Regulation of vitamin D-1alpha-hydroxylase in a human cortical collecting duct cell line

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Regulation of vitamin D-1alpha-hydroxylase in a human cortical collecting duct cell line. / Bland, Rosemary; Zehnder, Daniel; Hughes, Susan; Ronco, PM; Stewart, Paul; Hewison, Martin.

In: Kidney International, Vol. 60, No. 4, 21.10.2001, p. 1277-1286.

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@article{c35c53100f9b48199523c43b57faf64e,
title = "Regulation of vitamin D-1alpha-hydroxylase in a human cortical collecting duct cell line",
abstract = "BACKGROUND: Recent studies have shown that renal expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1alpha-OHase expression and activity in a human cortical collecting duct cell line (HCD). METHODS: Expression of 1alpha-OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D3 as the substrate; conversion to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 was then determined by thin-layer chromatography. RESULTS: HCD cells expressed mRNA and protein for 1alpha-OHase. However, basal 1,25(OH)2D3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)2D3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)2D3 itself decreased 1alpha-OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1alpha-OHase activity was observed in the HCD cells following incubation with lipopolysaccharide, which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4. CONCLUSIONS: These results highlight the capacity for synthesis of 1,25(OH)2D3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1alpha-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron.",
keywords = "parathyroid hormone, immunosuppression, steroid hormone, HKC-8 cell line, vitamin D metabolism., calcium homeostasis, HCD cell line, distal nephron",
author = "Rosemary Bland and Daniel Zehnder and Susan Hughes and PM Ronco and Paul Stewart and Martin Hewison",
year = "2001",
month = oct,
day = "21",
doi = "10.1046/j.1523-1755.2001.00966.x",
language = "English",
volume = "60",
pages = "1277--1286",
journal = "Kidney International",
issn = "0085-2538",
publisher = "Nature Publishing Group",
number = "4",

}

RIS

TY - JOUR

T1 - Regulation of vitamin D-1alpha-hydroxylase in a human cortical collecting duct cell line

AU - Bland, Rosemary

AU - Zehnder, Daniel

AU - Hughes, Susan

AU - Ronco, PM

AU - Stewart, Paul

AU - Hewison, Martin

PY - 2001/10/21

Y1 - 2001/10/21

N2 - BACKGROUND: Recent studies have shown that renal expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1alpha-OHase expression and activity in a human cortical collecting duct cell line (HCD). METHODS: Expression of 1alpha-OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D3 as the substrate; conversion to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 was then determined by thin-layer chromatography. RESULTS: HCD cells expressed mRNA and protein for 1alpha-OHase. However, basal 1,25(OH)2D3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)2D3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)2D3 itself decreased 1alpha-OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1alpha-OHase activity was observed in the HCD cells following incubation with lipopolysaccharide, which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4. CONCLUSIONS: These results highlight the capacity for synthesis of 1,25(OH)2D3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1alpha-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron.

AB - BACKGROUND: Recent studies have shown that renal expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1alpha-OHase expression and activity in a human cortical collecting duct cell line (HCD). METHODS: Expression of 1alpha-OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D3 as the substrate; conversion to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 was then determined by thin-layer chromatography. RESULTS: HCD cells expressed mRNA and protein for 1alpha-OHase. However, basal 1,25(OH)2D3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)2D3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)2D3 itself decreased 1alpha-OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1alpha-OHase activity was observed in the HCD cells following incubation with lipopolysaccharide, which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4. CONCLUSIONS: These results highlight the capacity for synthesis of 1,25(OH)2D3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1alpha-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron.

KW - parathyroid hormone

KW - immunosuppression

KW - steroid hormone

KW - HKC-8 cell line

KW - vitamin D metabolism.

KW - calcium homeostasis

KW - HCD cell line

KW - distal nephron

UR - http://www.scopus.com/inward/record.url?scp=0034799492&partnerID=8YFLogxK

U2 - 10.1046/j.1523-1755.2001.00966.x

DO - 10.1046/j.1523-1755.2001.00966.x

M3 - Article

C2 - 11576342

VL - 60

SP - 1277

EP - 1286

JO - Kidney International

JF - Kidney International

SN - 0085-2538

IS - 4

ER -