From the lists of microarray results 10 genes from dietary iron overload mice and 15 from Hfe knock out mice were selected for further analysis, because they were interesting and had notable fold change values (Table 6, Table 7). The expression levels of these genes were analyzed by Q-RT-PCR. The results from Q-RT-PCR correlated well with the results from microarray; only 2 genes from dietary iron overload mice (Xbp1 and Fbxw5) and 4 genes from Hfe knock out mice (Gdf15, Slc25a37, Tnfrsf12a and Fbxw5) turned out to be false positives (Table 6, Table 7). The regulated genes showed the same direction of change in their fold change values. Representations of these Q-RT-PCR results are shown in figures 7 and 8; the calculated fold change values differ slightly from those obtained from microarray data analysis, but altogether the changes of their fold change values are still significant.
Table 6. Genes that were selected to Q-RT-PCR analysis from dietary iron overload mice and their fold change values obtained from both methods used.
Gene name Symbol Accession. Fold change
microarray
Fold change Q-RT-PCR cytochrome P450, family 4, subfamily a, polypeptide 14 Cyp4a14 NM_007822 33.21 45.3
angiopoietin-like 4 Angptl4 NM_020581 2.46 2.1
acyl-CoA thioesterase 3 Acot3 NM_134246 2.18 2.7
acetyl-Coenzyme A acyltransferase 1B Acaa1b NM_146230 1.89 2.4
F-box and WD-40 domain protein 5 Fbxw5 NM_013908 -7.98 NS
transthyrethin Ttr NM_013697 -5.74 -5.9
heat shock protein 1A Hspa1a NM_010479 -2.70 -7.3
DnaJ (Hsp40) homolog, subfamily B, member 1 Dnajb1 NM_018808 -2.69 -2.7
heat shock protein 105 Hsp105 NM_013559 -2.56 -2.7
X-box binding protein Xbp1 NM_013842 -1.83 NS
Table 7. Genes that were selected to Q-RT-PCR analysis from Hfe knock out mice and their fold change values obtained from both methods used.
Gene name Symbol Accession. Fold change
microarray
Fold change Q-RT-PCR
cytokine inducible SH2-containing protein Cish NM_009895 3.14 1.9
complement factor D (adipsin) Cfd NM_013459 2.77 13.6
haptoglobin Hp NM_017370 2.05 3.9
uncoupling protein 1 (mitochondrial, proton carrier) Ucp1 NM_009463 1.74 4.5
transthyrethin Ttr NM_013697 1.55 1.9
DnaJ (Hsp40) homolog, subfamily B, member 1 Dnajb1 NM_018808 1.54 1.5
glutathione peroxidase 6 Gpx6 NM_145451 -10.83 -13.0
F-box and WD-40 domain protein 5 Fbxw5 NM_013908 -7.35 NS
cytochrome P450, family 26, subfamily b, polypeptide 1 Cyp26b1 NM_175475 -5.29 -3.7
cysteine-rich with EGF-like domains 2 Creld2 NM_029720 -2.5 -2.3
heme oxygenase (decycling) 1 Hmox1 NM_010442 -2.06 -1.5
heat shock protein 105 Hsp105 NM_013559 -1.95 -1.7
tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a NM_013749 -1.87 NS
solute carrier family 25, member 37 Slc25a37 AK034948 -1.59 NS
growth differentiation factor 15 Gdf15 NM_011819 -1.54 NS
Expression patterns of selected iron-related genes were also studied; these genes include hepcidin1, hepcidin2, Hfe, Hjv, neogenin, Tfr2, Tfr1 and Irp1. None of these genes were differentially expressed according to microarray studies, but because one aim of this study was to explore the expression patterns of iron-related genes in the kidney, they were included in the Q-RT-PCR analysis. Hepcidin1 and hepcidin2 were studied in Hfe knock out mice and all three strains of dietary iron overload mice; DBA2, Balb/C, and C57BL6, whereas all other genes were studied in Hfe knock out mice and C57BL6 dietary iron overload mice. The expression of both hepcidin1 and hepcidin2 was not detectable in any of the mice studied (data not shown), thus it seems that hepcidin is not expressed in the kidney and therefore its expression in the kidney is not influenced
by iron overload. Also Hjv showed no expression in the kidney of both dietary iron overload and Hfe knock out mice (data not shown). The proposed receptor of HJV, neogenin, was expressed in the kidney of both studied mice strains, but its expression was not influenced by iron overload (data not shown). In Hfe knock out mice the only gene of which expression was regulated was Tfr2. Its fold change was calculated to be 1.5, thus its expression was slightly up-regulated. In dietary iron overload mice differential expression patterns of Tfr2, Hfe, Tfr1 and Irp1 were detected. Tfr2, Hfe and Irp1 showed a slight increase in their expression, while Tfr1 showed a slight decrease in its expression (Figure 9).
Figure 7. Regulated genes in dietary iron overload mice: normalized expression values and fold changes values obtained from Q-RT-PCR. * indicates p-value below 0.065, ** indicates p-value below 0.05 and *** indicates p-value below 0.02.
Figure 8. Regulated genes in Hfe knock out mice: normalized expression values and fold changes values obtained from Q-RT-PCR. * indicates p-value below 0.065, ** indicates p-value below 0.05 and *** indicates p-value below 0.02.
0
Cfd Hp Cish Dnajb1 Cyp26b1 Creld2 Hmox1 control
Figure 9. Normalized expression levels and fold change values of iron-related genes in both dietary iron overload mice and Hfe knock out mice. * indicates p-value below 0.065, ** indicates p-value below 0.05 and *** indicates p-value below 0.02.
0
Kidney iron and H FE ko iron-related genes
*
**
**
**
6 DISCUSSION
Although kidney is the main excreting organ, its role in iron homeostasis has not been studied widely. Its ability to adjust the concentration of various ions by affecting the amount of ions and the amount of water reabsorbed could provide a mechanism to overcome the changes in the body caused by iron overload. Like the liver, also the kidney has an important role in degrading various exogenous ligands such as pharmaceuticals and thus it has for example high cytochrome P450 activity. It is assumed that the oxidative stress caused by iron overload induces the expression of various acute phase proteins that protect cells from oxidative damage; also the expression of various iron-related proteins is assumed to be altered. The current microarray study revealed previously unknown expression patterns of certain genes during iron overload and it also indicated that certain pathways have high precendence in the lists obtained from microarray data analysis. The role of these genes and pathways during iron overload has to be inspected in detail to confirm their role and function during these circumstances.