The rhizomicrobiome is composed of microbes that live in association with plant roots. From nutrient cycling to carbon sequestration, soil microorganisms have provided a solid base for natural and agricultural ecosystems to function. The relationship between plant roots and soil microorganisms is especially relevant in food staples such as rice (Oryza sativa L.), as the various properties of these microbes can influence crop yield and plant health, thereby affecting a major portion of the food supply for an ever-growing world population. In this study, we used 16S rRNA gene-based metagenomic analysis to investigate the impact of crop rotation and soil cultivation methods (no-till or tillage) on rhizosphere bacterial diversity and composition in eight crop fields in Arkansas. Illumina MiSeq sequencing revealed 56 Phyla, with four major Phyla: Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes. Soil microbial communities in the samples studied were phylogenetically diverse but with a stable community structure. Crop rotation and tillage did not significantly affect bacterial diversity.

Cold storage (CS)-mediated inflammation, a reality of donor kidney processing and transplantation, can contribute to organ graft failure. However, the mechanisms by which this inflammation is perpetuated during and after CS remain unclear. Here, we examined the immunoregulatory roles of signal transducer and activator of transcription (STAT) family proteins, most notably STAT1 and STAT3, with our in vivo model of renal CS and transplant. Donor rat kidneys were exposed to 4 h or 18 h of CS, which was then followed by transplantation (CS + transplant). STAT total protein level and activity (phosphorylation) were evaluated via Western blot analysis and mRNA expression was tabulated using quantitative RT-PCR after organ harvest on day 1 or day 9 post-surgery. In vivo assays were further corroborated via similar analyses featuring in vitro models, specifically proximal tubular cells (human and rat) as well as macrophage cells (Raw 264.7). Strikingly, gene expression of IFN-γ (a pro-inflammatory cytokine inducer of STAT) and STAT1 were markedly increased after CS + transplant. STAT3 dephosphorylation was additionally observed after CS, a result suggestive of dysregulation of anti-inflammatory signaling as phosphorylated STAT3 acts as a transcription factor in the nucleus to increase the expression of anti-inflammatory signaling molecules. In vitro, IFN-γ gene expression as well as amplification of downstream STAT1 and inducible nitric oxide synthase (iNOS; a hallmark of ischemia reperfusion injury) was remarkably increased after CS + rewarming. Collectively, these results demonstrate that aberrant induction of STAT1 is sustained in vivo post-CS exposure and post-transplant. Thus, Jak/STAT signaling may be a viable therapeutic target during CS to mitigate poor graft outcomes when transplanting kidneys from deceased donors.

Ischemia‐reperfusion injury (IRI) is the central contributing factor to acute kidney injury (AKI). Kidney tissue that becomes necrotic during this process releases a variety of pro‐inflammatory factors, driving activation of the complement cascade. Complement 5 (C5), in particular, has become an important therapeutic target, yet pharmacologic targeting does not achieve complete inhibition nor target all variants of this abundant protein. Here, we have generated and characterized a novel rat model of CRISPR/Cas9‐mediated global C5 deletion (C5 ^−/− ). C5 ^−/− rats displayed no differences in growth, blood chemistry, or kidney morphology/function from wild‐type (C5 ^+/+ ) counterparts at baseline. Subsequently, we compared C5 ^−/− rats to C5 ^+/+ littermates in a renal IRI model to assess differences in the post‐injury response. Compared to C5 ^+/+ , C5 ^−/− rats displayed significantly improved kidney injury/function as well as the attenuation of the apoptotic pathway post‐IRI. The circulating immune cell composition was affected by C5 ^−/− post‐injury, with significantly increased NK cells, B cells, and CD8+ T‐cells compared to C5 ^+/+ , indicating altered inflammatory signaling. Similarly, renal sections showed a reduced level of immune cell infiltration, including macrophages and neutrophils. Collectively, these results demonstrate the generation of an effective rodent model of global C5 deletion and the role of C5 as an injury‐promoting molecule during kidney IRI.

Ischemia‐reperfusion injury (IRI) is a prevalent condition that predominantly afflicts hospitalized patients, inducing acute kidney injury (AKI). In recent years, complement 5 (C5) and its anaphylatoxin receptor C5aR1 have been implicated in driving kidney IRI and loss of function. Beyond this, prior studies suggest C5‐C5aR1 mediates mitochondrial ROS production, although its role in the mitochondria has never been fully characterized. Here, we leverage a previously generated model of C5 gene deletion (male C5 ^−/− rats) and the clinically relevant C5aR1 inhibitor Avacopan (AV) to investigate C5‐C5aR1 signaling in renal mitochondrial physiology and pathophysiology. For the first time, we report that C5‐C5aR1 axis inhibition modifies physiological mitochondrial protein levels, respiratory activity, and complexes/supercomplexes. We identified a novel relationship between the C5‐C5aR1 axis and ATPase Inhibitory Factor 1 (IF1), a potent regulator of the ATP synthase, using in vivo and in vitro approaches. Post‐IRI, C5‐C5aR1 axis inhibition improved kidney function/morphology and preserved ATP levels, despite IRI‐mediated disintegration of mitochondrial complexes and supercomplexes. We show in vitro that C5‐C5aR1 axis inhibition facilitated IF1‐dependent ATP recovery via the glycolysis pathway. Collectively, our results demonstrate a complex interplay between C5‐C5aR1 and IF1 in renal mitochondria, which contributes to mitochondrial pathophysiology during IRI.

Acute kidney injury, a broad term associated with diverse etiologies, is a common pathological condition that develops into chronic disease via mechanisms that have yet to be fully understood. Key processes that promote chronic disease transition include mitochondrial dysfunction and aberrant complement system activation, specifically inducing inflammation and accumulation of pro-fibrotic changes. Although emerging evidence strongly indicates that these two processes are closely intertwined, identification of appropriate therapeutic targets remains limited. Among complement proteins, terminal portions of the cascade, including complement 5 (C5), exert particularly robust effects on mitochondrial function across tissues, including the kidney. Moreover, C5 is the most terminal portion of the cascade to produce a highly pro-inflammatory anaphylatoxin, positioning C5 as an ideal clinical target during kidney injury/disease. In this review, we will hence summarize current knowledge regarding mitochondrial contributions to kidney pathophysiology through the lens of the close relationship between mitochondria and the complement system, particularly C5.

Background: Hypothermic machine perfusion (HMP) has been associated with reduced delayed graft function compared with static cold storage (SCS). However, the molecular mechanisms underlying these differences during cold preservation remain incompletely understood. This study compared cold-storage-related biochemical and histological changes in kidneys preserved by HMP versus SCS using a Lewis rat model prior to transplantation. Methods: Following isolation, rat kidneys were flushed with cold saline (4 °C). Left kidneys were preserved by HMP at constant flow using Belzer’s machine perfusion solution (MPS) at 4 °C, while right kidneys were stored using SCS in University of Wisconsin solution at 4 °C. After four hours of preservation, kidneys were processed for biochemical and histological analysis. Fresh biopsies were evaluated for mitochondrial complex respiration. Western blotting was performed to assess expression of NDUFS3, a complex I subunit. Histological staining for nitrotyrosine and kidney injury markers was compared across groups. Results: Mitochondrial complex respiration did not differ significantly between the SCS and HMP groups. Western blot analysis demonstrated significantly increased NDUFS3 expression in HMP-preserved kidneys compared with SCS and control kidneys. Histological evaluation revealed elevated tubular staining of nitrotyrosine and kidney injury markers in SCS kidneys relative to controls, whereas HMP preservation markedly attenuated these increases. Conclusions: HMP mitigates cold-storage-induced oxidative stress and reduces expression of kidney injury markers after four hours of preservation. These molecular findings suggest a protective effect of HMP during cold preservation. Future studies with longer preservation times and transplantation models are needed to determine whether these improvements translate into enhanced post-transplant kidney function.