels, H.; Lauwerys, R.; Witters, R.; Gielens, C.; Soumillion, A.; Van Damme, J.; De Ley, M. Human urinary protein 1: Evidence for identity with the Clara cell protein and occurrence in respiratory tract and urogenital secretions. Clin. Chim. Acta 1992, 207, 239-249.

4. Bernard, A.M.; Roels, H.; Cardenas, A.; Lauwerys, R. Assessment of urinary protein 1 and transferrin as early markers of cadmium nephrotoxicity. Br. J. Ind. Med. 1990, 47, 559-565.

5. Beamer, P.I.; Klimecki, W.T.; Loh, M.; Van Horne, Y.O.; Sugeng, A.J.; Lothrop, N.; Billheimer, D.; Guerra, S.; Lantz, R.C.; Canales, R.A.; et al. Association of children's urinary CC16 levels with arsenic concentrations in multiple environmental media. Int. J. Environ. Res. Public Health 2016, 13, 521.

6. Pearce, L.; Brown, W.H. Chemopathological studies with compounds of arsenic: II. Histological changes in arsenic kidneys. J. Exp. Med. 1915, 22, 525-534.

7. Brown, M.M.; Rhyne, B.C.; Goyer, R.A. Intracellular effects of chronic arsenic administration on renal proximal tubule cells. J. Toxicol. Environ. Health 1976, 1, 505-514.

8. Peraza, M.A.; Cromey, D.W.; Carolus, B.; Carter, D.E.; Gandolfi, A.J. Morphological and functional alterations in human proximal tubular cell line induced by low level inorganic arsenic: Evidence for targeting of mitochondria and initiated apoptosis. J. Appl. Toxicol. 2006, 26, 356-367.

9. Tokumoto, M.; Lee, J.Y.; Fujiwara, Y.; Uchiyama, M.; Satoh, M. Inorganic arsenic induces apoptosis through downregulation of Ube2d genes and p53 accumulation in rat proximal tubular cells. J. Toxicol. Sci. 2013, 38, 815-820.

10. Chen, J.W.; Chen, H.Y.; Li, W.F.; Liou, S.H.; Chen, C.J.; Wu, J.H.; Wang, S.L. The association between total urinary arsenic concentration and renal dysfunction in a community-based population from central Taiwan. Chemosphere 2011, 84, 17-24.

11. Robles-Osorio, M.L.; Pérez-Maldonado, I.N.; Martín del Campo, D.; Montero-Perea, D.; Avilés-Romo, I.; Sabath-Silva, E.; Sabath, E. Urinary arsenic levels and risk of renal injury in a cross-sectional study in open population. Rev. Investig. Clin. 2012, 64, 609-614.

12. Martín-Granado, A.; Vázquez-Moncholí, C.; Luis-Yanes, M.I.; López-Méndez, M.; García-Nieto, V. Determination of Clara cell protein urinary elimination as a marker of tubular dysfunction. Pediatr. Nephrol. 2009, 24, 747-52.

13. Pérez Rodríguez, A.; Mancini, D.R.; Aracil Hernández, D.; Ferreiro Diaz-Velis, V.; Rodríguez Lorenzo, T.; García Nieto, V.M. La proteína de las células de Clara y diabetes mellitus tipo 1. Estudio longitudinal. Can. Pediatr. 2016, 40, 119.

Víctor García-Nieto *, Domenico Mancini and Eva Rodríguez-Carrasco

Hospital Nuestra Señora de Candelaria, Santa Cruz de Tenerife, 38010, Spain; [email protected] (D.M.); [email protected] (E.R.-C.)

* Correspondence: [email protected]

Academic Editor: Helena Solo-Gabriele and Alesia Ferguson

Received: 02 August 2016; Accepted: 19 September 2016; Published: 4 October 2016

Reply

Response to García-Nieto et al. Comments on Beamer et al. Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media. Int. J. Environ. Res. Public Health 2016, 13, 521.

We would like to thank the editors for providing us with the opportunity to respond to the points raised by Dr. García Nieto.

Our group and others have demonstrated that lower levels of circulating CC16 are associated with lower lung function, greater airflow limitation, and increased mortality [1,2]. More recently, work from our group has demonstrated that lower levels of circulating CC16 at six years of age is associated with decreased lung function by the teenage years in multiple birth cohorts [3]. Another group has reported that the odds of a child having asthma decreased with increasing CC16 levels in their urine, and their forced vital capacity increased with increasing levels of urinary CC16 [4]. Similarly, in a prospective cohort, higher urinary CC16 levels in infants at the time of an acute lower respiratory tract infection were associated with decreased odds of subsequent childhood wheeze [5]. Taken together, these findings indicate that CC16 may be an important biomarker of early life epithelial and airway damage from potentially preventable environmental exposures.

Because others have demonstrated that decreased levels of CC16 are associated with increasing arsenic exposure [6-8], we sought to determine if this association persisted in children. We reported that decreased levels of urinary CC16 in children were most strongly associated with the concentration of arsenic in their soil and that this may demonstrate that localized arsenic exposure in the lungs could damage the airway epithelium [9].

Arsenic exposure is associated with adverse health effects in virtually every physiological system in the body [10], including the respiratory system and, as Dr. García Nieto indicates, the renal system. Specifically, he highlights that arsenic exposure can cause proximal tubular dysfunction and thus reduce the amount of CC16 that can be reabsorbed by the kidneys, resulting in increased urinary CC16 levels in those with increased arsenic exposure [11]. However, individuals exposed to arsenic have reduced levels of CC16 in their serum, and it is not clear if arsenic-induced proximal tubular dysfunction would compensate for that difference.

In occupational cohorts, arsenic exposure was associated with decreased serum CC16 and increased urinary ß2-microglobulin, yet no significant differences in serum ß2-microglobulin were reported [6,7]. Unfortunately, we did not have serum samples to measure CC16 for this population. However, none of the children in our study reported any history of kidney injury, disease, or failure. If their arsenic exposure had reduced their kidneys' ability to reabsorb CC16 from their urine, then, if anything, we have underestimated the potential effect of arsenic exposure on CC16 production in the body.

We should, and do, acknowledge alternative interpretations of the data. This is particularly important because, even though there have been multitudinous studies of arsenic-exposed human populations, there have been relatively few studies involving urinary CC16 in the context of arsenic exposure. Future studies that measure both CC16 in serum and urine, as well as arsenic exposure, will be needed to better understand our findings and elucidate the competing effects of arsenic toxicity of the lungs and the kidneys on CC16 levels in children's urine. Our hope is that our results encourage further investigation of the relationship between arsenic exposure and CC16 in larger populations, particularly between high- and low-exposure areas.

Based upon his own work, Dr. García Nieto's second point is that detectable levels of CC16 in urine are very low among healthy children [11-13]. We also had a large number of undetectable values of CC16 in children's urine with the assay we used (Human Uteroglobulin Quantikine ELISA kit, R & D Systems Minneapolis, MN, USA) (Limit of Detection [LOD] = 0.8 ng/mL). We do not dispute that healthy children may have low urinary CC16 levels. However, this does not preclude the finding that, in our population, particularly in light of the number of censored values, there was a negative association between the concentration of arsenic in the children's soil and the levels of urinary CC16.

Unfortunately, one of Dr. García-Nieto's studies is not yet available from the journal [13,14], and the other study does not report the LOD of the ELISA kit used, or how censored values were treated in the analysis. In addition, children with respiratory disease, who may have even lower levels of urinary CC16 than otherwise healthy children, were excluded from that study [12]. Thus, it is difficult to fully compare results. However, it is noteworthy that, despite the low CC16 concentrations in urine, previous studies that measured urinary CC16 levels using different ELISA kits were also able to demonstrate inverse associations of urinary CC16 levels with concurrent asthma and lung function deficits in school-age children [4] and with subsequent risk of childhood wheezing in infants with lower respiratory tract infections [5].

In summary, both serum and urinary CC16 demonstrate promise as biomarkers that could be developed and validated as measures of early effects of lung epithelium injury or pulmonary permeability. However, given the complexities of this interesting protein, as discussed in Dr. GarcíaNieto's points, multiple studies are needed to better understand the role of environmental exposures, including arsenic, and disease in relation to its levels in serum and urine. We look forward to our study contributing to this process.

Acknowledgments: This work was supported by the University of Arizona Superfund Research Program (NIEHS P42 ES004940), the Southwest Environmental Health Sciences Center (NIEHS P30 ES006694). Paloma I. Beamer was supported by National Heart, Lung, and Blood Institute (K25 HL103970) and Yoshira Ornelas Van Horne was supported by National Institute of Environmental Health Sciences (T32 ES007091). This publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Guerra, S.; Vasquez, M.M.; Spangenberg, A.; Halonen, M.; Martinez, F.D. Serum concentrations of club cell secretory protein (Clara) and cancer mortality in adults: A population-based, prospective cohort study. Lancet Respir. Med. 2013, 1, 779-785.

2. Rava, M.; Tares, L.; Lavi, I.; Barreiro, E.; Zock, J.; Ferrer, A.; Muniozguren, N.; Nadif, R.; Cazzoletti, L.; Kauffmann, F. Serum levels of Clara cell secretory protein, asthma, and lung function in the adult general population. J. Allergy Clin. Immunol. 2013, 132, 230-232.

3. Guerra, S.; Halonen, M.; Vasquez, M.M.; Spangenberg, A.; Stern, D.A.; Morgan, W.J.; Wright, A.L.; Lavi, I.; Tarès, L.; Carsin, A.; et al. Relation between circulating CC16 concentrations, lung function, and development of chronic obstructive pulmonary disease across the lifespan: A prospective study. Lancet Respir. Med. 2015, 3, 613-620.

4. Ma, Y.N.; Wang, J.; Lee, Y.L.; Ren, W.; Lv, X.F.; He, Q.C.; Dong, G.H. Association of urine CC16 and lung function and asthma in Chinese children. Allergy Asthma Proc. 2015, 36, 59-64.

5. Rosas-Salazar, C.; Gebretsadik, T.; Carroll, K.N.; Reiss, S.; Wickersham, N.; Larkin, E.K.; James, K.M.; Miller, E.K.; Anderson, L.J.; Hartert, T.V. Urine Club Cell 16-kDa Secretory Protein and Childhood Wheezing Illnesses After Lower Respiratory Tract Infections in Infancy. Pediatr. Allergy Immunol. Pulmonol. 2015, 28, 158-164.

6. Halatek, T.; Sinczuk-Walczak, H.; Rabieh, S.; Wasowicz, W. Association between occupational exposure to arsenic and neurological, respiratory and renal effects. Toxicol. Appl. Pharmacol. 2009, 239, 193-199.

7. Halatek, T.; Sinczuk-Walczak, H.; Janasik, B.; Trzcinka-Ochocka, M.; Winnicka, R.; Wasowicz, W. Health effects and arsenic species in urine of copper smelter workers. J. Environ. Sci. Health Part A 2014, 49, 787-797.

8. Parvez, F.; Chen, Y.; Brandt-Rauf, P.W.; Bernard, A.; Dumont, X.; Slavkovich, V.; Argos, M.; D'Armiento, J.; Foronjy, R.; Hasan, M.R. Nonmalignant respiratory effects of chronic arsenic exposure from drinking water among never-smokers in Bangladesh. Environ. Health Perspect. 2008, 116, 190-195.

9. Beamer, P.I.; Klimecki, W.T.; Loh, M.; Van Horne, Y.O.; Sugeng, A.J.; Lothrop, N.; Billheimer, D.; Guerra, S.; Lantz, R.C.; Canales, R.A. Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media. Int. J. Enviorn. Res. Public Health 2016, 13, 521.

10. Naujokas, M.F.; Anderson, B.; Ahsan, H.; Aposhian, H.V.; Graziano, J.H.; Thompson, C.; Suk, W.A. The broad scope of health effects from chronic arsenic exposure: Update on a worldwide public health problem. Environ. Health Perspect. 2013, 121, 295-302.

11. García Nieto, V.; Mancini, D.; Rodríguez-Carrasco, E. Normal Levels of Urinary CC16 Protein. Comments on Beamer et al. Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media. Int. J. Environ. Res. Public Health 2016, 13, 521. Int. J. Enviorn. Res. Public Health 2016, 13, 977.

12. Martín-Granado, A.; Vázquez-Moncholí, C.; Luis-Yanes, M.I.; López-Méndez, M.; García-Nieto, V. Determination of Clara cell protein urinary elimination as a marker of tubular dysfunction. Pediatr. Nephrol. 2009, 24, 747-752.

13. Pérez Rodríguez, A.; Mancini, D.; Aracil Hernández, D.; Ferreiro Diaz-Velis, V.; Rodríguez Lorenzo, T.; García Nieto, V. La proteína de las células de Clara y diabetes mellitus tip 1. Estudio longitudinal. Canar. Pediátr. 2016, 40, 119.

14. Canarias Pediátrica, Available online: https://dialnet.unirioja.es/servlet/revista?codigo=8174 (Accessed on: 27 September 2016).

Paloma I. Beamer 12 3 *, Walter T. Klimecki 2 4, Miranda Loh 25, Yoshira Ornelas Van Horne 2, Anastasia J. Sugeng 2, Nathan Lothrop 2, Dean Billheimer 12, Stefano Guerra 1, Robert Clark Lantz 26, Robert A. Canales 2 and Fernando D. Martinez 1

1 Asthma and Airway Disease Research Center, 1501 N. Campbell Ave., University of Arizona, Tucson, AZ 85724, USA; [email protected] (D.B.); [email protected] (S.G.); [email protected] (F.D.M.)

2 Mel and Enid Zuckerman College of Public Health, University of Arizona, 1295 N. Martin Ave., Tucson, AZ 85724, USA; [email protected] (W.T.K.); [email protected] (M.L.); [email protected] (Y.O.VH.); [email protected] (A.J.S.); [email protected] (N.L.); [email protected] (R.C.L.); [email protected] (R.A.C.)

3 Department of Chemical and Environmental Engineering, University of Arizona, 1133 E. James E. Rogers Way, Tucson, AZ 85721, USA

4 Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, P.O. Box 210207, Tucson, AZ 85724, USA

5 Institute of Occupational Medicine, Research Avenue North, Riccarton, Edinburgh EH14 4AP, UK

6 Department of Cellular and Molecular Medicine, University of Arizona, P.O. Box 245044, Tucson, AZ 85724, USA

* Correspondence: [email protected]; Tel.: [phone]

Academic Editor: Helena Solo-Gabriele and Alesia Ferguson

Received: 02 September 2016; Accepted: 19 September 2016; Published: 4 October 2016