Limits...
Renal Mitochondrial Lipid Peroxidation during Sepsis.

Singh P, Parajuli N, Mayeux PR, MacMillan-Crow LA - J Kidney (2016)

Bottom Line: However, few studies have attempted to dissect specific renal targets and/or types of oxidative injury using the cecal ligation and puncture (CLP) murine model of sepsis.Our results show that CLP induced increased 4-hydroxy-nonenal protein adduction (marker of lipid peroxidation) in renal homogenates and mitochondrial fractions.This supports our prior report showing renal complex III inactivation following CLP.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, AR, USA.

ABSTRACT

Sepsis can provoke kidney injury, which increases mortality. Human and animal studies have documented increased renal oxidative injury and mitochondrial damage during sepsis. However, few studies have attempted to dissect specific renal targets and/or types of oxidative injury using the cecal ligation and puncture (CLP) murine model of sepsis. The purpose of this short communication is to examine the extent of lipid peroxidation within renal mitochondria using CLP and blue native gel electrophoresis which separates intact mitochondrial respiratory complexes. Our results show that CLP induced increased 4-hydroxy-nonenal protein adduction (marker of lipid peroxidation) in renal homogenates and mitochondrial fractions. Blue native gel electrophoresis revealed that respiratory complex III was selectively targeted within mitochondrial fractions. This supports our prior report showing renal complex III inactivation following CLP. Future studies will identify specific renal proteins within complex III that are modified during sepsis to provide mechanistic insight on how mitochondrial respiration is inhibited during sepsis.

No MeSH data available.


Related in: MedlinePlus

4-HNE adduction of respiratory complex III. A. Representative 4-HNE western blot (WB) of blue native gel electrophoresis (BN-PAGE) of renal mitochondria isolated from mice subjected to CLP (sepsis) or sham surgery. B. Membrane from A was stripped and probed with antibody to Core-2 (as marker of complex III), showing equal levels of respiratory complex III in sham and CLP samples. Densitometry of band intensity 4-HNE/Core2 revealed increased 4-HNE adduction of complex III compared to sham animals *P<0.05 vs. sham; n = 4/group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836864&req=5

Figure 2: 4-HNE adduction of respiratory complex III. A. Representative 4-HNE western blot (WB) of blue native gel electrophoresis (BN-PAGE) of renal mitochondria isolated from mice subjected to CLP (sepsis) or sham surgery. B. Membrane from A was stripped and probed with antibody to Core-2 (as marker of complex III), showing equal levels of respiratory complex III in sham and CLP samples. Densitometry of band intensity 4-HNE/Core2 revealed increased 4-HNE adduction of complex III compared to sham animals *P<0.05 vs. sham; n = 4/group.

Mentions: Next, we wanted to determine whether lipid peroxidation was localized to the mitochondrial respiratory complexes, since our earlier studies showed that complex III activity was significantly declined at this time point (18 hr) post CLP [9]. Blue-native gel electrophoresis (BN-PAGE) was used to resolve different respiratory complexes without dissociating critical subunits [19,20]. To assess lipid peroxidation within renal mitochondrial respiratory complexes following CLP, solubilized renal mitochondrial extracts were resolved on a BN-PAGE gel followed by western blotting for 4-HNE. 4-HNE staining was increased within a single band corresponding to the molecular weight of complex III (~500 kD) in CLP, but not in Sham groups (Figure 2). The membrane was stripped and reprobed with an antibody to Core 2 (Complex III subunit) which showed equal loading of mitochondrial complex III in all samples. Densitometry revealed significant increases in 4-HNE protein adduction when compared to Core-2.


Renal Mitochondrial Lipid Peroxidation during Sepsis.

Singh P, Parajuli N, Mayeux PR, MacMillan-Crow LA - J Kidney (2016)

4-HNE adduction of respiratory complex III. A. Representative 4-HNE western blot (WB) of blue native gel electrophoresis (BN-PAGE) of renal mitochondria isolated from mice subjected to CLP (sepsis) or sham surgery. B. Membrane from A was stripped and probed with antibody to Core-2 (as marker of complex III), showing equal levels of respiratory complex III in sham and CLP samples. Densitometry of band intensity 4-HNE/Core2 revealed increased 4-HNE adduction of complex III compared to sham animals *P<0.05 vs. sham; n = 4/group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836864&req=5

Figure 2: 4-HNE adduction of respiratory complex III. A. Representative 4-HNE western blot (WB) of blue native gel electrophoresis (BN-PAGE) of renal mitochondria isolated from mice subjected to CLP (sepsis) or sham surgery. B. Membrane from A was stripped and probed with antibody to Core-2 (as marker of complex III), showing equal levels of respiratory complex III in sham and CLP samples. Densitometry of band intensity 4-HNE/Core2 revealed increased 4-HNE adduction of complex III compared to sham animals *P<0.05 vs. sham; n = 4/group.
Mentions: Next, we wanted to determine whether lipid peroxidation was localized to the mitochondrial respiratory complexes, since our earlier studies showed that complex III activity was significantly declined at this time point (18 hr) post CLP [9]. Blue-native gel electrophoresis (BN-PAGE) was used to resolve different respiratory complexes without dissociating critical subunits [19,20]. To assess lipid peroxidation within renal mitochondrial respiratory complexes following CLP, solubilized renal mitochondrial extracts were resolved on a BN-PAGE gel followed by western blotting for 4-HNE. 4-HNE staining was increased within a single band corresponding to the molecular weight of complex III (~500 kD) in CLP, but not in Sham groups (Figure 2). The membrane was stripped and reprobed with an antibody to Core 2 (Complex III subunit) which showed equal loading of mitochondrial complex III in all samples. Densitometry revealed significant increases in 4-HNE protein adduction when compared to Core-2.

Bottom Line: However, few studies have attempted to dissect specific renal targets and/or types of oxidative injury using the cecal ligation and puncture (CLP) murine model of sepsis.Our results show that CLP induced increased 4-hydroxy-nonenal protein adduction (marker of lipid peroxidation) in renal homogenates and mitochondrial fractions.This supports our prior report showing renal complex III inactivation following CLP.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, AR, USA.

ABSTRACT

Sepsis can provoke kidney injury, which increases mortality. Human and animal studies have documented increased renal oxidative injury and mitochondrial damage during sepsis. However, few studies have attempted to dissect specific renal targets and/or types of oxidative injury using the cecal ligation and puncture (CLP) murine model of sepsis. The purpose of this short communication is to examine the extent of lipid peroxidation within renal mitochondria using CLP and blue native gel electrophoresis which separates intact mitochondrial respiratory complexes. Our results show that CLP induced increased 4-hydroxy-nonenal protein adduction (marker of lipid peroxidation) in renal homogenates and mitochondrial fractions. Blue native gel electrophoresis revealed that respiratory complex III was selectively targeted within mitochondrial fractions. This supports our prior report showing renal complex III inactivation following CLP. Future studies will identify specific renal proteins within complex III that are modified during sepsis to provide mechanistic insight on how mitochondrial respiration is inhibited during sepsis.

No MeSH data available.


Related in: MedlinePlus