Nephropathy Is Aggravated by Fatty Acids in Diabetic Kidney Disease through Tubular Epithelial Cell Necroptosis and Is Alleviated by an RIPK-1 Inhibitor.

Introduction: Diabetic kidney disease (DKD), one of the leading causes of end-stage renal disease, has complex pathogenic mechanisms and few effective clinical therapies. DKD progression is accompanied by the loss of renal resident cells, followed by chronic inflammation and extracellular matrix deposition. Necroptosis is a newly discovered form of regulated cell death and is a major form of intrinsic cell loss in certain diabetic complications such as cardiomyopathy, intestinal disease, and retinal neuropathy; however, its significance in DKD is largely unknown. Methods: In this study, the expression of necroptosis marker phosphorylated MLKL (p-MLKL) in renal biopsy tissues of patients with DKD was detected using immunofluorescence and semiquantified using immunohistochemistry. The effects of different disease-causing factors on necroptosis activation in human HK-2 cells were evaluated using immunofluorescence and Western blotting. db/db diabetic mice were fed a high-fat diet to establish an animal model of DKD with significant renal tubule damage. Mice were treated with the RIPK1 inhibitor RIPA-56 to evaluate its renal protective effects. mRNA transcriptome sequencing was used to explore the changes in signaling pathways after RIPA-56 treatment. Oil red O staining and electron macroscopy were used to observe lipid droplet accumulation in renal biopsy tissues and mouse kidney tissues. Results: Immunostaining of phosphorylated RIPK1/RIPK3/MLKL verified the occurrence of necroptosis in renal tubular epithelial cells of patients with DKD. The level of the necroptosis marker p-MLKL correlated positively with the severity of renal functional, pathological damages, and lipid droplet accumulation in patients with DKD. High glucose and fatty acids were the main factors causing necroptosis in human renal tubular HK-2 cells. Renal function deterioration and renal pathological injury were accelerated, and the necroptosis pathway was activated in db/db mice fed a high-fat diet. Application of RIPA-56 effectively reduced the degree of renal injury, inhibited the necroptosis pathway activation, and reduced necroinflammation and lipid droplet accumulation in the renal tissues of db/db mice fed a high-fat diet. Conclusion: The present study revealed the role of necroptosis in the progression of DKD and might provide a new therapeutic target for the treatment of DKD.

Generation of functionally competent hepatic stellate cells from human stem cells to model liver fibrosis in vitro.

The detailed understanding of fibrogenesis has been hampered by a lack of important functional quiescence characteristics and an in vitro model to recapitulate hepatic stellate cell (HSC) activation. In our study, we establish robust endoderm- and mesoderm-sourced quiescent-like induced HSCs (iHSCs) derived from human pluripotent stem cells. Notably, iHSCs present features of mature HSCs, including accumulation of vitamin A in the lipid droplets and maintained quiescent features. In addition, iHSCs display a fibrogenic response and secrete collagen I in response to hepatoxicity caused by thioacetamide, acetaminophen, and hepatitis B and C virus infection. Antiviral therapy attenuated virally induced iHSC activation. Interestingly, endoderm- and mesoderm-derived iHSCs showed similar iHSC phenotypes. Therefore, we provide a novel and robust method to efficiently generate functional iHSCs from hESC and iPSC differentiation, which could be used as a model for hepatocyte toxicity prediction, anti-liver-fibrosis drug screening, and viral hepatitis-induced liver fibrosis.

TRIM56 impairs HBV infection and replication by inhibiting HBV core promoter activity.

Members of the tripartite motif (TRIM) protein family strongly induced by interferons (IFNs) are parts of the innate immune system with antiviral activity. However, it is still unclear which TRIMs could play important roles in hepatitis B virus (HBV) inhibition. Here, we identified that TRIM56 expression responded in IFN-treated HepG2-NTCP cells and HBV-infected liver tissues, which was a potent IFN-inducible inhibitor of HBV replication. Mechanistically, TRIM56 suppressed HBV replication via its Ring and C-terminal domain. C-terminal domain was essential for TRIM56 translocating from cytoplasm to nucleus during HBV infection. Further analysis revealed that TRIM56's Ring domain targeted IκBα for ubiquitination. This modification induced phosphorylation of p65, which subsequently inhibited HBV core promoter activity, resulting in the inhibition of HBV replication. The p65 was found to be necessary for NF-κB signal pathway to inhibit HBV replication. We verified our findings using HepG2-NTCP and primary human hepatocytes. Our findings reveal that TRIM56 is a critical antiviral immune effector and exerts an anti-HBV activity via NF-κB signal pathway, which is essential for inhibiting transcription of HBV covalently closed circular DNA.

Human Amnion Epithelial Cells and Their Derived Exosomes Alleviate Sepsis-Associated Acute Kidney Injury via Mitigating Endothelial Dysfunction.

Background: Sepsis is characterized by organ dysfunction resulting from a patient's dysregulated response to infection. Sepsis-associated acute kidney injury (S-AKI) is the most frequent complication contributing to the morbidity and mortality of sepsis. The prevention and treatment of S-AKI remains a significant challenge worldwide. In the recent years, human amnion epithelial cells (hAECs) have drawn much attention in regenerative medicine, yet the therapeutic efficiency of hAECs in S-AKI has not been evaluated. Methods: Septic mice were induced by cecal ligation and puncture (CLP) operation. hAECs and their derived exosomes (EXOs) were injected into the mice via tail vein right after CLP surgery. The 7-day survival rate was observed. Serum creatinine level was measured and H&E staining of tissue sections were performed 16 h after CLP. Transmission electron microscopy was used to examine the renal endothelial integrity in CLP mice. Human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) and EXOs. Zonula occludens-1 (ZO-1) localization was observed by immunofluorescence staining. Expression of phosphor-p65 (p-p65), p65, vascular cell adhesion molecule-1 (VCAM-1), and ZO-1 in the kidney were determined by Western blot. Results: hAECs decreased the mortality of CLP mice, ameliorated septic injury in the kidney, and improved kidney function. More precisely, hAECs suppressed systemic inflammation and maintained the renal endothelial integrity in septic animals. EXOs from hAECs exhibited similar renal protective effects as their parental cells. EXOs maintained endothelial cell adhesion junction in vitro and inhibited endothelial cell hyperactivation in vivo. Mechanistically, EXOs suppressed proinflammatory nuclear factor kappa B (NF-κB) pathway activation in LPS-treated HUVECs and in CLP mice kidneys. Conclusion: Our results indicate that hAECs and their derived EXOs may ameliorate S-AKI via the prevention of endothelial dysfunction in the early stage of sepsis in mice. Stem cell or exosome-based therapy targeting endothelial disorders may be a promising alternative for treatment of S-AKI.

Single Cell RNA Sequencing Identifies a Unique Inflammatory Macrophage Subset as a Druggable Target for Alleviating Acute Kidney Injury.

Acute kidney injury (AKI) is a complex clinical disorder associated with poor outcomes. Targeted regulation of the degree of inflammation has been a potential strategy for AKI management. Macrophages are the main effector cells of kidney inflammation. However, macrophage heterogeneity in ischemia reperfusion injury induced AKI (IRI-AKI) remains unclear. Using single-cell RNA sequencing of the mononuclear phagocytic system in the murine IRI model, the authors demonstrate the complementary roles of kidney resident macrophages (KRMs) and monocyte-derived infiltrated macrophages (IMs) in modulating tissue inflammation and promoting tissue repair. A unique population of S100a9hi Ly6chi IMs is identified as an early responder to AKI, mediating the initiation and amplification of kidney inflammation. Kidney infiltration of S100A8/A9+ macrophages and the relevance of renal S100A8/A9 to tissue injury is confirmed in human AKI. Targeting the S100a8/a9 signaling with small-molecule inhibitors exhibits renal protective effects represented by improved renal function and reduced mortality in bilateral IRI model, and decreased inflammatory response, ameliorated kidney injury, and improved long-term outcome with decreased renal fibrosis in the unilateral IRI model. The findings support S100A8/A9 blockade as a feasible and clinically relevant therapy potentially waiting for translation in human AKI.

Human Amniotic Epithelial Cells and Their Derived Exosomes Protect Against Cisplatin-Induced Acute Kidney Injury Without Compromising Its Antitumor Activity in Mice.

Background: Cisplatin is a widely used chemotherapeutic drug, whereas the clinical application is greatly limited by its nephrotoxic side effect. Currently, there has been no effective treatment to prevent cisplatin-induced acute kidney injury (cisplatin-AKI). Human amniotic epithelial cells (hAECs) and their derived exosomes (EXOs) have been proven to effectively protect against ischemia reperfusion-induced AKI, yet their roles in cisplatin-AKI are still unknown. Methods: C57BL/6J mice were given two doses of cisplatin at 20 or 15 mg/kg of body weight to induce AKI with or without mortality. hAECs or EXOs were injected via tail vein 1 day after cisplatin administration. Serum and kidney tissues were collected on the fourth day after 15 mg/kg cisplatin treatment to explore the nephro-protective effects of hAECs and EXOs on cisplatin-AKI. Lung cancer xenograft model was built by subcutaneous injection of A549 cells into BALB/c nude mice to evaluate the effect of hAECs or EXOs on cisplatin chemotherapy. Results: Cisplatin nephrotoxicity was significantly attenuated by hAECs and EXOs as evidenced by reduced mortality rate and decreased serum creatinine (sCr) and reduced tubular injury score. hAECs or EXOs exerted the nephro-protective effects via suppression of TNF-α/MAPK and caspase signaling pathways. In the A549 lung cancer xenograft mouse model, administration of hAECs or EXOs did not promote tumor growth or compromise the therapeutic effects of cisplatin on tumors. Conclusion: This study is the first to demonstrate that hAECs and their derived exosomes have nephro-protective effects in cisplatin-AKI in vivo. Importantly, neither hAECs nor EXOs compromise the antitumor activity of cisplatin. These results potentially support the use of hAECs and their derived EXOs as nephro-protectors against cisplatin-induced nephrotoxicity clinically.

A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts.

Terminally differentiated cells can be generated by lineage reprogramming, which is, however, hindered by incomplete conversion with residual initial cell identity and partial functionality. Here, we demonstrate a new reprogramming strategy by mimicking the natural regeneration route, which permits generating expandable hepatic progenitor cells and functionally competent human hepatocytes. Fibroblasts were first induced into human hepatic progenitor-like cells (hHPLCs), which could robustly expand in vitro and efficiently engraft in vivo. Moreover, hHPLCs could be efficiently induced into mature human hepatocytes (hiHeps) in vitro, whose molecular identity highly resembles primary human hepatocytes (PHHs). Most importantly, hiHeps could be generated in large quantity and were functionally competent to replace PHHs for drug-metabolism estimation, toxicity prediction and hepatitis B virus infection modeling. Our results highlight the advantages of the progenitor stage for successful lineage reprogramming. This strategy is promising for generating other mature human cell types by lineage reprogramming.

Long-term functional maintenance of primary human hepatocytes in vitro.

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts.

Conventional embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) derived from primates resemble mouse epiblast stem cells, raising an intriguing question regarding whether the naive pluripotent state resembling mouse embryonic stem cells (mESCs) exists in primates and how to capture it in vitro. Here we identified several specific signaling modulators that are sufficient to generate rhesus monkey fibroblast-derived iPSCs with the features of naive pluripotency in terms of growth properties, gene expression profiles, self-renewal signaling, X-reactivation, and the potential to generate cross-species chimeric embryos. Interestingly, together with recent reports of naive human pluripotent stem cells, our findings suggest several conserved signaling pathways shared with rodents and specific to primates, providing significant insights for acquiring naive pluripotency from other species. In addition, the derivation of rhesus monkey naive iPSCs also provides a valuable cell source for use in preclinical research and disease modeling.

Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.

Obtaining fully functional cell types is a major challenge for drug discovery and regenerative medicine. Currently, a fundamental solution to this key problem is still lacking. Here, we show that functional human induced hepatocytes (hiHeps) can be generated from fibroblasts by overexpressing the hepatic fate conversion factors HNF1A, HNF4A, and HNF6 along with the maturation factors ATF5, PROX1, and CEBPA. hiHeps express a spectrum of phase I and II drug-metabolizing enzymes and phase III drug transporters. Importantly, the metabolic activities of CYP3A4, CYP1A2, CYP2B6, CYP2C9, and CYP2C19 are comparable between hiHeps and freshly isolated primary human hepatocytes. Transplanted hiHeps repopulate up to 30% of the livers of Tet-uPA/Rag2(-/-)/γc(-/-) mice and secrete more than 300 µg/ml human ALBUMIN in vivo. Our data demonstrate that human hepatocytes with drug metabolic function can be generated by lineage reprogramming, thus providing a cell resource for pharmaceutical applications.