Transient Increase in Cellular Dehydrogenase Activity After Cadmium Treatment Precedes Enhanced Production of Reactive Oxygen Species in Human Proximal Tubular Kidney Cells

Abstract

Cadmium is a heavy metal causing toxicity especially in kidney cells. The toxicity is linked also with enhanced oxidative stress leading to cell death. On the other hand, our recent experiments have shown that an increase of total intracellular dehydrogenases activity can also occur in kidney cells before declining until cell death. The aim of the present study, therefore, was to evaluate this transient enhancement in cell viability after cadmium treatment. The human kidney HK-2 cell line was treated with CdCl2 at concentrations 0-200 µM for 2-24 h and intracellular dehydrogenase activity was tested. In addition, we measured reactive oxygen species (ROS) production, glutathione levels, mitochondrial membrane potential, and C-Jun-N-terminal kinase (JNK) activation. We found that significantly increased dehydrogenase activity could occur in cells treated with 25, 100, and 200 µM CdCl2. Moreover, the results showed an increase in ROS production linked with JNK activation following the enhancement of dehydrogenase activity. Other tests detected no relationship with the increased in intracellular dehydrogenase activity. Hence, the transient increase in dehydrogenase activity in HK-2 cells preceded the enhancement of ROS production and our finding provides new evidence in cadmium kidney toxicity.

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

“…Human kidney, HK-2 cells (ATCC CRL-2190), a proximal tubular epithelial cell line derived from normal adult human kidney cells immortalized by transduction with human papillomavirus (HPV 16) DNA fragment 67 , were purchased from the ATCC (Manassas, VA, USA). The cells were cultured in supplemented Dulbecco’s modified Eagle’s medium (DMEM/F12 = 1:1) with 5% (v/v) fetal bovine serum, 1 mM pyruvate, 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL sodium selenite, 50 µg/mL penicillin, 50 µg/mL streptomycin, and 5 ng/mL epidermal growth factor according to a published protocol 68 , 69 . All the experiments were conducted using the HK-2 cells in passages 5–15.…”

Cadmium-Induced Renal Cell Toxicity Is Associated With MicroRNA Deregulation

“…In addition, to assess the robustness of the data observed in RPTEC/ hTERT cells and to further support the validity of our profiling approach, the miRNA deregulation of 6 miRNAs was evaluated in the widely used HK-2 cell line. 53 Interestingly, despite limited differences that may be caused by basal miRNA expression and cadmium concentrations, a similar miRNA upregulation was also globally observed. Taken together and with acknowledgement of intrinsic limitations of in vitro studies, these findings demonstrate that cadmium significantly modulates the miRNA expression profile in renal proximal tubular cells and suggest that miRNA dysregulation may participate in the pathogenesis of cadmium-induced kidney injury.…”

“…In order to clarify mechanisms underlying miRNA relevance, we have first chosen to explore miRNA expression profile in RPTEC/ hTERT, a cellular model that has been proven to be reliable in several nephrotoxicity studies. 28,53,44,62 In particular, this cell line may be viewed as an improved tool for in vitro nephrotoxicity assessments. 44 Indeed, by using only the catalytic subunit of the endogenous hTERT enzyme for immortalization, telomere length is stabilized, and then, the RPTEC/hTERT cell line does not undergo replicative senescence, 37 a well-known limitation of primary RPTECs.…”

“…We also aimed to further assess the relevance of the cadmiuminduced miRNA dysregulation that we observed in RPTEC/ hTERT cells in the commonly used HK-2 cell line. [45][46][47][48][49][50][51][52][53] The HK-2 cells also exhibited typical features of cadmium toxicity, including a decreased cell viability and a morphological modification. However, HK-2 cells were less sensitive to cadmium than RPTEC/hTERT, as suggested by a higher IC50 (IC50 ¼ 36.42 + 7.10 mmol/L versus IC50 ¼ 26.01 + 6.38 mmol/L; Supplemental Figure 1).…”

Quantitative Spectrofluorometric Assay Detecting Nuclear Condensation and Fragmentation in Intact Cells

“…Human kidney, HK-2 cells (ATCC® CRL-2190™), a proximal tubular epithelial cell line derived from normal adult human kidney cells immortalized by transduction with human papillomavirus (HPV 16) DNA fragment 64 , were purchased from the ATCC (Manassas, VA, USA). The cells were cultured in supplemented Dulbecco's modi ed Eagle's medium (DMEM/F12 = 1:1) with 5% (v/v) fetal bovine serum, 1 mM pyruvate, 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL sodium selenite, 50 µg/mL penicillin, 50 µg/mL streptomycin, and 5 ng/mL epidermal growth factor according to a published protocol 65,66 . All the experiments were conducted using the HK-2 cells in passages 5-15.…”