**4. Discussion**

Allograft kidney transplantation, which triggers innate host cellular- and antibodymediated rejection of the kidney, is a major contributor to short and long-term kidney damage during transplant, and the associated Delayed Graft Function seen in up to 50% of transplanted kidneys. We undertook this study to determine whether PrC-210 would be effective in suppressing the severity of the damage induced following allograft kidney transplant in a rat model that largely eliminates transplant ischemic time and its associated oxidative stress. Our assumption was that this approach should allow us to see the impact of PrC-210 on the post-transplantation inflammation insult with minimal ischemia interference.

The increase in TNF-alpha and substantial mononuclear infiltration demonstrate that allograft kidney transplantation induces pronounced acute inflammation in the 20 h after transplantation, and this was correlated with the damage of the kidney tubular cells seen in the kidney histology (Banff Scores). TNF-alpha is mainly produced by activated macrophages and is a cell signaling protein involved in acute inflammation. It is closely associated with the pathogenesis of acute and chronic allograft rejection [24].

In contrast to the above findings in non-treated BN kidneys, PrC-210 given as part of the UW Solution and administered systemically in the post-transplant rats reduced both TNF-alpha level and kidney infiltration by mononuclear cells, which are both signs of reduced acute inflammation. PrC-210 reduced the kidney damage as seen in the histological kidney injury scores (Banff Scores) to untreated background levels and lowered levels of both kidney pathology functional scores, creatinine and BUN.

Inflammation in untreated BN kidneys was associated with an increase in both TIMP-1 and MIP-3A/CCL20. By comparison, we saw that PrC-210 treatment significantly reduced the TIMP-1 level and significantly increased the MIP-3A/CCL20 level.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix (ECM) synthesis and degradation. Excess ECM accumulation is the main pathological mechanism of fibrosis development during and after acute kidney injury. There is essentially no expression of TIMP-1 in normal kidney tissue [25], an observation which is corroborated in our Figure 6A,B, but TIMP-1 is known to be expressed in injured kidneys, mainly in renal tubular epithelial cells, renal tubular basement membrane and the cytoplasm of interstitial cells. Increased TIMP-1 expression was positively correlated with the simultaneous deterioration of renal function [26]. Rats treated with PrC-210 showed a profound reduction in TIMP-1 levels (*p* = 0.001), both in kidney homogenate and plasma; this implies that PrC-210 exerts a strong protective effect against transplantation-induced reorganization of the kidney extracellular matrix.

The chemokine MIP-3a/CCL20 activates the CCR6 receptor, which is expressed especially on regulatory T-cells (Tregs). CCL20 is expressed by tubular endothelial and interstitial cells and is also upregulated in kidneys with acute kidney injury. The CCL20– CCR6 pathway plays a vital role in Treg-mediated T-cell recruitment to the kidney, and Tregs have been described to have a positive role in kidney repair, transplant tolerance, and kidney survival. Both antibody blocking of the CCL20–CCR6 pathway, as well as the use of CCR6-deficient mice in acute kidney injury experiments, were shown to increase the severity of kidney failure and mortality [27]. This suggests, that clinically, CCL20–CCR6 pathway enhancement and Treg activation may be a possible therapeutic route to limit acute and chronic kidney injury [28]. In our study (Figure 6D), the MIP-3a/CCL20 level was significantly higher in PrC-210-treated rats than in untreated rats. We speculate that this is one of the reasons for both the (i) significantly lower recruitment of mononuclear cells to kidneys (Figure 3C) and (ii) the significantly reduced kidney damage (Figure 2) in the PrC-210-treated rats.

Normal kidney mitochondrial function, and importantly, insults to it during the kidney storage, implant, and post-implant inflammation steps are significant determinants of ROS injury, and kidney failure during transplant. It was thus significant that PrC-210 was shown to confer complete suppression of mitochondrial DNA fragmentation (Figure 7) at concentrations (2–4 mM) that have been achieved in the plasma of both mice and rats that were given either intraperitoneal or oral systemic 0.5 MTD doses of PrC-210 that were tolerated with no detectable toxicities [29].

In our earlier kidney transplant-related studies [16,17], we saw substantial increases in activated caspase in kidneys exposed to "cold ischemia" and "ischemia-reperfusion" injury. These ischemia-induced insults to the kidneys were reduced to background by treatment with PrC-210 (Figure 8). In the studies of this manuscript (Figure 5), in which cold ischemia and ischemia-reperfusion were essentially eliminated by immediate transplant, there was no increase in activated caspase in transplanted kidneys. Rather, activated caspase was significantly reduced at +20 h in "No Drug Treatment" controls, and PrC-210 treatment completely eliminated this caspase reduction in +20 h rats and kept the caspase level stable. Our interpretation of these interesting results is that absent any significant ischemia-induced free radical insult through ROS and RNS to the post-transplant kidneys, there is no associated cell death and apoptosis markers like activated caspases. Rather, in these allograft kidney transplants, inflammatory signals from newly expressed cytoand chemokines now regulate cell metabolism, which includes influencing the apoptosis

pathway. The literature describes that overexpression of TIMP-1 leads to suppression of apoptosis [26]. Our caspase results (Figure 5) support this described TIMP-1 effect, and they imply that TIMP-1 is important in regulating the pathophysiology of cell damage after kidney allograft transplantation. In corroboration of the earlier PrC-210 suppression of TIMP-1 expression (Figure 6A,B), PrC-210 treatment completely ablated the caspase change, keeping the caspase levels at the same level seen in the control "0 h" kidneys. Because the reduced PrC-210 serum TIMP-1 levels at +20 h (Figure 6B) accurately reflect the significant suppression of allograft: (i) apoptosis (Figure 5), (ii) histologic pathology (Figures 2 and 3) and (iii) inflammatory cell infiltration (Figure 3), we expect that monitoring serum TIMP-1 levels in human kidney allograft recipients will be a logical way to monitor PrC-210 clinical efficacy in future clinical trials.

**Figure 8.** Schematic summarizing PrC-210 efficacy in suppressing each of the three organ insults that occur during kidney transplantation.

In our work to date [16,17], we have shown that PrC-210 is able to protect transplanted kidneys against both the cold-ischemia and ischemia-reperfusion insults. In this study, we now see that PrC-210 also protects allograft kidneys from the non-ischemia inflammatory insults that occur after kidney implant. PrC-210 significantly reduces levels of acute inflammatory cytokines, such as TNF-alpha, and suppresses expression of the TIMP-1 chemokine. Both of these events, and potentially, further supported by additional CCL20 expression, would be expected to: (i) reduce allograft kidney damage, (ii) suppress T-cell recruitment to the kidney, and (iii) suppress activation of the innate and adaptive immune system. In Figure 8, we summarize these findings to support the role that we feel PrC-210 can play in human kidney transplantation; it suppresses: (i) cold ischemia reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage to background [17], (ii) ischemia-reperfusion ROS damage to background [16], and (iii) allograft inflammation damage substantially, in some cases, to background.

Since the primary PrC-210 mechanism of action for PrC-210 is scavenging oxygen and nitrogen free radicals, this implies that these free radicals are an important contributor to the kidney damage seen in non-ischemic conditions, i.e., the allograft-associated inflammation studied in this manuscript.

In summary, this suggests that PrC-210 could provide broadly applicable organ protection for many allograft transplantation conditions; it could protect transplanted kidneys during and after all stages of the transplantation process—from organ donation, through transportation, re-implantation and the post-operative inflammation—to minimize acute and chronic rejection.

**Author Contributions:** N.A.W., W.Z. (Weifeng Zeng), W.Z. (Weixiong Zhong), B.M.V. and M.M.C.G. participated in performance of the research and data analysis; T.R.G. and N.A.W. participated in research design and data analysis; W.E.F. and T.R.G. participated in research design, performance of the research, data analysis, and writing of the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the UW Institute for Clinical and Translational Research and grant UL1TR002373 to UW ICTR from NIH/NCATS, American Society of Transplant Surgeons (133 AAA1552), American College of Surgeons (133 AAB2176), and grant support to W.E.F. (#R03CA176799).

**Institutional Review Board Statement:** This research was prospectively approved by School of Medicine and Public Health Institutional Animal Care and Use Committee at the University of Wisconsin (Animal Protocol #M005204).

**Data Availability Statement:** The study data are fully available within this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

