Effects of caffeine at oxidative damage and crystallization in cellular model of oxalate urolithiasis of proximal renal tubules LLC-PK1

Cilj istraživanja: Utvrditi toksični učinak kalcijevog oksalata na epitelnim stanicama proksimalnih bubrežnih tubula staničnog tipa LLC-PK tip I i procijeniti učinkovitost različitih koncentracija kofeina u zaustavljanju kristalizacije kalcijevog oksalata u staničnom modelu oksalatne urolitijaze. Odrediti razinu opterećenja stanica slobodnim radikalima mjerenjem koncentracija glutationa (GSH) nakon tretmana kalcijevim oksalatom i kofeinom. Nacrt studije: Stručna istraživačka studija Materijali i metode: Korištena stanična kultura je Lilly Laboratories Cell Porcine Kidney, stanična linija proizvedena iz bubrega normalne, zdrave muške svinje (Sus scrofa) između 3. i 4. tjedna starosti. Od kemikalija korišten je kofein (Sigma) te kalcijev oksalat (Sigma-Aldrich). Stanice su uzgajane u pločama sa šest jažica te su tretirane kalcijevim oksalatom i kofeinom u odgovarajućim koncentracijama. Nakon inkubacije svjetlosnom mikroskopijom pratio se nastanak kristalizacije i utjecaj djelovanja kofeina nakon jednog, tri i 24 sata te su se učinile mikrofotografije EVOSTM FL Color mikroskopom. Koncentracija ukupnog glutationa izmjerena je spektrofotometrijski pomoću komercijalno dostupnog kita (Glutathione Assay Kit, Sigma-Aldrich, Saint Louis, MO, SAD). Vijabilnost stanica u suspenziji utvrdila se ekskluzijskim testom Trypan Blue bojom (Sigma-Aldrich, St. Louis, SAD) uz pomoću Neubauerovog hemocitometra svjetlosnom mikroskopijom. Rezultati: Proporcionalnim povećanjem koncentracije, kofein zaustavlja kristalizaciju kalcijevog oksalata u staničnom modelu oksalatne urolitijaze proksimalnih bubrežnih tubula – LLC-PK tip I. Kofein kao potencijalna zaštitna terapija uzrokuje značajno preživljenje stanica. Ekspresija ukupnog glutationa pokazala je da oksidativno oštećenje stanica koje su tretirane samo kalcijevim oksalatom veće u odnosu na stanice koje su tretirane, osim kalcijevim oksalatom, i kofeinom. Zaključak: Kofein sprječava nastanak kristalizacije kalcijevog oksalatnog monohidrata u staničnom modelu oksalatne urolitijaze proksimalnih bubrežnih tubula – LLC-PK tip I.Objectives: The aim of this study was to determine the toxicity of calcium oxalate on epithelial cells of proximal kidney types LLC-PK type I, evaluate the effect of different caffeine concentrations in stopping the crystallization of calcium oxalate in the cellular model of oxalate urolithiasis of proximal kidney tubules – LLC-PK type I. Furthermore, the aim was to determine the level of cell loading by free radicals by measuring the glutathione concentration (GSH) after the treatment with calcium oxalate and caffeine and to determine cell viability with increased caffeine concentrations. Study design: Laboratory research study Materials and methods: The used cell culture is the Lilly Laboratories Cell Porcine Kidney, a cell line produced from the kidney of a normal, healthy male pig (Sus scrofa) between 3 and 4 weeks of age. The chemicals used included caffeine (Sigma) and calcium oxalate (Sigma-Aldrich). The cells were cultured in six-well plates and treated with calcium oxalate and caffeine at appropriate concentrations. After incubation at 37 °C, 5 % CO2, light microscopy followed the formation of crystallization and the effect of caffeine activity after one, three and 24 hours, and EVOSTM FL Color microscope was used to make microphotographs. Total Glutathione Concentration (GSH) was measured spectrophotometrically by using a commercially available kit (Glutathione Assay Kit, Sigma-Aldrich, Saint Louis, MO, USA). The cell susceptibility in suspension was determined by an exclusive Trypan Blue dye (Sigma-Aldrich, St. Louis, USA) using Neubauer's hemocytometer by means of medium-light microscopy. Results: By the proportional increase in concentration, caffeine stops the crystallization of calcium oxalate in the cellular model of oxalate urolithiasis of proximal kidney tubules – LLC-PK type I. Caffeine as a potential protective therapy causes significant cell survival. Overall glutathione expression showed that oxidative damage to cells treated only with calcium oxalate was higher compared to cells treated by both calcium oxalate and caffeine. Conclusion: Caffeine prevents crystallization of calcium oxalate monohydrate in the cellular model of oxalate urolithiasis of proximal kidney tubules LLC-PK type I