Background/Objectives: Obesity-associated non-alcoholic fatty liver disease (NAFLD) drives systemic metabolic stress and accelerates chronic kidney disease, yet the mechanistic links remain unclear. Mitochondrial dysfunction has emerged as a central mediator of obesity-induced organ injury. Here, we investigated renal mitochondrial remodeling in a rat model of obesity-associated NAFLD (Ob-NAFLD) and examined the effects of metformin. Methods: Female Zucker rats (obese fa/fa and lean Fa/Fa) were fed an AIN-93G diet for eight weeks, followed by 10 weeks of metformin treatment in designated groups. Kidney tissues were analyzed using biochemical assays, immunoblotting, blue native PAGE, in-gel activity assays, and histological evaluation. Results: In Ob-NAFLD rats, renal ATP levels were elevated despite reduced electron transport chain (ETC) Complex III and increased Complex V expression, reflecting compensatory ATP synthase hyperactivity uncoupled from efficient oxidative phosphorylation. Mitochondrial dynamics were disrupted such that inhibitory phosphorylation of DRP1 was reduced, promoting fission, and total OPA1 expression was decreased with a shift in short-to-long isoform balance, indicating impaired fusion and cristae remodeling. Notably, ATPase inhibitory factor 1 (IF1), a checkpoint that limits ATP synthase overdrive, remained stably expressed, suggesting an adaptive ceiling or failed protective control under chronic metabolic stress. Metformin partially alleviated bioenergetic stress by lowering ATP and modestly restoring Complex III, yet ETC imbalance and structural remodeling persisted, revealing the limitations of metabolic modulation alone. Conclusions: These findings position entrenched mitochondrial dysregulation as a mechanistic bridge linking obesity-driven liver disease to kidney injury. Therapeutic strategies combining metabolic interventions with targeted restoration of ETC coordination, mitochondrial dynamics, and regulatory checkpoints such as IF1 may be required to fully restore renal mitochondrial health and prevent the progression of metabolic kidney disease.

Prolonged cold storage (CS) of donor kidneys results in poor outcomes after transplantation. We reported earlier that cold storage (CS) of rat kidneys for 18 h followed by transplantation (CS + Tx) reduces proteasome function, disrupts protein homeostasis, and compromises graft function. The goal of the present study was to define the contribution of specific heat shock proteins (Hsp) to CS-induced disruption of renal graft function and determine the benefit conferred by their pharmacological inhibition. We subjected kidneys isolated from donor Lewis rats to 18-h CS with or without pharmacological inhibition of heat shock protein 72 (Hsp72), a stress-inducible member of the Hsp70 family. Subsequently, the donor kidneys were transplanted into Lewis rats (CS + Tx). Hsp72 was upregulated in kidney grafts after CS + Tx, and this finding was coupled to a reciprocal loss of cognate Hsc70 and profound tubular injury. Knockdown of Hsc70 in renal cells increased Hsp72, compromised proteasome function, and increased mitochondrial oxidative stress. The addition of HS-72, a Hsp72-specific inhibitor, to the CS solution restored proteasome function and improved renal injury/function after transplantation. Our study shows that CS + Tx dysregulates heat shock proteins in the kidney, and targeting a single disrupted protein, Hsp72, can improve graft function.NEW & NOTEWORTHY Our study using a rat renal transplant model shows that cold storage (CS)-induced injury involves dysregulation of heat shock proteins in the kidney and provides proof of concept that targeting a single disrupted protein, heat shock protein 72 (Hsp72), can improve graft function.

Abstract Ischemia–reperfusion injury (IRI) is a major driver of acute kidney injury and development of chronic kidney disease. Although complement activation worsens IRI, the roles of upstream (C3) versus downstream (C5) components remain unclear. Renal IRI was surgically induced in C3 knockout (C3 − / − ) and C5 knockout (C5 − / − ) Lewis rats, and the renal function as well as histopathology were systematically assessed. Further, quantitative proteomics coupled with pathway enrichment analysis was performed to define complement‐dependent mechanisms. C3 and C5 deficiency conferred strong protection against renal IRI with improved renal function, reduced tubular necrosis, and lower expression of injury markers (KIM‐1, NGAL). Post‐IRI, C3 −/− enhanced mitochondrial, metabolic, and purine pathways while suppressing immune and extra‐cellular matrix programs. C5 −/− affected extracellular matrix remodeling and structural pathways with modest immune suppression. We demonstrate for the first time that upstream C3 −/− impacts a diverse range of renal injury and repair mechanisms compared to C5 −/− alone, although both interventions successfully reduced IRI‐mediated injury. Together, these findings highlight strategies for future complement‐based therapies targeting the upstream or downstream cascade.

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.

Abstract 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.

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.

Background. Prolonged cold storage (CS) of kidneys results in poor long-term outcomes after transplantation (Tx). We reported previously that CS of rat kidneys for 18 h before transplant impaired proteasome function, disrupted protein homeostasis, and reduced graft function. The goal of the present study was to identify the renal proteins, including phosphoproteins, that are dysregulated by this CS injury. Methods. Isolated donor Lewis rat kidneys were subject to 18 h CS and transplanted into recipient Lewis rats (CS + Tx). Autotransplantation (transplant with 0 h CS) or Sham (right nephrectomy) surgeries served as controls. The proteome of kidney homogenates was analyzed with tandem mass-tag mass spectrometry to identify CS-induced abnormalities in kidney grafts. Results. CS injury disrupted the renal proteome/phosphoproteome landscape in kidney grafts and dysregulated numerous signaling pathways. We identified 3217 phosphopeptides (with 1398 novel phosphosites) that were significantly dysregulated in a CS-specific manner. In particular, proteins and pathways such as complement system and mitogen-activated protein kinases, including p38MAPK, were upregulated, whereas antioxidant/metabolic pathways, such as glutathione, were suppressed in CS + Tx groups compared with autotransplantation and sham controls. Conclusions. This study provides deeper insight into the disruption of the renal proteome/phosphoproteome caused by CS injury and provides a novel set of pathways and molecules, including p38MAPK, that can be investigated to delineate their specific role in renal transplant outcomes, ultimately improving outcomes for patients with end-stage kidney disease.