Friedrich 2010 Abstract MiP2010

From Bioblast
Kidney cortex mitochondria are non-functional in a potassium-based media whereas heart mitochondria improve with increasing potassium concentration. Mitochondr Physiol Network 15.6.

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Friederich-Persson M, Wilcox CS, Palm F (2010)

Event: MiP2010

A medium of containing high levels of potassium chloride (KCl) is commonly used when assessing respiratory function of isolated mitochondria from various tissues. However, the measured intracellular [K+] in kidney proximal tubular cells is about 60 mM and in cardiac myocytes approximately 130 mM. Therefore, the use of a similar media [K+] for all tissues seems unsupported. Here we investigated the effect of different [K+] on respiratory function in mitochondria isolated from kidney cortex and heart of healthy male Sprague-Dawley rats.

Oxygen consumptions and the respiratory control ratios (RCR) were measured using respiratory medias containing [K+] of 15, 37, 81, 111 and 146 mM. In all measurements, the media contained (in mM): 1 EGTA, 20 HEPES, 5 MgCl2, 5 KPO4- and 1 g/l bovine serum albumin. pH was adjusted to 7.4 and the osmolarity to 330 mosm/kg HK2O using a 1:3 ratio of sucrose and mannitol.

The RCR of kidney cortex mitochondria decreased when the [K+] was elevated compared to the media containing 15 mM K+ (5.2Β±0.2 vs. 2.5Β±0.2, 3.7Β±0.2, 3.9Β±0.2, 3.0Β±0.1, respectively). However, RCR of heart mitochondria was lowest at 37 mM (3.9Β±0.3) and was highest at 146 mM K+ (10.1Β±0.45). A two-way ANOVA showed that kidney cortex mitochondria have a different sensitivity towards K+ compared to heart mitochondria (interaction P<0.05, treatment P<0.05, group P<0.05). Glibenclamide (100 Β΅M), an inhibitor of the ATP-sensitive K+ channel, increased RCR in kidney cortex mitochondria at 15 mM K+ (+32%), but significantly more at 146 mM K+ (+47%). Blockade of the voltage-gated K+ channel by 4-aminopyridine (4-AP, 1 mM) together with glibenclamide improved RCR by +73% at 146 mM K+. Neither of the applied K+-channel blockers had any effect on the RCR of heart mitochondria. Mitochondria swelling at increasing [K+] were observed in kidney cortex mitochondria, measured as loss of absorbance at 540 nm.

Kidney cortex mitochondria in K+-based media are non-functional in [K+] ranging from 37-146 mM. Heart mitochondria do not display K+-sensitivity to the same degree, but rather increase respiratory function with increasing [K+]. Furthermore, we demonstrated that a tissue specific difference in mitochondria K+-channels may explain these differences. The present study therefore demonstrates the importance of choosing a correct in vitro media to ensure a high quality of mitochondria research.


β€’ O2k-Network Lab: SE Uppsala Liss P, US CO Denver Van Hove J


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Organism: Rat  Tissue;cell: Heart, Kidney, Other cell lines  Preparation: Isolated mitochondria 



HRR: Oxygraph-2k 

MiP2010 

Affiliation

1-Dept Med Cell Biol, Uppsala Univ, Sweden.

2-Dept Med, Division Hypertension Nephrology, Georgetown Univ Medical Center, Washington DC, USA. - malou.friederich@mcb.uu.se

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