title: Renal effects of metallothionein induction by zinc in vitro and in vivo.
abstract: BACKGROUND
  Metallothionein (MTT) is an endogenous antioxidant that can be induced by both zinc (Zn) and ischemia. In kidneys, increased MTT expression exerts a putative protective role in diabetes and hypoxia. Our goal was to further investigate the behavior of MTT under the influence of Zn and hypoxia in vitro and in vivo.
  
  
  METHODS
  MTT expression was measured in vitro in cell cultures of proximal tubular cells (LCC-PK1) by immune-histochemistry and real-time PCR after incubation with increasing concentrations of Zn under hypoxic and non-hypoxic conditions. In addition, in vivo studies were carried out in 54 patients to study MTT induction through Zn. This is a sub-study of a prospective, randomized, double-blind trial on prevention of contrast-media-induced nephropathy using Placebo, Zn and N-Acetylcysteine. Blood samples were obtained before and after 2 days p.o. treatment with or without Zn (60 mg). ELISA-based MTT level measurements were done to evaluate the effects of Zn administration. For in vivo analysis, we considered the ratio of MTT to baseline MTT (MTT1/MTT0) and the ratio of eGFR (eGFR1/eGFR0), correspondingly.
  
  
  RESULTS
  In vitro quantitative immuno-histochemical analysis (IHC) and real-time PCR showed that at increasing levels of Zn (5, 10, and 15 μg/ml) led to a progressive increase of MTTs: Median (IQR) expression of IHC also increased progressively from 0.10 (0.09-0.12), 0.15 (0.12-0.18), 0.25 (0.25-0.27), 0.59 (0.48-0.70) (p < 0.0001). Median (IQR) expression of PCR: 0.59 (0.51-1.72), 1.62 (1.38-4.70), 3.58 (3.06-10.42) and 10.81 (9.24-31.47) (p < 0.0001). In contrast, hypoxia did not change MTT-levels in vitro (p > 0.05). In vivo no significant differences (p = 0.96) occurred in MTT-levels after 2 days of Zn administration compared with no Zn intake. Nevertheless, there was a significant correlation between MTT (MTT1/MTT0) and eGFR (eGFR1/eGFR0) in case of Zn administration (rho = -0.49; 95%-CI: -0.78 to -0.03; p = 0.04).
  
  
  CONCLUSIONS
  We found that Zn did induce MTTs in vitro, whereas hypoxia had no significant impact. In contrast, no significant increase of MTTs was detected after in vivo administration of Zn. However, there was a significant negative correlation between MTT and eGFR in vivo in case of Zn administration, this could indicate a protective role of MTTs in a setting of reduced kidney function, which is possibly influenced by Zn.
  
  
  BACKGROUND
  ClinicalTrials.gov Identifier: NCT00399256 . Retrospectively registered 11/13/2006.
authors.affiliation: Department of Internal Medicine, Division of General Internal Medicine and Nephrology, Robert-Bosch Hospital Stuttgart, Auerbachstraße 110, 70376, Stuttgart, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Schanz
authors.affiliation: Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
authors.forename: Lea
authors.initials: L
authors.lastname: Schaaf
authors.affiliation: Department of Mathematics, University of Stuttgart, Stuttgart, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Dippon
authors.affiliation: Department of Internal Medicine, Division of General Internal Medicine and Nephrology, Robert-Bosch Hospital Stuttgart, Auerbachstraße 110, 70376, Stuttgart, Germany.|Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Biegger
authors.affiliation: Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Fritz
authors.affiliation: Department of Internal Medicine, Division of General Internal Medicine and Nephrology, Robert-Bosch Hospital Stuttgart, Auerbachstraße 110, 70376, Stuttgart, Germany.
authors.forename: Mark Dominik
authors.initials: MD
authors.lastname: Alscher
authors.affiliation: Department of Internal Medicine, Division of General Internal Medicine and Nephrology, Robert-Bosch Hospital Stuttgart, Auerbachstraße 110, 70376, Stuttgart, Germany. martin.kimmel@rbk.de.
authors.forename: Martin
authors.identifier: http://orcid.org/0000-0003-3368-1108
authors.initials: M
authors.lastname: Kimmel
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015687:Cell Hypoxia / Q000187:drug effects* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004311:Double-Blind Method; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D008668:Metallothionein / Q000097:blood* / Q000378:metabolism*; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult; D015032:Zinc / Q000008:administration & dosage*
chemical_list: D008668:Metallothionein; D015032:Zinc
keywords: Cell culture; Contrast medium; Hypoxia; Induction; Metallothionein; Zinc