Friedrich 2010 Abstract MiP2010

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

Link:

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⁺ (5.2±0.2 vs. 2.5±0.2, 3.7±0.2, 3.9±0.2, 3.0±0.1, respectively). However, RCR of heart mitochondria was lowest at 37 mM (3.9±0.3) and was highest at 146 mM K⁺ (10.1±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 µM), 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.

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

Labels:

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. - [email protected]