APOL1 risk variants affect podocyte lipid homeostasis and energy production in focal segmental glomerulosclerosis.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Lycium Barbarum Exerts Protection against Glaucoma-Like Injury Via Inhibition of MMP-9 Signaling In Vitro.

BACKGROUND The phytochemical ingredients of berries have been used in the treatment of various bodily ailments; while their roles in preventing the severity of glaucoma are poorly understood. Hence, the present study was framed to investigate whether ethanolic extracts of Lycium barbarum exerts protection against the onset of glaucoma using cultured PC12 neuronal cells by modulating the expression of extracellular matrix proteins. MATERIAL AND METHODS In order to develop glaucoma like condition in cells, cultured PC12 cells were subjected to 50 and 100 mmHg hydrostatic pressure for 24 hours. The pressure exposed cells were analyzed for the expression of glaucoma markers such as ANGPTL7 and the expressions of extracellular matrix proteins in the presence and absence of L. barbarum, matrix metalloproteinase (MMP)-9 inhibitor, and latanoprost, a current drug for the treatment of glaucoma. RESULTS PC12 cells exposed to hydrostatic pressures (50 and 100 mmHg) increased the expression of glaucoma marker, ANGPTL7. Moreover, results have demonstrated the significant changes in the expression of MMP-2, MMP-9, collagen I, and TGF-ß at the gene level. In contrast, cells pretreated with L. barbarum extracts showed reduced expression of ANGPTL7 and extracellular matrix proteins compared to control. Furthermore, to elucidate the role of MMP-9 in the onset of glaucoma, cells were silenced using MMP-9 inhibitor along with L. barbarum demonstrated a significant reduction in the glaucoma marker ANGPTL7 while improving the expression of caveolin-1 expression in cells subjected to pressure. CONCLUSIONS The extract of L. barbarum protects the cells from intraocular pressure by activating caveolin-1 dependent pathway via inhibition of MMP-9 expression.

Cardiolipin-targeted peptides rejuvenate mitochondrial function, remodel mitochondria, and promote tissue regeneration during aging.

It has been proposed that a loss of bioenergetic capacity of cells contributes to the progressive loss of biological function with age. Aging is associated with loss of mitochondrial cristae membranes and inhibition of ATP production. Despite the many approaches being pursued for improving mitochondrial function, none of them directly targets the electron transport chain to improve ATP production. Recent studies have brought attention to cardiolipin as a unique target for promoting mitochondrial efficiency. Cardiolipin is important for cristae curvatures and is necessary for optimal activity of the respiratory complexes and the assembly of supercomplexes. Here we describe the discovery of a class of cell-penetrating aromatic-cationic tetrapeptides that selectively target cardiolipin and increase coupling efficiency while reducing reactive oxygen species production. These compounds can rejuvenate mitochondrial bioenergetics, remodel mitochondrial cristae structure, repair cellular structure, and restore organ function during aging.

Mitochondria Protection after Acute Ischemia Prevents Prolonged Upregulation of IL-1ß and IL-18 and Arrests CKD.

The innate immune system has been implicated in both AKI and CKD. Damaged mitochondria release danger molecules, such as reactive oxygen species, DNA, and cardiolipin, which can cause NLRP3 inflammasome activation and upregulation of IL-18 and IL-1ß It is not known if mitochondrial damage persists long after ischemia to sustain chronic inflammasome activation. We conducted a 9-month study in Sprague-Dawley rats after 45 minutes of bilateral renal ischemia. We detected glomerular and peritubular capillary rarefaction, macrophage infiltration, and fibrosis at 1 month. Transmission electron microscopy revealed mitochondrial degeneration, mitophagy, and deformed foot processes in podocytes. These changes progressed over the study period, with a persistent increase in renal cortical expression of IL-18, IL-1ß, and TGF-ß, despite a gradual decline in TNF-α expression and macrophage infiltration. Treatment with a mitoprotective agent (SS-31; elamipretide) for 6 weeks, starting 1 month after ischemia, preserved mitochondrial integrity, ameliorated expression levels of all inflammatory markers, restored glomerular capillaries and podocyte structure, and arrested glomerulosclerosis and interstitial fibrosis. Further, helium ion microscopy vividly demonstrated the restoration of podocyte structure by SS-31. The protection by SS-31 was sustained for ≥6 months after treatment ended, with normalization of IL-18 and IL-1ß expression. These results support a role for mitochondrial damage in inflammasome activation and CKD and suggest mitochondrial protection as a novel therapeutic approach that can arrest the progression of CKD. Notably, SS-31 is effective when given long after AKI and provides persistent protection after termination of drug treatment.

Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia.

BACKGROUND: It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe(2+), which inhibits electron transport (ET) and promotes oxygenase activity. METHODS: SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met(80)-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. RESULTS: Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met(80)-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. CONCLUSION: Our results show that ischemia disrupts the Met(80)-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe(2+) that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. GENERAL SIGNIFICANCE: Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met(80)-Fe ligation, minimizes ischemic injury and promotes ATP recovery.

Protection of mitochondria prevents high-fat diet-induced glomerulopathy and proximal tubular injury.

Obesity is a major risk factor for the development of chronic kidney disease, even independent of its association with hypertension, diabetes, and dyslipidemia. The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis. Proposed mechanisms leading to renal pathology include abnormal lipid metabolism, lipotoxicity, inhibition of AMP kinase, and endoplasmic reticulum stress. Here we report dramatic changes in mitochondrial structure in glomerular endothelial cells, podocytes, and proximal tubular epithelial cells after 28 weeks of a high-fat diet in C57BL/6 mice. Treatment with SS-31, a tetrapeptide that targets cardiolipin and protects mitochondrial cristae structure, during high-fat diet preserved normal mitochondrial structure in all kidney cells, restored renal AMP kinase activity, and prevented intracellular lipid accumulation, endoplasmic reticulum stress, and apoptosis. SS-31 had no effect on weight gain, insulin resistance or hyperglycemia. However, SS-31 prevented loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory (TNF-α, MCP-1, NF-κB) and profibrotic (TGF-ß) cytokines. Thus, mitochondria protection can overcome lipotoxicity in the kidney and represent a novel upstream target for therapeutic development.

Improving mitochondrial bioenergetics under ischemic conditions increases warm ischemia tolerance in the kidney.

Ischemia time during partial nephrectomy is strongly associated with acute and chronic renal injury. ATP depletion during warm ischemia inhibits ATP-dependent processes, resulting in cell swelling, cytoskeletal breakdown, and cell death. The duration of ischemia tolerated by the kidney depends on the amount of ATP that can be produced with residual substrates and oxygen in the tissue to sustain cell function. We previously reported that the rat can tolerate 30-min ischemia quite well but 45-min ischemia results in acute kidney injury and progressive interstitial fibrosis. Here, we report that pretreatment with SS-20 30 min before warm ischemia in the rat increased ischemia tolerance from 30 to 45 min. Histological examination of kidney tissues revealed that SS-20 reduced cytoskeletal breakdown and cell swelling after 45-min ischemia. Electron microscopy showed that SS-20 reduced mitochondrial matrix swelling and preserved cristae membranes, suggesting that SS-20 enhanced mitochondrial ATP synthesis under ischemic conditions. Studies with isolated kidney mitochondria showed dramatic reduction in state 3 respiration and respiratory control ratio after 45-min ischemia, and this was significantly improved by SS-20 treatment. These results suggest that SS-20 increases efficiency of the electron transport chain and improves coupling of oxidative phosphorylation. SS-20 treatment after ischemia also significantly reduced interstitial fibrosis. These new findings reveal that enhancing mitochondrial bioenergetics may be an important target for improving ischemia tolerance, and SS-20 may serve well for minimizing acute kidney injury and chronic kidney disease following surgical procedures such as partial nephrectomy and transplantation.

Novel cardiolipin therapeutic protects endothelial mitochondria during renal ischemia and mitigates microvascular rarefaction, inflammation, and fibrosis.

Microvascular rarefaction, or loss of microvascular density, is increasingly implicated in the progression from acute ischemic kidney injury to chronic kidney disease. Microvascular dropout results in chronic tissue hypoxia, interstitial inflammation, and fibrosis. There is currently no therapeutic intervention for microvascular rarefaction. We hypothesize that capillary dropout begins with ischemic damage to endothelial mitochondria due to cardiolipin peroxidation, resulting in loss of cristae and the failure to regenerate ATP upon reperfusion. SS-31 is a cell-permeable peptide that targets the inner mitochondrial membrane and binds selectively to cardiolipin. It was recently shown to inhibit cardiolipin peroxidation by cytochrome c peroxidase activity, and it has been shown to protect mitochondrial cristae in proximal tubular cells during ischemia, and accelerated ATP recovery upon reperfusion. We found mitochondrial swelling and loss of cristae membranes in endothelial and medullary tubular epithelial cells after 45-min ischemia in the rat. The loss of cristae membranes limited the ability of these cells to regenerate ATP upon reperfusion and led to loss of vascular integrity and to tubular cell swelling. SS-31 prevented mitochondria swelling and protected cristae membranes in both endothelial and epithelial cells. By minimizing endothelial and epithelial cell injury, SS-31 prevented "no-reflow" after ischemia and significantly reduced the loss of peritubular capillaries and cortical arterioles, interstitial inflammation, and fibrosis at 4 wk after ischemia. These results suggest that mitochondria protection represents an upstream target for pharmacological intervention in microvascular rarefaction and fibrosis.

The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin.

Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.

A Delayed Spiking Neural Membrane System for Adaptive Nearest Neighbor-Based Density Peak Clustering.

Although the density peak clustering (DPC) algorithm can effectively distribute samples and quickly identify noise points, it lacks adaptability and cannot consider the local data structure. In addition, clustering algorithms generally suffer from high time complexity. Prior research suggests that clustering algorithms grounded in P systems can mitigate time complexity concerns. Within the realm of membrane systems (P systems), spiking neural P systems (SN P systems), inspired by biological nervous systems, are third-generation neural networks that possess intricate structures and offer substantial parallelism advantages. Thus, this study first improved the DPC by introducing the maximum nearest neighbor distance and K-nearest neighbors (KNN). Moreover, a method based on delayed spiking neural P systems (DSN P systems) was proposed to improve the performance of the algorithm. Subsequently, the DSNP-ANDPC algorithm was proposed. The effectiveness of DSNP-ANDPC was evaluated through comprehensive evaluations across four synthetic datasets and 10 real-world datasets. The proposed method outperformed the other comparison methods in most cases.