Rapamycin Alternatively Modifies Mitochondrial Dynamics in Dendritic Cells to Reduce Kidney Ischemic Reperfusion Injury.

Dendritic cells (DCs) are unique immune cells that can link innate and adaptive immune responses and Immunometabolism greatly impacts their phenotype. Rapamycin is a macrolide compound that has immunosuppressant functions and is used to prevent graft loss in kidney transplantation. The current study evaluated the therapeutic potential of ex-vivo rapamycin treated DCs to protect kidneys in a mouse model of acute kidney injury (AKI). For the rapamycin single (S) treatment (Rapa-S-DC), Veh-DCs were treated with rapamycin (10 ng/mL) for 1 h before LPS. In contrast, rapamycin multiple (M) treatment (Rapa-M-DC) were exposed to 3 treatments over 7 days. Only multiple ex-vivo rapamycin treatments of DCs induced a persistent reprogramming of mitochondrial metabolism. These DCs had 18-fold more mitochondria, had almost 4-fold higher oxygen consumption rates, and produced more ATP compared to Veh-DCs (Veh treated control DCs). Pathway analysis showed IL10 signaling as a major contributing pathway to the altered immunophenotype after Rapamycin treatment compared to vehicle with significantly lower cytokines Tnfa, Il1b, and Il6, while regulators of mitochondrial content Pgc1a, Tfam, and Ho1 remained elevated. Critically, adoptive transfer of rapamycin-treated DCs to WT recipients 24 h before bilateral kidney ischemia significantly protected the kidneys from injury with a significant 3-fold improvement in kidney function. Last, the infusion of DCs containing higher mitochondria numbers (treated ex-vivo with healthy isolated mitochondria (10 µg/mL) one day before) also partially protected the kidneys from IRI. These studies demonstrate that pre-emptive infusion of ex-vivo reprogrammed DCs that have higher mitochondria content has therapeutic capacity to induce an anti-inflammatory regulatory phenotype to protect kidneys from injury.

An optimized protocol for single nuclei isolation from clinical biopsies for RNA-seq.

Single nuclei RNA sequencing (snRNA-seq) has evolved as a powerful tool to study complex human diseases. Single cell resolution enables the study of novel cell types, biological processes, cell trajectories, and cell-cell signaling pathways. snRNA-seq largely relies on the dissociation of intact nuclei from human tissues. However, the study of complex tissues using small core biopsies presents many technical challenges. Here, an optimized protocol for single nuclei isolation is presented for frozen and RNAlater preserved human kidney biopsies. The described protocol is fast, low cost, and time effective due to the elimination of cell sorting and ultra-centrifugation. Samples can be processed in 90 min or less. This method is effective for obtaining normal nuclei morphology without signs of structural damage. Using snRNA-seq, 16 distinct kidney cell clusters were recovered from normal and peri-transplant acute kidney injury allograft samples, including immune cell clusters. Quality control measurements demonstrated that these optimizations eliminated cellular debris and allowed for a high yield of high-quality nuclei and RNA for library preparation and sequencing. Cellular disassociation did not induce cellular stress responses, which recapitulated transcriptional patterns associated with standardized methods of nuclei isolation. Future applications of this protocol will allow for thorough investigations of small biobank biopsies, identifying cell-specific injury pathways and driving the discovery of novel diagnostics and therapeutic targets.

The cellular landscape of the normal kidney allograft: Main players balancing the alloimmune response.

Despite recent advances made in short-term outcomes; minimal improvements have been observed in long-term kidney transplantation outcomes. Due to an imbalance between organ transplant availability and patient waiting list, expanding kidney allograft longevity is a critical need in the field. Prior studies have either focused on early ischemic and immunological conditions affecting kidney allografts (e.g., delayed graft function, acute rejection) or late stage chronic injury when interventions are no longer feasible. However, studies characterizing kidney allografts with normal function by its cellular distribution, cell-cell interactions, and associated molecular pathways are lacking. Herein, we used single nuclei RNA-sequencing to uncover the cellular landscape and transcriptome of the normal kidney allograft. We profiled 40,950 nuclei from seven human kidney biopsies (normal native, N = 3; normal allograft, N = 4); normal allograft protocol biopsies were collected ≥15-months post-transplant. A total of 17 distinct cell clusters were identified with proximal tubules (25.70 and 21.01%), distal tubules (15.22 and 18.20%), and endothelial cells (EC) (4.26 and 9.94%) constituting the major cell populations of normal native and normal allograft kidneys, respectively. A large proportion of cycling cells from normal native kidneys were in G1-phase (43.96%) whereas cells from normal allograft were predominantly in S-phase (32.69%). This result suggests that transcriptional differences between normal native and normal allograft biopsies are dependent on the new host environment, immunosuppression, and injury-affliction. In the normal allograft, EC-specific genes upregulated metabolism, the immune response, and cellular growth, emphasizing their role in maintaining homeostasis during the ongoing alloreactive stress response. Immune cells, including B (2.81%), macrophages (24.96%), monocytes (15.29%), natural killer (NK) (12.83%), neutrophils (8.44%), and T cells (14.41%, were increased in normal allografts despite lack of histological or clinical evidence of acute rejection. Phenotypic characterization of immune cell markers supported lymphocyte activation and proinflammatory cytokines signaling pathways (i.e., IL-15, IL-32). The activation of B, NK, and T cells reveals potential immune cells underlying subclinical inflammation and repair. These single nuclei analyses provide novel insights into kidney and immune cell associated signaling pathways that portray kidney grafts with normal allograft function beyond 2-years post-transplant, revealing a novel perspective in understanding long-term allograft graft survival.

The gut mycobiome of healthy mice is shaped by the environment and correlates with metabolic outcomes in response to diet.

As an active interface between the host and their diet, the gut microbiota influences host metabolic adaptation; however, the contributions of fungi have been overlooked. Here, we investigate whether variations in gut mycobiome abundance and composition correlate with key features of host metabolism. We obtained animals from four commercial sources in parallel to test if differing starting mycobiomes can shape host adaptation in response to processed diets. We show that the gut mycobiome of healthy mice is shaped by the environment, including diet, and significantly correlates with metabolic outcomes. We demonstrate that exposure to processed diet leads to persistent differences in fungal communities that significantly associate with differential deposition of body mass in male mice compared to mice fed standardized diet. Fat deposition in the liver, transcriptional adaptation of metabolically active tissues and serum metabolic biomarker levels are linked with alterations in fungal community diversity and composition. Specifically, variation in fungi from the genera Thermomyces and Saccharomyces most strongly associate with metabolic disturbance and weight gain. These data suggest that host-microbe metabolic interactions may be influenced by variability in the mycobiome. This work highlights the potential significance of the gut mycobiome in health and has implications for human and experimental metabolic studies.

FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury.

Dendritic cells (DCs) are central in regulating immune responses of kidney ischemia-reperfusion injury (IRI), and strategies to alter DC function may provide new therapeutic opportunities. Sphingosine 1-phosphate (S1P) modulates immunity through binding to its receptors (S1P1-5), and protection from kidney IRI occurs in mice treated with S1PR agonist, FTY720 (FTY). We tested if ex vivo propagation of DCs with FTY could be used as cellular therapy to limit the off-target effects associated with systemic FTY administration in kidney IRI. DCs have the ability of regulate innate and adaptive responses and we posited that treatment of DC with FTY may underlie improvements in kidney IRI. Herein, it was observed that treatment of bone marrow derived dendritic cells (BMDCs) with FTY induced mitochondrial biogenesis, FTY-treated BMDCs (FTY-DCs) showed significantly higher oxygen consumption rate and ATP production compared to vehicle treated BMDCs (Veh-DCs). Adoptive transfer of FTY-DCs to mice 24 h before or 4 h after IRI significantly protected the kidneys from injury compared to mice treated with Veh-DCs. Additionally, allogeneic adoptive transfer of C57BL/6J FTY-DCs into BALB/c mice equally protected the kidneys from IRI. FTY-DCs propagated from S1pr1-deficient DCs derived from CD11cCreS1pr1fl/fl mice as well as blunting mitochondrial oxidation in wildtype (WT) FTY-DCs prior to transfer abrogated the protection observed by FTY-DCs. We queried if DC mitochondrial content alters kidney responses after IRI, a novel but little studied phenomenon shown to be integral to regulation of the immune response. Transfer of mitochondria rich FTY-DCs protects kidneys from IRI as transferred FTY-DCs donated their mitochondria to recipient splenocytes (i.e., macrophages) and prior splenectomy abrogated this protection. Adoptive transfer of FTY-DCs either prior to or after ischemic injury protects kidneys from IRI demonstrating a potent role for donor DC-mitochondria in FTY's efficacy. This is the first evidence, to our knowledge, that DCs have the potential to protect against kidney injury by donating mitochondria to splenic macrophages to alter their bioenergetics thus making them anti-inflammatory. In conclusion, the results support that ex vivo FTY720-induction of the regulatory DC phenotype could have therapeutic relevance that can be preventively infused to reduce acute kidney injury.