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Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells.

Henzi T, Schwaller B - PLoS ONE (2015)

Bottom Line: With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated.Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected.In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.

No MeSH data available.


Related in: MedlinePlus

Western blots for COX1 and ATP synthase subunit β in DCT lysates from 4 control (WT) and 4 PV-/- mice.Values are mean ± sem of 3 independent experiments. In PV-/- mice the expression of COX1 was augmented by 31.2 ± 7.0% compared to WT (*p = 0.03706), while no changes were observed for ATP synthase (n.s.). Representative Western blot signals are shown in the upper part.
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pone.0142005.g003: Western blots for COX1 and ATP synthase subunit β in DCT lysates from 4 control (WT) and 4 PV-/- mice.Values are mean ± sem of 3 independent experiments. In PV-/- mice the expression of COX1 was augmented by 31.2 ± 7.0% compared to WT (*p = 0.03706), while no changes were observed for ATP synthase (n.s.). Representative Western blot signals are shown in the upper part.

Mentions: DCT segments from mice expressing EGFP under the control of the Pvalb promoter [20], either control mice with PV expression (PV+/+) or PV-deficient mice (PV-/-) were isolated using a Complex Object Parametric Analyzer and Sorter (COPAS), an established method to isolate specific tubular fragments of the nephron [15,21,26]. Beforehand, in order to verify that EGFP-expressing tubule segments are identical to segments with endogenous PV expression, kidney sections from EGFP-PV+/+ mice were stained for either EGFP or PV (Fig 1); the merged images demonstrated essentially 100% overlap. The EGFP expression pattern was the same in EGFP-PV-/- mice, but evidently no specific PV signal was observed (Fig 1). RNA from both genotypes (3 mice per group) was extracted and used for Affymetrix Gene Chip Analysis. Initial analyzes revealed a vast number of signals to be significantly up- or downregulated in DCT of PV-/- mice (S1 Table). As previously reported for fast-twitch muscle and PV-overexpressing myotubes [5,9], signals for several genes coding for mitochondrial proteins (Ucp2, Efhd1, Micu1, Mcu, Mcur1, COX1, Atp5b) were increased. In order to validate the Gene Chip data, total RNA was extracted from a second set of COPAS-isolated DCT fragments of 4 PV+/+ and 4 PV-/- animals and mRNA levels of seven genes coding for mitochondrial proteins were determined by quantitative RT-PCR. Besides prototypical mitochondrial genes including cytochrome c oxidase subunit 1 (COX1), ATP synthase subunit β (Atp5b), uncoupling protein 2 (Ucp2), we focused on genes implicated in mitochondrial Ca2+ handling: mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu) and mitochondrial calcium uniporter regulator 1 (Mcur1). In comparison to PV+/+ mice, mRNA levels in PV-/- DCT were upregulated for all investigated genes (Fig 2). The same set of genes was also analyzed in TA of mice with or without PV expression. All seven genes were also upregulated in TA at the level of mRNA (S1 Fig). With respect to protein expression, the mitochondrial encoded cytochrome oxidase c subunit 1 (COX1), part of complex IV that participates in the transport of protons across the inner mitochondrial membrane and in establishing the mitochondrial membrane potential (ΔΨm), was increased by 31% ± 7% in the DCT of PV-/- mice (Fig 3), while protein levels of ATP synthase subunit β were unchanged. Thus, results from qRT-PCR and Western blot analyses suggested that the absence of the cytoplasmic Ca2+ buffer PV altered the mitochondrial protein composition: up-regulating proteins implicated in Ca2+ handling and generation of ΔΨm, likely without increasing ATP production evidenced by unchanged ATP synthase subunit β and also by increased Ucp2 mRNA levels. Thus, we postulated that in the absence of PV, mitochondria are modified to better uphold ΔΨm necessary to drive mitochondrial Ca2+ uptake, thus emphasizing the role of mitochondria as temporary Ca2+ stores and involved in the shaping of Ca2+ transients [27]. To further gain insight in mechanisms of PV and mitochondria regulation, we used a model system, i.e. MDCK cells representing dog kidney distal nephron cells [24].


Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells.

Henzi T, Schwaller B - PLoS ONE (2015)

Western blots for COX1 and ATP synthase subunit β in DCT lysates from 4 control (WT) and 4 PV-/- mice.Values are mean ± sem of 3 independent experiments. In PV-/- mice the expression of COX1 was augmented by 31.2 ± 7.0% compared to WT (*p = 0.03706), while no changes were observed for ATP synthase (n.s.). Representative Western blot signals are shown in the upper part.
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pone.0142005.g003: Western blots for COX1 and ATP synthase subunit β in DCT lysates from 4 control (WT) and 4 PV-/- mice.Values are mean ± sem of 3 independent experiments. In PV-/- mice the expression of COX1 was augmented by 31.2 ± 7.0% compared to WT (*p = 0.03706), while no changes were observed for ATP synthase (n.s.). Representative Western blot signals are shown in the upper part.
Mentions: DCT segments from mice expressing EGFP under the control of the Pvalb promoter [20], either control mice with PV expression (PV+/+) or PV-deficient mice (PV-/-) were isolated using a Complex Object Parametric Analyzer and Sorter (COPAS), an established method to isolate specific tubular fragments of the nephron [15,21,26]. Beforehand, in order to verify that EGFP-expressing tubule segments are identical to segments with endogenous PV expression, kidney sections from EGFP-PV+/+ mice were stained for either EGFP or PV (Fig 1); the merged images demonstrated essentially 100% overlap. The EGFP expression pattern was the same in EGFP-PV-/- mice, but evidently no specific PV signal was observed (Fig 1). RNA from both genotypes (3 mice per group) was extracted and used for Affymetrix Gene Chip Analysis. Initial analyzes revealed a vast number of signals to be significantly up- or downregulated in DCT of PV-/- mice (S1 Table). As previously reported for fast-twitch muscle and PV-overexpressing myotubes [5,9], signals for several genes coding for mitochondrial proteins (Ucp2, Efhd1, Micu1, Mcu, Mcur1, COX1, Atp5b) were increased. In order to validate the Gene Chip data, total RNA was extracted from a second set of COPAS-isolated DCT fragments of 4 PV+/+ and 4 PV-/- animals and mRNA levels of seven genes coding for mitochondrial proteins were determined by quantitative RT-PCR. Besides prototypical mitochondrial genes including cytochrome c oxidase subunit 1 (COX1), ATP synthase subunit β (Atp5b), uncoupling protein 2 (Ucp2), we focused on genes implicated in mitochondrial Ca2+ handling: mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu) and mitochondrial calcium uniporter regulator 1 (Mcur1). In comparison to PV+/+ mice, mRNA levels in PV-/- DCT were upregulated for all investigated genes (Fig 2). The same set of genes was also analyzed in TA of mice with or without PV expression. All seven genes were also upregulated in TA at the level of mRNA (S1 Fig). With respect to protein expression, the mitochondrial encoded cytochrome oxidase c subunit 1 (COX1), part of complex IV that participates in the transport of protons across the inner mitochondrial membrane and in establishing the mitochondrial membrane potential (ΔΨm), was increased by 31% ± 7% in the DCT of PV-/- mice (Fig 3), while protein levels of ATP synthase subunit β were unchanged. Thus, results from qRT-PCR and Western blot analyses suggested that the absence of the cytoplasmic Ca2+ buffer PV altered the mitochondrial protein composition: up-regulating proteins implicated in Ca2+ handling and generation of ΔΨm, likely without increasing ATP production evidenced by unchanged ATP synthase subunit β and also by increased Ucp2 mRNA levels. Thus, we postulated that in the absence of PV, mitochondria are modified to better uphold ΔΨm necessary to drive mitochondrial Ca2+ uptake, thus emphasizing the role of mitochondria as temporary Ca2+ stores and involved in the shaping of Ca2+ transients [27]. To further gain insight in mechanisms of PV and mitochondria regulation, we used a model system, i.e. MDCK cells representing dog kidney distal nephron cells [24].

Bottom Line: With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated.Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected.In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.

No MeSH data available.


Related in: MedlinePlus