Diabetic nephropathy cases, which have doubled in the past decade, account for approximately half of all end-stage renal disease cases [1
]. Although oxygen is a necessary component for complex organisms that demand high energy, overproduction of reactive oxygen species (ROS) may cause DNA damage, cell death, and protein modifications that result in mitochondrial and cellular dysfunction [3
]. Diabetes, a common metabolic disorder, can trigger excess generation of ROS and plays an important role in increasing OS oxidative stress (OS) in various tissues, including the kidney. The increased OS during diabetes exacerbates the progression of disease and its associated complications such as renal vascular and proximal tubule dysfunction [1
]. However, the temporal and spatial aspects of these changes and, more specifically, the time of onset of OS during diabetes remain unknown.
The bcl-2 gene plays a central role in maintaining the mitochondrial oxidative homeostasis and its expression is protective during hyperglycemia-induced lipid peroxidation and advanced glycation end product modifications in endothelial cells [9
]. Thus, its absence causes a more oxidized state in tissue and, as such, the mitochondria is more oxidized in bcl-2–/–
mice as compared with their controls. In addition, bcl-2 is an anti-apoptotic protein whose expression decreases significantly during diabetes, and thus is of direct interest for this study.
mice develop type 1 diabetes as early as 4 weeks of age. Enzymatic and non-enzymatic sources contribute to ROS observed in the diabetic kidneys, including advanced glycation, mitochondrial respiration chain deficiencies, and NAD(P)H oxidase. OS occurs due to the inability of cells to detoxify excess amounts of ROS or loss of the cellular anti-oxidant defense [1
]. Thus, the tissue metabolic state is an indicator of cellular oxygen consumption, and it can be extracted from fluorescence images [13
Thrombospondin-1 (TSP1) is a potent endogenous inhibitor of angiogenesis, whose expression is dramatically down regulated during diabetes. Our hypothesis is that decreased production of TSP1 promotes the development and progression of diabetic nephropathy. The Akita/+;TSP1–/– mouse is a novel diabetes model developed in Dr. Sheibani’s lab that exhibits severe nephropathies with a relatively short duration of diabetes compared with the parental Akita/+ mice. Thus, the mice that carry the Akita mutation and lack TSP1 (TSP1–/–) serve as a model for severe diabetic nephropathy. Here we propose to determine the impact of the combination of these genetic modifications on the mitochondrial redox state associated with the onset and progression of diabetes. To verify that TSP1–/– genotype itself does not contribute to a more oxidized mitochondria, we compared TSP1–/– mice with Akita/+;TSP1–/– mice.
Fluorescence imaging provides specific information on tissue using intrinsic fluorophores or exogenous tagged proteins. Since some of the molecules in the cell have intrinsic fluorophores and are able to fluoresce when excited with the appropriate wavelength, a growing field of fluorescence microscopy techniques relies on autofluorescent fluorophores. Fluorescence-based techniques are widely used in biomedical applications as diagnostic/therapeutic tools for early detection of various diseases such as cancers or heart disease. Optical fluorescence techniques have the potential to diagnose tissue metabolic states in intact organs. These techniques are widely used in biomedical applications and have been shown to have a high sensitivity and specificity for discriminating between diseased and non-diseased tissue [15
Mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and Flavoprotein Adenine Dinocleotide (FADH2
) are the primary electron carriers in oxidative phosphorylation. NADH and FAD (the oxidized form of FADH2
) are autofluorescent and can be monitored without exogenous labels through the use of optical techniques. These coenzymes are beneficial in that NADH is primarily fluorescent in its reduced biochemical state, whereas FAD is only fluorescent in its oxidized form. Therefore, by imaging these two coenzymes, we can probe the oxidative state of the metabolism in tissue. The fluorescent signals of these intrinsic fluorophores have been used as indicators of tissue metabolism in injuries due to hypoxia, ischemia, and cell death [11
]. In addition, by evaluating the ratio of these two coenzymes, some of the confounding factors in determining this oxidative state can be removed, such as absorbers, including hemoglobin and collagen, as well as scattering effects. Our studies have demonstrated that the normalized ratio of these fluorophores, (NADH/FAD), called the mitochondrial redox ratio (RR), acts as a novel marker of the mitochondrial redox and metabolic state of tissue ex vivo
and in vivo
. Although this ratio is not a direct measure of the concentrations of these fluorophores, the fluorescence intensity measured is a relative measure of their concentrations. To date, several groups have used optical fluorescence imaging to probe the biochemical and morphological characteristics of tissues [15
]. Here we employed 3D cryo fluorescence redox imaging to delineate the temporal distribution of OS in kidneys from mice with different durations of diabetes.
We have used fluorescent imaging of these two proteins to determine the mitochondrial oxidative state in mice with the above genotypes, resulting in a total of 6 categories of mice, as follows. First, bcl-2–/– mice, which are more sensitive to OS and thus are expected to have a lower RR, were compared with their bcl-2+/+ controls to verify the ability of the system to detect differences in OS. Next, Akita/+ and their wild type (WT) controls were studied to determine whether a change in OS is correlated with the presence of diabetes. Similar to bcl-2–/–, Akita/+ mice are more susceptible to OS and are thus expected to have a lower RR. Finally, Akita/+;TSP1–/– mice were compared with their control, TSP1–/–, in order to evaluate the severity of diabetic nephropathy in these mice using the mitochondrial oxidative state as a quantitative marker. It should be noted that the name of each group indicates the only modification to the mouse, and thus Akita/+, Akita/+;TSP1–/– and TSP1–/– are not bcl-2 deficient.