Okratoksin A izaziva apoptozu u LLC-PK1 stanicama aktivirajući JNK i p38 MAPK

Abstract

Ochratoxin A (OTA) is a potential inducer of a tubular-interstitial nephropathy in humans and animals. In our study we addressed the question of involvement of apoptosis in the development of OTA-provoked nephrotoxicity. LLC-PK1 kidney cells were treated with nanomolar and micromolar concentrations of OTA for different lengths of time. The apoptotic process was estimated by morphological (haematoxylin/eosin staining, fluorescent staining of DNA free ends – TUNEL assay) and biochemical (MAPKs and Hsps) changes of cells. Forty-eight hours of treatment with 5 10–6 M OTA significantly decreased cell viability and induced apoptosis in 30.7 % of cells. In addition, a transient activation of ERK was observed as well as a strong and prolonged activation of stress kinases, JNK and p38 MAPK, after 12 and 48 hours of treatment. Expression of Hsp72 and Hsp27 was not affected by OTA. The results suggest that apoptosis mediated by activation of JNK and p38 MAPK might play an important role in OTA-induced nephrotoxicity in LLC-PK1 cells.Okratoksin A (OTA) može izazvati tubularno-intersticijsku nefropatiju u ljudi i životinja. Cilj našeg istra- živanja bio je ispitati ulogu apoptoze u nastanku nefrotoksičnosti koju uzrokuje OTA. Naš eksperimentalni model bile su LLC-PK1 bubrežne stanice izložene djelovanju OTA, od nanomolarnih do mikromolarnih koncentracija kroz različita vremenska razdoblja. Apoptoza je u stanicama utvrđena na temelju morfoloških (bojanje stanica hematoksilin/eozinom, fluorescentno bojanje slobodnih krajeva DNA tzv. TUNEL) i biokemijskih (MAP kinaze, Hsps) promjena. 48-satno izlaganje stanica 5 x 10–6 M OTA značajno je smanjilo staničnu vijabilnost i potaknulo apoptozu u 30,7 % stanica. Osim toga, utvrđena je kratkotrajna aktivacija ERK te snažna i dugotrajna aktivacija JNK i p38 MAPK nakon 6-, 12- i 48-satnoga tretiranja. OTA nije utjecao na razinu Hsp72 i Hsp27. Rezultati istraživanja ukazuju da je apoptoza posredovana aktivacijom JNK i p38 MAPK važan događaj u razvoju nefrotoksičnosti izazvanoj djelovanjem OTA na LLC-PK1 stanice