Complement Blockade in Recipients Prevents Delayed Graft Function and Delays Antibody-mediated Rejection in a Nonhuman Primate Model of Kidney Transplantation.

BACKGROUND: Complement activation in kidney transplantation is implicated in the pathogenesis of delayed graft function (DGF). This study evaluated the therapeutic efficacy of high-dose recombinant human C1 esterase inhibitor (rhC1INH) to prevent DGF in a nonhuman primate model of kidney transplantation after brain death and prolonged cold ischemia. METHODS: Brain death donors underwent 20 h of conventional management. Procured kidneys were stored on ice for 44-48 h, then transplanted into ABO-compatible major histocompatibility complex-mismatched recipients. Recipients were treated with vehicle (n = 5) or rhC1INH 500 U/kg plus heparin 40 U/kg (n = 8) before reperfusion, 12 h, and 24 h posttransplant. Recipients were followed up for 120 d. RESULTS: Of vehicle-treated recipients, 80% (4 of 5) developed DGF versus 12.5% (1 of 8) rhC1INH-treated recipients (P = 0.015). rhC1INH-treated recipients had faster creatinine recovery, superior urinary output, and reduced urinary neutrophil gelatinase-associated lipocalin and tissue inhibitor of metalloproteinases 2-insulin-like growth factor-binding protein 7 throughout the first week, indicating reduced allograft injury. Treated recipients presented lower postreperfusion plasma interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, and IL-18, lower day 4 monocyte chemoattractant protein 1, and trended toward lower C5. Treated recipients exhibited less C3b/C5b-9 deposition on day 7 biopsies. rhC1INH-treated animals also trended toward prolonged mediated rejection-free survival. CONCLUSIONS: Our results recommend high-dose C1INH complement blockade in transplant recipients as an effective strategy to reduce kidney injury and inflammation, prevent DGF, delay antibody-mediated rejection development, and improve transplant outcomes.

Human Placental Transcriptome Reveals Critical Alterations in Inflammation and Energy Metabolism with Fetal Sex Differences in Spontaneous Preterm Birth.

A well-functioning placenta is crucial for normal gestation and regulates the nutrient, gas, and waste exchanges between the maternal and fetal circulations and is an important endocrine organ producing hormones that regulate both the maternal and fetal physiologies during pregnancy. Placental insufficiency is implicated in spontaneous preterm birth (SPTB). We proposed that deficits in the capacity of the placenta to maintain bioenergetic and metabolic stability during pregnancy may ultimately result in SPTB. To explore our hypothesis, we performed a RNA-seq study in male and female placentas from women with SPTB (<36 weeks gestation) compared to normal pregnancies (≥38 weeks gestation) to assess the alterations in the gene expression profiles. We focused exclusively on Black women (cases and controls), who are at the highest risk of SPTB. Six hundred and seventy differentially expressed genes were identified in male SPTB placentas. Among them, 313 and 357 transcripts were increased and decreased, respectively. In contrast, only 61 differentially expressed genes were identified in female SPTB placenta. The ingenuity pathway analysis showed alterations in the genes and canonical pathways critical for regulating inflammation, oxidative stress, detoxification, mitochondrial function, energy metabolism, and the extracellular matrix. Many upstream regulators and master regulators important for nutrient-sensing and metabolism were also altered in SPTB placentas, including the PI3K complex, TGFB1/SMADs, SMARCA4, TP63, CDKN2A, BRCA1, and NFAT. The transcriptome was integrated with published human placental metabolome to assess the interactions of altered genes and metabolites. Collectively, significant and biologically relevant alterations in the transcriptome were identified in SPTB placentas with fetal sex disparities. Altered energy metabolism, mitochondrial function, inflammation, and detoxification may underly the mechanisms of placental dysfunction in SPTB.

Combinatorial glucose, nicotinic acid and N-acetylcysteine therapy has synergistic effect in preclinical C. elegans and zebrafish models of mitochondrial complex I disease.

Mitochondrial respiratory chain disorders are empirically managed with variable antioxidant, cofactor and vitamin 'cocktails'. However, clinical trial validated and approved compounds, or doses, do not exist for any single or combinatorial mitochondrial disease therapy. Here, we sought to pre-clinically evaluate whether rationally designed mitochondrial medicine combinatorial regimens might synergistically improve survival, health and physiology in translational animal models of respiratory chain complex I disease. Having previously demonstrated that gas-1(fc21) complex I subunit ndufs2-/-C. elegans have short lifespan that can be significantly rescued with 17 different metabolic modifiers, signaling modifiers or antioxidants, here we evaluated 11 random combinations of these three treatment classes on gas-1(fc21) lifespan. Synergistic rescue occurred only with glucose, nicotinic acid and N-acetylcysteine (Glu + NA + NAC), yielding improved mitochondrial membrane potential that reflects integrated respiratory chain function, without exacerbating oxidative stress, and while reducing mitochondrial stress (UPRmt) and improving intermediary metabolic disruptions at the levels of the transcriptome, steady-state metabolites and intermediary metabolic flux. Equimolar Glu + NA + NAC dosing in a zebrafish vertebrate model of rotenone-based complex I inhibition synergistically rescued larval activity, brain death, lactate, ATP and glutathione levels. Overall, these data provide objective preclinical evidence in two evolutionary-divergent animal models of mitochondrial complex I disease to demonstrate that combinatorial Glu + NA + NAC therapy significantly improved animal resiliency, even in the face of stressors that cause severe metabolic deficiency, thereby preventing acute neurologic and biochemical decompensation. Clinical trials are warranted to evaluate the efficacy of this lead combinatorial therapy regimen to improve resiliency and health outcomes in human subjects with mitochondrial disease.

Pre-clinical evaluation of cysteamine bitartrate as a therapeutic agent for mitochondrial respiratory chain disease.

Cysteamine bitartrate is a US Food and Drug Administration-approved therapy for nephropathic cystinosis also postulated to enhance glutathione biosynthesis. We hypothesized this antioxidant effect may reduce oxidative stress in primary mitochondrial respiratory chain (RC) disease, improving cellular viability and organismal health. Here, we systematically evaluated the therapeutic potential of cysteamine bitartrate in RC disease models spanning three evolutionarily distinct species. These pre-clinical studies demonstrated the narrow therapeutic window of cysteamine bitartrate, with toxicity at millimolar levels directly correlating with marked induction of hydrogen peroxide production. Micromolar range cysteamine bitartrate treatment in Caenorhabditis elegans gas-1(fc21) RC complex I (NDUFS2-/-) disease invertebrate worms significantly improved mitochondrial membrane potential and oxidative stress, with corresponding modest improvement in fecundity but not lifespan. At 10 to 100 µm concentrations, cysteamine bitartrate improved multiple RC complex disease FBXL4 human fibroblast survival, and protected both complex I (rotenone) and complex IV (azide) Danio rerio vertebrate zebrafish disease models from brain death. Mechanistic profiling of cysteamine bitartrate effects showed it increases aspartate levels and flux, without increasing total glutathione levels. Transcriptional normalization of broadly dysregulated intermediary metabolic, glutathione, cell defense, DNA, and immune pathways was greater in RC disease human cells than in C. elegans, with similar rescue in both models of downregulated ribosomal and proteasomal pathway expression. Overall, these data suggest cysteamine bitartrate may hold therapeutic potential in RC disease, although not through obvious modulation of total glutathione levels. Careful consideration is required to determine safe and effective cysteamine bitartrate concentrations to further evaluate in clinical trials of human subjects with primary mitochondrial RC disease.

N-acetylcysteine and vitamin E rescue animal longevity and cellular oxidative stress in pre-clinical models of mitochondrial complex I disease.

Oxidative stress is a known contributing factor in mitochondrial respiratory chain (RC) disease pathogenesis. Yet, no efficient means exists to objectively evaluate the comparative therapeutic efficacy or toxicity of different antioxidant compounds empirically used in human RC disease. We postulated that pre-clinical comparative analysis of diverse antioxidant drugs having suggested utility in primary RC disease using animal and cellular models of RC dysfunction may improve understanding of their integrated effects and physiologic mechanisms, and enable prioritization of lead antioxidant molecules to pursue in human clinical trials. Here, lifespan effects of N-acetylcysteine (NAC), vitamin E, vitamin C, coenzyme Q10 (CoQ10), mitochondrial-targeted CoQ10 (MS010), lipoate, and orotate were evaluated as the primary outcome in a well-established, short-lived C. elegans gas-1(fc21) animal model of RC complex I disease. Healthspan effects were interrogated to assess potential reversal of their globally disrupted in vivo mitochondrial physiology, transcriptome profiles, and intermediary metabolic flux. NAC or vitamin E fully rescued, and coenzyme Q, lipoic acid, orotic acid, and vitamin C partially rescued gas-1(fc21) lifespan toward that of wild-type N2 Bristol worms. MS010 and CoQ10 largely reversed biochemical pathway expression changes in gas-1(fc21) worms. While nearly all drugs normalized the upregulated expression of the "cellular antioxidant pathway", they failed to rescue the mutant worms' increased in vivo mitochondrial oxidant burden. NAC and vitamin E therapeutic efficacy were validated in human fibroblast and/or zebrafish complex I disease models. Remarkably, rotenone-induced zebrafish brain death was preventable partially with NAC and fully with vitamin E. Overall, these pre-clinical model animal data demonstrate that several classical antioxidant drugs do yield significant benefit on viability and survival in primary mitochondrial disease, where their major therapeutic benefit appears to result from targeting global cellular, rather than intramitochondria-specific, oxidative stress. Clinical trials are needed to evaluate whether the two antioxidants, NAC and vitamin E, that show greatest efficacy in translational model animals significantly improve the survival, function, and feeling of human subjects with primary mitochondrial RC disease.

Propionyl-CoA carboxylase pcca-1 and pccb-1 gene deletions in Caenorhabditis elegans globally impair mitochondrial energy metabolism.

Propionic acidemia (PA) is a classical inborn error of metabolism with high morbidity that results from the inability of the propionyl-CoA carboxylase (PCC) enzyme to convert propionyl-CoA to methylmalonyl-CoA. PA is inherited in an autosomal recessive fashion due to functional loss of both alleles of either PCCA or PCCB. These genes are highly conserved across evolutionarily diverse species and share extensive similarity with pcca-1 and pccb-1 in the nematode, Caenorhabditis elegans. Here, we report the global metabolic effects of deletion in a single PCC gene, either pcca-1 or pccb-1, in C. elegans. Animal lifespan was significantly reduced relative to wild-type worms in both mutant strains, although to a greater degree in pcca-1. Mitochondrial oxidative phosphorylation (OXPHOS) capacity and efficiency as determined by direct polarography of isolated mitochondria were also significantly reduced in both mutant strains. While in vivo quantitation of mitochondrial physiology was normal in pccb-1 mutants, pcca-1 deletion mutants had significantly increased mitochondrial matrix oxidant burden as well as significantly decreased mitochondrial membrane potential and mitochondrial content. Whole worm steady-state free amino acid profiling by UPLC revealed reduced levels in both mutant strains of the glutathione precursor cysteine, possibly suggestive of increased oxidative stress. Intermediary metabolic flux analysis by GC/MS with 1,6-13C2-glucose further showed both PCC deletion strains had decreased accumulation of a distal tricarboxylic acid (TCA) cycle metabolic intermediate (+1 malate), isotopic enrichment in a proximal TCA cycle intermediate (+1 citrate), and increased +1 lactate accumulation. GC/MS analysis further revealed accumulation in the PCC mutants of a small amount of 3-hydroxypropionate, which appeared to be metabolized in C. elegans to oxalate through a unique metabolic pathway. Collectively, these detailed metabolic investigations in translational PA model animals with genetic-based PCC deficiency reveal their significantly dysregulated energy metabolism at multiple levels, including reduced mitochondrial OXPHOS capacity, increased oxidative stress, and inhibition of distal TCA cycle flux, culminating in reduced animal lifespan. These findings demonstrate that the pathophysiology of PA extends well beyond what has classically been understood as a single PCC enzyme deficiency with toxic precursor accumulation, and suggest that therapeutically targeting the globally disrupted energy metabolism may offer novel treatment opportunities for PA. SUMMARY: Two C. elegans model animals of propionic acidemia with single-gene pcca-1 or pccb-1 deletions have reduced lifespan with significantly reduced mitochondrial energy metabolism and increased oxidative stress, reflecting the disease's broader pathophysiology beyond a single enzyme deficiency with toxic precursor accumulation.

Leucine-rich pentatricopeptide-repeat containing protein regulates mitochondrial transcription.

Mitochondrial function depends upon the coordinated expression of the mitochondrial and nuclear genomes. Although the basal factors that carry out the process of mitochondrial transcription are known, the regulation of this process is incompletely understood. To further our understanding of mitochondrial gene regulation, we identified proteins that bound to the previously described point of termination for the major mRNA-coding transcript H2. One was the leucine-rich pentatricopeptide-repeat containing protein (LRPPRC), which has been linked to the French-Canadian variant of Leigh syndrome. Cells with reduced expression of LRPPRC had a reduction in oxygen consumption. The expression of mitochondrial mRNA and tRNA was dependent upon LRPPRC levels, but reductions in LRPPRC did not affect the expression of mitochondrial rRNA. Reduction of LRPPRC levels interfered with mitochondrial transcription in vitro but did not affect the stability of mitochondrial mRNAs or alter the expression of nuclear genes responsible for mitochondrial transcription in vivo. These findings demonstrate the control of mitochondrial mRNA synthesis by a protein that has an established role in regulating nuclear transcription and a link to mitochondrial disease.