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Light and heavy ferritin chain expression in the liver and kidneys of Wistar rats: aging, sex differences, and impact of gonadectomy

Mirela Pavić Vulinović

Mirela Pavić Vulinović

University of Zagreb Faculty of Veterinary Medicine, Department of Anatomy, Histology and EmbryologyZagreb, Croatia
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Vulinović, Mirela Pavić
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Petra Turčić

Petra Turčić

University of Zagreb Faculty of Pharmacy and Biochemistry, Department of PharmacologyZagreb, Croatia
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Turčić, Petra
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Vedran Micek

Vedran Micek

Institute for Medical Research and Occupational HealthZagreb, Croatia
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Micek, Vedran
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Marija Ljubojević

Marija Ljubojević

Institute for Medical Research and Occupational HealthZagreb, Croatia
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Ljubojević, Marija
  
07 apr 2022
Light and heavy ferritin chain expression in the liver and kidneys of Wistar rats: aging, sex differences, and impact of gonadectomy's Cover Image
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Categoria dell'articolo: Original article
Pubblicato online: 07 apr 2022
Pagine: 48 - 61
Ricevuto: 01 dic 2021
Accettato: 01 mar 2022
DOI: https://doi.org/10.2478/aiht-2022-73-3621
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© 2022 Mirela Pavić Vulinović, Petra Turčić, Vedran Micek, Marija Ljubojević, published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Liver ferritin immunochemistry conditions during procedure optimisation. (A) Sample preparation for Western blotting in reducing and nonreducing conditions at three temperatures that all yielded a strong and sharp protein band of 20 kDa for FtL and 21 kDa for FtH; (B) Western blotting for protein load – livers of 3-month-old male and female stained with Coomassie blue staining (CBS). PVDF membrane was incubated only with secondary antibody (secAb) followed by FtH antibody reincubation. Densitometric result between CBS samples and secondary antibody nonspecific band (NB) staining did not show difference vs protein band after FtH staining; (C) Antigen retrieval conditions for immunofluorescent staining for both ferritin antibodies. No staining was observed either without retrieval or with sodium dodecyl sulphate (SDS). Microwave cooking in citrate buffer (CT) with different pH gave best results for both ferritin antibodies at pH 6. Sham deparaffinisation with organic solutions and alcohol prior to cooking in citrate buffer (DCT) did not increase staining intensity
Liver ferritin immunochemistry conditions during procedure optimisation. (A) Sample preparation for Western blotting in reducing and nonreducing conditions at three temperatures that all yielded a strong and sharp protein band of 20 kDa for FtL and 21 kDa for FtH; (B) Western blotting for protein load – livers of 3-month-old male and female stained with Coomassie blue staining (CBS). PVDF membrane was incubated only with secondary antibody (secAb) followed by FtH antibody reincubation. Densitometric result between CBS samples and secondary antibody nonspecific band (NB) staining did not show difference vs protein band after FtH staining; (C) Antigen retrieval conditions for immunofluorescent staining for both ferritin antibodies. No staining was observed either without retrieval or with sodium dodecyl sulphate (SDS). Microwave cooking in citrate buffer (CT) with different pH gave best results for both ferritin antibodies at pH 6. Sham deparaffinisation with organic solutions and alcohol prior to cooking in citrate buffer (DCT) did not increase staining intensity

Figure 2

Kidney panels with parallel ferritin staining in consecutive cryosections of female kidney. Immunofluorescence of FtL (a) and FtH (b) shows stronger staining in epithelial cells of proximal tubules throughout the cortex and much weaker and mosaic staining in the outer stripe of kidney zones. Proximal tubules showed homogenous intracellular staining, whereas some S1 and S2 segments showed relatively dotty and faint (a, FtL) to strong (b, FtH) staining. Arrowheads on both panels show S1 segment of the proximal tubule (a, b) leaving juxtamedullar glomeruli (G). On the b panel S2 segments show homogenous and strong staining (arrow in G proximity). Faint (a,b) and mosaic staining (a) was observed in S3 segments (bottom of panels below G). Many proximal tubules contained ferritin-positive vesicles in the cells and in the lumen (asterisk in the S2 segment). This was found for both chains in the same cells/tubules of consecutive cryosections (a and b panels). However, FtH was more prominently stained in structures such as intercalated cells in the collecting duct (ladder) and weakly in the thick ascending limb of Henley (tubule to the left above the ladder). Bar=50 μm
Kidney panels with parallel ferritin staining in consecutive cryosections of female kidney. Immunofluorescence of FtL (a) and FtH (b) shows stronger staining in epithelial cells of proximal tubules throughout the cortex and much weaker and mosaic staining in the outer stripe of kidney zones. Proximal tubules showed homogenous intracellular staining, whereas some S1 and S2 segments showed relatively dotty and faint (a, FtL) to strong (b, FtH) staining. Arrowheads on both panels show S1 segment of the proximal tubule (a, b) leaving juxtamedullar glomeruli (G). On the b panel S2 segments show homogenous and strong staining (arrow in G proximity). Faint (a,b) and mosaic staining (a) was observed in S3 segments (bottom of panels below G). Many proximal tubules contained ferritin-positive vesicles in the cells and in the lumen (asterisk in the S2 segment). This was found for both chains in the same cells/tubules of consecutive cryosections (a and b panels). However, FtH was more prominently stained in structures such as intercalated cells in the collecting duct (ladder) and weakly in the thick ascending limb of Henley (tubule to the left above the ladder). Bar=50 μm

Figure 3

Details of ferritin chain expression in the kidneys. Panel a, b, and c show FtL and panels d, e, and f FtH staining in specific nephron segments. Intracellular staining was mostly homogenous in epithelial cells of the proximal tubule (PT). Staining in the S1 segment leaving glomeruli (G) was relatively faint for FtL (a) and stronger for FtH (d) (asterisk in the S1 segment of PT leaving G). Ferritin-positive were specific structures of macula densa with both antibodies (arrowhead in b, e below G). Intracellular vesicles can be seen in all PT tubules (arrow in b and e) and in the PT lumen (arrow in c and f). Both ferritin chains were positive in intercalated cells of the collecting duct (asterisk in c, f). Panels d, e, and f show some positive FtH staining in glomeruli, possibly in the podocytes, mesangial cells, and/or parietal layer. This was not prominent with FtL (G in a, b). Bar=20 μm
Details of ferritin chain expression in the kidneys. Panel a, b, and c show FtL and panels d, e, and f FtH staining in specific nephron segments. Intracellular staining was mostly homogenous in epithelial cells of the proximal tubule (PT). Staining in the S1 segment leaving glomeruli (G) was relatively faint for FtL (a) and stronger for FtH (d) (asterisk in the S1 segment of PT leaving G). Ferritin-positive were specific structures of macula densa with both antibodies (arrowhead in b, e below G). Intracellular vesicles can be seen in all PT tubules (arrow in b and e) and in the PT lumen (arrow in c and f). Both ferritin chains were positive in intercalated cells of the collecting duct (asterisk in c, f). Panels d, e, and f show some positive FtH staining in glomeruli, possibly in the podocytes, mesangial cells, and/or parietal layer. This was not prominent with FtL (G in a, b). Bar=20 μm

Figure 4

Immunochemistry of the light ferritin chain in the liver. Western blotting with D-1 antibody against FtL (A) followed by densitometric evaluation (B) of the protein bands labeled in liver tissue homogenates. FtL immunostaining in cryosections of the liver tissues of three-month (3m) and two-year (2y) old male (M) and female (F) rats (C). The strong protein band of ~ 20 kDa represents the FtL chain and was used for densitometric evaluation in relation to NB (A). Each band represents FtL protein expression in tissue homogenates from individual animals. Higher expression of FtL is visible in female rats regardless of age and in 2-year-old male and female animals. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G) as unused housekeeping proteins. Densitometric evaluation of protein bands (B) is shown by bars, each representing mean ± SEM of band density of four animals per group. Statistics: vs respective M or 3-month-old animals, **P<0.01. Immunohistochemical images of FtL in liver tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b) and 2y old F vs 2y old M (c, d), 2y old M vs 3m old M (a, c), and 2y old F (b, d) vs 3m old F. Bar=30 μm
Immunochemistry of the light ferritin chain in the liver. Western blotting with D-1 antibody against FtL (A) followed by densitometric evaluation (B) of the protein bands labeled in liver tissue homogenates. FtL immunostaining in cryosections of the liver tissues of three-month (3m) and two-year (2y) old male (M) and female (F) rats (C). The strong protein band of ~ 20 kDa represents the FtL chain and was used for densitometric evaluation in relation to NB (A). Each band represents FtL protein expression in tissue homogenates from individual animals. Higher expression of FtL is visible in female rats regardless of age and in 2-year-old male and female animals. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G) as unused housekeeping proteins. Densitometric evaluation of protein bands (B) is shown by bars, each representing mean ± SEM of band density of four animals per group. Statistics: vs respective M or 3-month-old animals, **P<0.01. Immunohistochemical images of FtL in liver tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b) and 2y old F vs 2y old M (c, d), 2y old M vs 3m old M (a, c), and 2y old F (b, d) vs 3m old F. Bar=30 μm

Figure 5

Immunochemistry of the heavy ferritin chain in the liver. Western blotting with B-12 antibody against FtH (A) and densitometric evaluation (B) of the protein bands. FtH immunostaining in cryosections of the liver tissues of three-month (3m) and two-year (2y) old male (M) and female (F) rats (C). The strong protein band of ~21 kDa represents the FtH and was used for densitometric evaluation in relation to NB. Each band represents FtH protein expression in tissue homogenates from individual animals. Higher expression of FtH is visible in female rats regardless of age and in 2-year-old male and female animals in M and F. Densitometric evaluation of the protein bands (B) is shown by bars, each representing mean ± SEM of band density of four animals per group. Statistics: vs respective males or 3-month-old animals , * * P < 0 . 0 1 . Immunohistochemical images of FtH in liver tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b) and in 2y old F vs 2y old M (c, d) and in 2y old M vs 3m old M (a, c) and in 2y old F vs 3m old F (b, d). Bar=30 μm
Immunochemistry of the heavy ferritin chain in the liver. Western blotting with B-12 antibody against FtH (A) and densitometric evaluation (B) of the protein bands. FtH immunostaining in cryosections of the liver tissues of three-month (3m) and two-year (2y) old male (M) and female (F) rats (C). The strong protein band of ~21 kDa represents the FtH and was used for densitometric evaluation in relation to NB. Each band represents FtH protein expression in tissue homogenates from individual animals. Higher expression of FtH is visible in female rats regardless of age and in 2-year-old male and female animals in M and F. Densitometric evaluation of the protein bands (B) is shown by bars, each representing mean ± SEM of band density of four animals per group. Statistics: vs respective males or 3-month-old animals , * * P < 0 . 0 1 . Immunohistochemical images of FtH in liver tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b) and in 2y old F vs 2y old M (c, d) and in 2y old M vs 3m old M (a, c) and in 2y old F vs 3m old F (b, d). Bar=30 μm

Figure 6

Immunochemistry of the light ferritin chain in the kidney. Western blotting with D-1 antibody against FtL (A) followed by densitometric evaluation (B) of the protein bands labeled in kidney tissue homogenates. F t L immunostaining in cryosections of the kidney tissues of three-month (3m) and two-year (2y) old male (M) and female (F) rats (C). The strong protein band represents FtL and was used for densitometric evaluation in relation to beta-actin (bA). Each band represents FtL protein expression in tissue homogenates from individual animals. Higher expression of FtL is visible in female rats regardless of age and in 2-year-old male and female animals. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G) as housekeeping proteins. Densitometric evaluation of protein bands (B) is shown by bars, each representing mean ± SEM of band density of four animals per group. Statistics: vs respective M or 3m old animals, **P <0.01. Immunohistochemical images of FtL in kidney tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b), 2y old F vs 2y old M (c, d), 2y old M (a, c) vs 3m old M, and 2y old F (b, d) vs 3m old F. Glomeruli (G) are marked on panel (C) and arrowhead in c marks macula densa, whereas arrows mark positive interstitial staining, probably macrophages. Bar=20 μm
Immunochemistry of the light ferritin chain in the kidney. Western blotting with D-1 antibody against FtL (A) followed by densitometric evaluation (B) of the protein bands labeled in kidney tissue homogenates. F t L immunostaining in cryosections of the kidney tissues of three-month (3m) and two-year (2y) old male (M) and female (F) rats (C). The strong protein band represents FtL and was used for densitometric evaluation in relation to beta-actin (bA). Each band represents FtL protein expression in tissue homogenates from individual animals. Higher expression of FtL is visible in female rats regardless of age and in 2-year-old male and female animals. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G) as housekeeping proteins. Densitometric evaluation of protein bands (B) is shown by bars, each representing mean ± SEM of band density of four animals per group. Statistics: vs respective M or 3m old animals, **P <0.01. Immunohistochemical images of FtL in kidney tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b), 2y old F vs 2y old M (c, d), 2y old M (a, c) vs 3m old M, and 2y old F (b, d) vs 3m old F. Glomeruli (G) are marked on panel (C) and arrowhead in c marks macula densa, whereas arrows mark positive interstitial staining, probably macrophages. Bar=20 μm

Figure 7

Immunochemistry of the heavy ferritin chain in the kidney. Western blotting with B-12 antibody against FtH (A), and densitometric evaluation (B) of the protein bands. FtH immunostaining in cryosections of the kidney tissue of three-month (3m) and two-year (2y) old male (M) and female (F) (C). The strong protein represents FtH and was used for densitometric evaluation in relation to beta-actin (bA) (A). Higher expression of FtH is visible in female rats regardless of age and in 2-year-old male and female animals. Each band represents FtH protein expression in tissue homogenates from individual animals. Densitometric evaluation of protein bands (B) is shown by bars, each bar representing mean ± SEM of band density of four animals per group. Statistics: vs respective M or 3m old animals, **P <0.01. Immunohistochemical images of FtH in kidney tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b), 2y old F vs 2y old M (c, d), 2y old M vs 3m old M (a, c), and 2y old F vs 3m old F (b, d). Glomeruli (G) are marked on panel (C) and in c (2y M) and d (2y F) and are better stained in 3m M and F (a, b) (possibly present in podocytes, mesangial cells or/and parietal layer). Bar=20 μm
Immunochemistry of the heavy ferritin chain in the kidney. Western blotting with B-12 antibody against FtH (A), and densitometric evaluation (B) of the protein bands. FtH immunostaining in cryosections of the kidney tissue of three-month (3m) and two-year (2y) old male (M) and female (F) (C). The strong protein represents FtH and was used for densitometric evaluation in relation to beta-actin (bA) (A). Higher expression of FtH is visible in female rats regardless of age and in 2-year-old male and female animals. Each band represents FtH protein expression in tissue homogenates from individual animals. Densitometric evaluation of protein bands (B) is shown by bars, each bar representing mean ± SEM of band density of four animals per group. Statistics: vs respective M or 3m old animals, **P <0.01. Immunohistochemical images of FtH in kidney tissue cryosections (C) show similar findings in 3m (a, b) and 2y (c, d) old M (a, c) and F (b, d) rats. Intracellular staining is stronger in 3m old F vs 3m old M (a, b), 2y old F vs 2y old M (c, d), 2y old M vs 3m old M (a, c), and 2y old F vs 3m old F (b, d). Glomeruli (G) are marked on panel (C) and in c (2y M) and d (2y F) and are better stained in 3m M and F (a, b) (possibly present in podocytes, mesangial cells or/and parietal layer). Bar=20 μm

Figure 8

Effect of gonadectomy on both ferritin chain expression in the liver. Expression of FtL and FtH protein (A, B) in the liver by western blotting. Each band represents FtL or FtH expression in the tissue of individual animals. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G). Each bar represents mean ± SEM of band density from four animals per group. Data were analysed with the t-test. Ovariectomy statistically decreased the expression of both ferritin chains and castration increased their expression. Statistics: sham-gonadectomised (SG) vs castrated males **P<0.01 (A); SG vs ovariectomised females *P<0.05 (B)
Effect of gonadectomy on both ferritin chain expression in the liver. Expression of FtL and FtH protein (A, B) in the liver by western blotting. Each band represents FtL or FtH expression in the tissue of individual animals. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G). Each bar represents mean ± SEM of band density from four animals per group. Data were analysed with the t-test. Ovariectomy statistically decreased the expression of both ferritin chains and castration increased their expression. Statistics: sham-gonadectomised (SG) vs castrated males **P<0.01 (A); SG vs ovariectomised females *P<0.05 (B)

Figure 9

Effect of gonadectomy on both ferritin chain expression in the kidney. Expression of FtL and FtH protein (A, B) in the kidney by western blotting. Each band represents FtL or FtH expression in the tissue of individual animals. Each bar represents mean ± SEM of band density from four animals per group. Data were analysed with the t-test. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G). Ovariectomy statistically decreased the expression of both ferritin chains and castration increased their expression. Statistics: sham-gonadectomised (SG) vs castrated males **P <0.01 (A); SG vs ovariectomised females *P < 0.05 (B)
Effect of gonadectomy on both ferritin chain expression in the kidney. Expression of FtL and FtH protein (A, B) in the kidney by western blotting. Each band represents FtL or FtH expression in the tissue of individual animals. Each bar represents mean ± SEM of band density from four animals per group. Data were analysed with the t-test. Additional protein bands are from alpha-tubulin (aT), beta-actin (bA), and GAPDH (G). Ovariectomy statistically decreased the expression of both ferritin chains and castration increased their expression. Statistics: sham-gonadectomised (SG) vs castrated males **P <0.01 (A); SG vs ovariectomised females *P < 0.05 (B)
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