Protein lysates from the cells stimulated with basal conditions and those stimulated with rMIF and rMCP-1 were analysed.
4.5.2.1 Phospho p38 MAPK
Phospho p38 MAPK is found in podocytes in all conditions. There are low levels of phospho p38 MAPK produced by podocytes in glycated albumin, compared to cytokine stimulation. There was no effect on cell viability as the glycated albumin podocytes had the same number of cells with or without cytokine stimulus. Stimulation of the other basal conditions with rMIF showed a slight reduction in phospho p38 MAPK compared to rMCP-1 stimulated conditions, see Fig 4.5.2.1.
Phospho p38 MAPK levels were higher in podocytes with conditions with rMIF compared to unstimulated conditions. MCP-1 is known to act through phospho p38 MAPK however whether the degree of protein production of this activated form is maintained at 48hours in podocytes is unknown.
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A B
C
Figure 4.5.2.1 A: p38 MAPK (40kDa) from podocytes stimulated with normal glucose, mannitol and high glucose. B: p38 MAPK(40kDa) from podocytes stimulated with glycated albumin.
C: Phospho p38 MAPK
in stimulated
Podocytes. Key; N – normal glucose, M – mannitol, H – high glucose, A2 – glycated albumin;
Prefix’s M – rMCP-1 (20ng/ml) and H – rMIF (20ng/ml). The blue bars indicate the 0ng/ml basal conditions at 48 hours, the red bars are podocytes stimulated with 20ng/ml rMIF, the green bars are podocytes stimulated with 20ng/ml rMCP-1.
4.5.2.2 Phospho p44/42 MAPK (ERK1/2)
There was a higher level of phospho p44/42 MAPK in podocytes stimulated for 48 hours in glycated albumin, see Fig 4.5.2.2. This rise was abrogated with stimulation with rMIF or rMCP-1 in this environment. Other conditions and stimuli had lower levels of this activated form of p44/42 MAPK.
MIF is known to use p44/42 MAPK pathways in cell signalling however, whether other mechanisms are employed to continue its effects are unknown. The activity would need to be compared with the total protein to determine whether the lower levels seen of phospho p44/42 MAPK in other conditions are still significantly higher than would be expected.
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A B
C
Figure 4.5.2.2 A:
p44/42 MAPK (42, 44kDa) from podocytes stimulated with normal glucose, mannitol and high glucose. B: p44/42 MAPK (42, 44kDa) from podocytes stimulated with glycated albumin. C:
Phospho p44/42 MAPK in stimulated Podocytes. Key; N – normal glucose, M – mannitol, H – high glucose, A2 – glycated albumin; Prefix’s M – rMCP-1 (20ng/ml) and H – rMIF (20ng/ml). The blue bars indicate the 0ng/ml basal conditions at 48 hours, the red bars are podocytes stimulated with 20ng/ml rMIF, the green bars are podocytes stimulated with 20ng/ml rMCP-1.
Summary of Podocyte cell signalling
There is a marked reduction of activated phospho p38 MAPK in glycated albumin regardless of cytokine stimulation. rMIF and rMCP-1 stimulated conditions appear to increase the levels of phospho p38 MAPK at 48 hours in podocytes in normal glucose, mannitol and high glucose.
rMIF and rMCP-1 may therefore use p38 MAPK in these conditions to induce their effects. These pathways however, may have already been switched on following the effects induced by the cytokines and other pathways may have been employed at 48 hours. Short time interval signalling experiments would need to be done to determine whether p38 MAPK is the main cell signalling pathway used by rMIF and rMCP-1 in normal glucose, mannitol and high glucose. Phospho p44/42 MAPK was higher in glycated albumin conditions with less activity seen in podocytes
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stimulated with rMIF and glycated albumin. There was no significant change in cell viability to explain these different findings as per the MTT assay.
4.5.2.3 Caspase 3
The findings of caspase 3 suggest glycated albumin has higher levels of cleaved caspase (17, and 19kDa), however, the membrane would need to be reblotted to determine this. Other conditions tended to have a single band with little change of the level of protein found following cytokine stimulation in the same condition. The exception is that of normal glucose where cytokine stimulation appeared to have lower levels of caspase 3 than in normal glucose alone. Mannitol had no lower detectable bands suggesting no cleaved caspase 3, see Fig 4.5.2.3. The MTT assay shows similar cell survival suggesting this change did not affect cell survival.
A B
C
Figure 4.5.2.3 A:
Caspase 3
(17,19,35kDa) from podocytes stimulated with normal glucose, mannitol and high glucose. B: Caspase 3 (17,19,35kDa) from podocytes stimulated with glycated albumin.
C: Caspase 3 in stimulated Podocytes. Key; N – normal glucose, M – mannitol, H – high glucose, A2 – glycated albumin; Prefix’s M – rMCP-1 (20ng/ml) and H – rMIF (20ng/ml). The blue bars indicate the
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0ng/ml basal conditions at 48 hours, the red bars are podocytes stimulated with 20ng/ml rMIF, the green bars are podocytes stimulated with 20ng/ml rMCP-1.
4.5.2.4 Caspase 7
Unfortunately there was insufficient cell lysate for Caspase 7 to be analysed in glycated albumin.
Time limitations did not allow the entire analysis to be repeated in all the experiments to ensure no sample bias. All these results need to be confirmed with further analysis, however, there is a suggestion of lower levels of caspase 7 in normal glucose, mannitol and high glucose following rMIF stimulation, see Fig 4.5.2.4. There was equivalent cell viability in this experiment between conditions. The MTT assay in podocytes stimulated with rMIF in mannitol and high glucose showed improved cell survival with little difference following rMCP-1 stimulation. 20kDa caspase 7 bands were not identified. The levels of caspase 7 were less in podocytes with these stimuli than those produced by HK2 cells.
A
B
Figure 4.5.2.4 A:
Caspase 7 (20, 35kDa) from podocytes stimulated with normal glucose, mannitol and high glucose. B:
Caspase 7 in stimulated Podocytes. Key; N – normal glucose, M – mannitol, H – high glucose, A2 – glycated
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albumin; Prefix’s M – rMCP-1 (20ng/ml) and H – rMIF (20ng/ml). The blue bars indicate the 0ng/ml basal conditions at 48 hours, the red bars are podocytes stimulated with 20ng/ml rMIF, the green bars are podocytes stimulated with 20ng/ml rMCP-1.
4.5.2.5 Caspase 9
An increase in 3 of the total 4 bands of caspase 9 in podocytes with glycated albumin, was detected that remained constant following rMIF or rMCP-1. Cleaved caspase 9 are found at 17 and 37kDa.
Bands 1 and 4 only are present in other stimulated conditions with bands at 1 and 3 suggesting the presence of cleaved caspase 9. The membrane would need to be reblotted with cleaved caspase 9 to determine its activation. High glucose resulted in higher protein levels of caspase 9 in podocytes stimulated with rMIF that did not affect cell survival, see Fig 4.5.2.5.
A B
C
Figure 4.5.2.5 A:
Caspase 9 (17,35, 37, 47kDa) from podocytes stimulated with normal glucose, mannitol and high glucose. B:
Caspase 9 (17,35, 37, 47kDa) from podocytes stimulated with glycated albumin. C:
Caspase 9 in stimulated Podocytes. Key; N – normal glucose, M – mannitol, H – high glucose, A2 – glycated albumin;
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Prefix’s M – rMCP-1 (20ng/ml) and H – rMIF (20ng/ml). The blue bars indicate the 0ng/ml basal conditions at 48 hours, the red bars are podocytes stimulated with 20ng/ml rMIF, the green bars are podocytes stimulated with 20ng/ml rMCP-1. The four bands seen in total caspase 9 are at 17, 35, 37, and 47kDa presented as 1 to 4 per condition. The cleaved forms are 17, and 37kDa, bands 1 and 3 respectively.
Summary of results of Caspases in Podocytes
Caspase 3 is increased in podocytes in glycated albumin and in high glucose with rMIF. There