movilidad urbana sostenible
3. Plan de Movilidad Urbana Sostenible de Bilbao 2030 ( PMUS 2030)
HEK293 cells (2.1.7.2) - human embryonic kidney cells – were used as a cell model to analyse if cellular protein levels and localisation of members of the Trx protein family depend on the environmental oxygen concentration. Cells were cultivated in an atmosphere containing distinct concentrations of oxygen for 24 h: 20 % O2, a concentration
commonly used for cell culture, 1 % and 0.1 %. Cells were washed with PBS buffer, harvested and lysed in NP40-lysis buffer. Protein levels of Trx family proteins were analysed by the Western blot technique using 10-20 µg of cell extracts, reduced with 100 mM DTT and 50 mM TCEP as described above. Various proteins, commonly used as loading controls, i.e the cytosceletal components tubulin and actin, as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the enzyme catalysing the sixth step of glycolysis, were also analysed. All proteins responded to the hypoxic insult. GAPDH for instance, was upregulated with decreasing O2 concentrations (Figure 23).
However, the overall protein concentration of all samples was thoroughly analysed by Bradford. Figure 23 summarizes the results of the Western blot analysis. Generally, Trx family proteins showed a complex response to the hypoxic insults. Protein levels of Grx1, Grx5, Prx2, TrxR1 and Nrx increased,
Figure 23: Protein expression of Trx family proteins is sensitive to oxygen concentrations. HEK293 cells were grown in
an atmosphere containing distinct O2 (0.1 %,
1 %, 20 %) concentrations. Protein levels were analysed by Western blot, using 20 µg of total cell extracts, preincubated with 100 mM DTT and 50 mM TCEP.
3. Results 55 whereas the levels of Prx1, Prx5 and TrxR2 decreased. Grx3, Trx1 and Trx2 showed similar levels at 0.1 % and 20 %, but were downregulated at 1 %, whereas Prx4 was upregulated at 1 %. Prx3 and Prx6 did not display any significant changes. We subsequently analysed Trx family proteins in a mouse model, where renal ischemia/reperfusion injury (2.1.8.2) was induced by clamping the pedicle of the right kidney for 30 min and releasing it afterwards for another 24 h. This animal model was established in the AG Lillig by Dr. José Godoy, who performed all animal experiments. Blood and urine specimens, as well as the ischemic and the contralateral kidney were used for analysis. Sham operated mice, animals where the kidney was exposed for 30 min, but was not clamped, were used as control. Numerous parameters including consistent body weight, decreased urea elimination, proteinuria, increased segmented nuclei cells, indicating the start of an inflammation as well as histological changes of the kidney, such as loss of brush borders, tubular cell flattening and luminal obstruction indicated the success of the procedure (Godoy et al. 2011b, Table 9, Figure 33).
Figure 24: Expression levels of Trx family proteins in a model for renal ischemia/reperfusion injury.
The I/R (right kidney), contralateral (left kidney) and sham kidneys were analysed for protein levels by Western blot and in the case of Grx2 by sandwich ELISA. Data were quantified using ImageJ and state the fold-increase in percent as means +/- SEM, comparing I/R and contralateral kidneys to the control sham kidneys (Godoy et al. 2011b, compare to 4.3).
Protein levels of members of the Trx family were analysed in the ischemic and the contralateral kidney by Western blot and ELISA. Figure 24 summarizes the results, representing the means +/- SEM, stating the fold-increase in percent of each protein of the I/R or the contralateral kidney with respect to the control sham operated kidneys. Most of the proteins did not show any significant changes, neither in the ischemic, nor in the contralateral kidney. However, the levels of Grx5, Trx1 and Trx2 were significantly increased to approximately 140-150 %, following the I/R insult. Grx1 was decreased to 82 %. In contralateral kidneys Grx5 was upregulated to more than 150 % and Prx6 to 138 %. TrxR2, Prx4 and Prx5 were downregulated compared to the control kidneys. Immunohistochemical analysis of the distribution of redoxins in distinct regions of the kidney revealed that Grx2, Prx3 and Prx6 were the only redoxins which were exclusively overexpressed in proximal tubule cells, a cell type which can regenerate after an I/R insult (Sutton et al. 2002), (Nony and Schnellmann, 2003). Members of the Trx family have
been shown to affect the cell cycle, regulating proliferation, differentiation and apoptosis (Powis et al. 1998), (Lillig et al. 2004) (Enoksson et al. 2005). To analyse if this is generally also valid for hypoxic conditions, Grx2, Prx3 and Prx6 were overexpressed in HEK293 cells using pExpress plasmids (compare to Figure 10 and 3.1.3). HEK293 cells were chemically transfected using lipofectamin (described in 2.2.2.6.2). Six hours after transfection, cells were transferred into the hypoxic incubator at 0.1 % O2 for 24 h, followed by a
reoxygenation period at 20 % O2
for another 24 h. The efficiency of chemical transfection was
Figure 25: Overexpression of Grx2, Prx3 and Prx6 in HEK293 cells exposed to hypoxia. Cells were transiently
transfected with redoxins and an empty plasmid as control (ctrl) using lipofectamin. Cells were incubated at 0.1 % O2
for 24 h and reoxygenated at 20 % O2 for another 24 h. The
cell number was determined using an automatic cell counter. The average of ≥ 5 independent experiments is illustrated ± the standard error of the mean (adapted from Godoy et al. 2011b).
3. Results 57 generally similar to the physical approach of electroporation, analysed by Western blot and ELISA, leading to a two to three fold overexpression, compared to control cells transfected with an empty pExpress plasmid (data not shown). Overexpression of all three redoxins in HEK293 cells led to an increase in cell number compared to control cells, analysed using an automatic cell counter (Cellometer, PeqLab). Figure 25 depicts the increase in cell number normalised to the control. Overexpression of the mitochondrial hGrx2 increased the cell number about 1.5 times, hPrx3 overexpression about 1.8 times and Prx6 more than two times.