**1. Introduction**

End-stage renal failure causes greater than 1.2 million deaths annually worldwide [1]. Kidney transplantation is the preferred treatment for patients with end-stage renal disease. Over 90,000 kidney transplants are performed each year worldwide.

The transplant process, itself, induces significant cellular and organ injury to the kidney, which reduces long-term survival of the organ. The three primary insults to a kidney during an allograft transplant are (i) reactive oxygen and nitrogen species (ROS and RNS)-induced damage during cold ischemia ('cold-storage') [2], (ii) ROS-induced damage upon implant ('re-perfusion injury') [3], and (iii) post-allograft-transplant inflammation, which triggers the innate immune response and antibody-mediated rejection (ABMR) [4].

Neutrophils and macrophages migrate into the damaged transplant within 6 h of reperfusion and stimulate chemokine synthesis in resident dendritic cells that then activate

**Citation:** Goesch, T.R.; Wilson, N.A.; Zeng, W.; Verhoven, B.M.; Zhong, W.; Coumbe Gitter, M.M.; Fahl, W.E. Suppression of Inflammation-Associated Kidney Damage Post-Transplant Using the New PrC-210 Free Radical Scavenger in Rats. *Biomolecules* **2021**, *11*, 1054. https://doi.org/10.3390/ biom11071054

Academic Editor: Liang-Jun Yan

Received: 30 June 2021 Accepted: 12 July 2021 Published: 19 July 2021

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T lymphocytes and recruit adaptive immune cells. Once these immune cells infiltrate the proximal tubule epithelial cells, they produce myeloperoxidase in neutrophils and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in macrophages, both of which contribute to local free radical production. These inflammatory processes lead to an activation of the complement pathway and further cell remodeling and lysis in the kidney allograft [5].

ABMR can occur as a result of either, or both, preformed alloantibody against the graft or through the *de novo* development of donor-specific antibody (dnDSA) [5–7]. The acute (min/days), transitioning to chronic (days/weeks), inflammatory response within the allograft kidney, with continuous production of ROS and inflammatory cytokines, can establish a severe, self-perpetuating response that causes kidney organ failure.

To better understand cellular and molecular pathways involved in the pathogenesis of kidney allograft inflammation and rejection, we developed and characterized a rat model that replicates most of the clinical criteria of innate immune response, ABMR and kidney organ loss [8]. This model has been used to evaluate a number of novel post-allograft transplant strategies.

The two currently acknowledged approaches for reducing the acute and long-term immune response against the kidney allograft are: (i) to increase the chance of finding a cross-matched donor, and (ii) to remove preexisting antibodies against the kidney allograft using desensitization protocols [9,10].

In the work described in this manuscript, we asked whether a third approach to suppress acute and longer-term inflammation severity would be beneficial. We administered the immediate-acting, free radical scavenger, PrC-210, both to the implanted allograft kidney and to the recipient rat, to determine whether inflammation-associated ROS damage could be suppressed. Though the concept of suppressing inflammation-associated ROS in kidney transplant is not new, the use here of the new, immediate-acting PrC-210 ROS scavenger is. Both immediate and chronic scavenging and inactivation of inflammationgenerating, and generated, free radicals within the newly transplanted allograft kidney would significantly enhance the existing strategies to suppress allograft rejection and would provide another pathway to reduce post-transplant kidney cell damage, and with it, suppress Delayed Graft Function to improve survival of the kidney allograft.

PrC-210 is a new small-molecule, aminothiol, free radical scavenger [11]; it has no measurable nausea/emesis nor hypotension side effects [12]. Unlike traditional antioxidants that act *indirectly* over hours to days via NrF-2 to activate expression of protective genes [13], PrC-210 *directly* scavenges ROS to confer 100% protection in seconds [11]. PrC-210 was the most potent of the 13 commonly studied "antioxidants" screened in an assay that scored the ability of molecules to prevent x-ray-induced damage to naked DNA; the majority of the tested "antioxidants" showed no protection [14,15]. In a related assay, addition of PrC-210 30 s before a 60 s pulse of •OH to naked DNA provided complete protection against the •OH insult that induced >95% DNA damage in unprotected controls [16]. In two previous rodent kidney transplant studies [16,17], PrC-210 was shown to suppress ROS-induced kidney damage induced during (i) 30 h cold storage [17] and (ii) reperfusion injury upon implant [16] to *background* levels, thus removing two substantial sources of injury to the transplanted kidneys. The PrC-210 molecule has also been shown to suppress free radical-induced injury in several other organ settings [15,18]. Thus, we hypothesized that PrC-210 should also be able to protect an allograft against oxidative stress that is generated by BOTH (i) cellular- and (ii) antibody-mediated rejection processes that produce free radicals as a byproduct.

To explore this hypothesis, we developed a new rat model which avoided the induction of major ischemic and reperfusion events, and administered PrC-210 both pre- and postimplantation. Brown rat kidneys were flushed with UW solution containing PrC-210 and immediately transplanted into syngeneic Lewis rat recipients. Cold ischemic time was virtually eliminated. Immediately following implant, and for 8 h following kidney implant, recipient rats received systemic PrC-210 injections at doses that would enable continuous

free radical-scavenging within the transplanted kidney. Transplanted kidneys and blood plasma were then harvested 20 h following transplant to enable measurement of both PrC-210-conferred (i) suppression of inflammatory byproducts and (ii) kidney protection.
