*2.9. Rat Kidney Mitochondria*

The purified mitochondrial fraction was prepared from homogenized rat kidneys by a standard centrifugation technique [22]. The purified mitochondria were suspended in 0.15 M Tris HCl buffer, pH 7.4.

To determine whether the addition of exogenous PrC-210 suppresses ROS-induced fragmentation of mitochondrial DNA [22], in a 25 μL reaction volume (in a PCR tube), we added: 10 μL purified mitochondria, 5 μL PrC-210 dilution or water (PrC-210 was added 10 min before the Fe++ + ADP +H2O2 •OH generator), and 10 <sup>μ</sup>L containing FeCl2 (2.5 mM; FW:127), adenosine 5 -diphosphate sodium salt (10 mM; FW: 427) and H2O2 (0.003% final concentration). After 20 min at 37 ◦C, 10 μL of the reaction was mixed with 5 μL of 6× gel loading dye containing 0.3% SDS; tubes sat in 60 ◦C water for 1 min, 10 ul was then loaded into a well of a 1% agarose TAE gel, and after 60 min at 60 volts, gels were stained and photographed. A minimum of three replicates were done for each assay point to enable statistical comparison.

#### *2.10. Statistical Analysis*

Data are expressed as the means +/− STDs. Student's t-tests were used to determine statistical difference and *p* values using GraphPad Prism 7.03 software. *p*-values less than 0.05 were considered significant.

#### **3. Results**

#### *3.1. PrC-210 Suppression of Kidney Allograft Pathology*

At 20 h post-transplant, the histology of the transplanted BN kidneys (Figure 2A–D) treated with UW Solution alone clearly showed increased Banff scores for tubulitis and peritubular capillaritis (red and yellow arrows); the summed pathology scores are shown in panel E. BN kidneys perfused with PrC-210-containing UW Solution and receiving postimplant intraperitoneal systemic doses of PrC-210 (Figure 2D) showed clearly suppressed inflammatory pathology to the kidney and suppressed Banff scores for tubulitis and peritubular capillaritis equal to Banff scores of the "0 h" control BN group (Figure 2E).

**Figure 2.** Histology of (**A**) BN kidney upon removal from BN rat. (**B**–**D**) BN kidneys 20 h following transplant into LEW rats. The "20 h" control kidneys (**B**,**C**) were flushed with room temperature UW Solution prior to transplant, and +PrC-210 kidneys (**D**) were flushed with room temperature UW Solution containing 30 mM PrC-210 prior to transplant, and recipient LEW rat also received three intraperitoneal systemic PrC-210 doses in the eight hours following transplant. Panel (**B**) red arrows highlight tubulitis pathology, and Panel C yellow arrows highlight peritubular capillaritis pathology. Panel (**E**) shows summed tubulitis and peritubular capillaritis severity scores for three kidneys in each of the indicated groups.

The "20 h" histology of the BN kidneys only flushed with UW Solution showed a significantly reduced thickness of the renal tubule brush border epithelium (Figure 3B) in comparison to the "0 h" control BN kidneys just removed from a BN rat (Figure 3A). The histology of BN kidneys receiving PrC-210 via both the perfusing UW Solution and intraperitoneal injections, clearly showed a preserved integrity of the renal tubular brush border (Figure 3C).

The same blinded histology sections were examined for mononuclear white cell infiltration. BN kidneys at 20 h only flushed with UW Solution showed a significant increase in the number of blue nuclei (scored as blue pixels), which reflects the infiltration of mononuclear white blood cells. In contrast, mononuclear infiltration in the BN kidneys flushed and treated with PrC-210 was significantly suppressed versus the untreated BN kidneys (*p* = 0.011) and the Inflammatory Infiltration Score was statistically the same as the 0 h control group (Figure 3D).

Serum creatinine and serum BUN were also measured to assess function in the BN allograft kidneys 20 h after transplant (Figure 4).

**Figure 3.** (**A**–**C**) Kidney histology 20 h following transplant. Operator randomly collected sample images of renal tubules from each of the indicated kidney groups in panels (**A**–**C**). Then, using an ImageJ macro (see Methods), H/E images (e.g., Figure 3 panels (**A**–**C**)) were analyzed, and abovethreshold pink or blue pixels were selected and enumerated, and their ratio was then calculated and plotted to provide an estimate of kidney white blood cell infiltration (Panel (**D**)). In Panel (**D**), a,b,c designations are used to enable statistical comparisons between groups.

**Figure 4.** Effects of PrC-210 administration on kidney function 20 h after transplant of the BN kidney into a LEW rat. Rats either received no PrC-210 ("20 h") or three IP PrC-210 injections in the 20 h period following transplant ("20 h + PrC-210"), before recipient rat was euthanized and serum was collected. Serum BUN and creatinine levels were determined as described in Methods. *p* values are indicated. a,b,c designations are used to enable statistical comparisons between groups.

Administration of PrC-210 conferred significant reductions in both the creatine (*p* = 0.032) and BUN (*p* = 0.046) kidney damage markers.

#### *3.2. Activated Caspase Levels in Post-Transplant Kidneys*

Levels of activated caspase in BN kidney homogenates were significantly reduced in BN allograft kidneys that were not exposed to PrC-210 treatment during the 20 h following transplant (Figure 5). Perfusion of BN kidneys with PrC-210-containing UW Solution and transplant into Lewis rats that received systemic PrC-210 doses resulted in the same activated caspase activity to that seen in the "0 h" control kidneys.

**Figure 5.** Caspase activity in post-transplant kidneys. Kidney supernatant activated caspase activity was measured enzymatically as described in Materials and Methods in a 60 min reaction. a,b,c designations are used to enable statistical comparisons between groups.

#### *3.3. Inflammatory Cytokine Levels Following BN Kidney Allograft*

In screening experiments, kidney homogenate supernates from 0 h controls and 20 h No Treatment kidneys were screened with the Proteome Profiler 29 cytokine array to detect altered, inflammation-associated, cytokine and chemokine expression levels 20 h posttransplant. As shown in the two Figure 6 microarray insets, changes were seen in TIMP-1, TNF-alpha and MIP-3a/CCL20. Individual ELISA plates were then used to quantify these changes in kidney homogenates and sera, now including rats treated with PrC-210 as well. Both TIMP-1 and TNF-alpha levels were increased 20 h post-transplant, and in both cases, their levels were decreased in the presence of PrC-210 (Figure 6A–C). MIP-3a/CCL20 levels were increased at 20 h, but they increased significantly higher in PrC-210-treated rats (Figure 6D).
