*3.4. PrC-210 Protection of Rat Kidney Mitochondria*

Sustained mitochondrial function during and after kidney transplant is required for survival of the transplanted organ. ROS generated during transplant-associated ischemia, ischemia-reperfusion, and post-implant inflammation can all affect kidney mitochondrial performance and survival. Because of the important mitochondrial role, we isolated mitochondria from rat kidneys and determined whether PrC-210 at achievable pharmacologic concentrations could protect these organelles from an ROS insult.

In Figure 7, purified rat kidney mitochondria incubated with an •OH generator [23]. Following the brief reaction, we saw significant ROS fragmentation of rat kidney mitochondrial DNA. Following the ROS insult, an aliquot of the mitochondria was solubilized in SDS-containing gel-loading buffer, and mitochondrial DNA was separated and "sized" using agarose gel chromatography (Figure 7). The ROS insult clearly reduced the mean size of the rat kidney mitochondrial DNA (lane b), and addition of PrC-210 to the mitochondria prevented the DNA breakage (lanes c–g) in a PrC-210 concentration-dependent manner.

**Figure 6.** Effect of PrC-210 administration on serum and kidney homogenate cytokine levels 20 h after transplant of the BN kidney into a LEW rat. (**A**,**B**) TIMP-1, (**C**) TNF-alpha and (**D**) MIP-3A/CCL20 levels were determined by ELISA analysis. Serum collection and transplanted BN kidney harvest and homogenization were done as described in Methods. *p* values are indicated. a,b,c designations are used to enable statistical comparisons between groups.

**Figure 7.** Suppression of hydroxyl radical-induced rat kidney mitochondrial DNA fragmentation by PrC-210 in a dose-dependent manner. Ten minutes before addition of the hydroxyl radical generator, PrC-210 was added to the incubations at the indicated concentrations. Following 20 min incubation at 37 ◦C, a reaction aliquot (10 μL) was mixed with 0.1% SDS loading dye and heated to 60 ◦C (1 min). Electrophoresis was performed, and gels were stained with ethidium bromide.
