C-X-C chemokine receptor type 4 promotes tubular cell senescence and renal fibrosis through ß-catenin-inhibited fatty acid oxidation.

The prevalence of chronic kidney disease (CKD) is highly increasing. Renal fibrosis is a common pathological feature in various CKD. Previous studies showed tubular cell senescence is highly involved in the pathogenesis of renal fibrosis. However, the inducers of tubular senescence and the underlying mechanisms have not been fully investigated. C-X-C motif chemokine receptor 4 (CXCR4), a G-protein-coupled seven-span transmembrane receptor, increases renal fibrosis and plays an important role in tubular cell injury. Whereas, whether CXCR4 could induce tubular cell senescence and the detailed mechanisms have not studied yet. In this study, we adopted adriamycin nephropathy and 5/6 nephrectomy models, and cultured tubular cell line. Overexpression or knockdown of CXCR4 was obtained by injection of related plasmids. We identified CXCR4 increased in injury tubular cells. CXCR4 was expressed predominantly in renal tubular epithelial cells and co-localized with adipose differentiation-related protein (ADRP) as well as the senescence-related protein P16INK4A . Furthermore, we found overexpression of CXCR4 greatly induced the activation of ß-catenin, while knockdown of CXCR4 inhibited it. We also found that CXCR4 inhibited fatty acid oxidation and triggered lipid deposition in tubular cells. To inhibit ß-catenin by ICG-001, an inhibitor of ß-catenin, could significantly block CXCR4-suppressed fatty acid oxidation. Taken together, our results indicate that CXCR4 is a key mediator in tubular cell senescence and renal fibrosis. CXCR4 promotes tubular cell senescence and renal fibrosis by inducing ß-catenin and inhibiting fatty acid metabolism. Our findings provide a new theory for tubular cell injury in renal fibrosis.

Serum granulosa cell-derived TNF-α promotes inflammation and apoptosis of renal tubular cells and PCOS-related kidney injury through NF-κB signaling.

Polycystic ovary syndrome (PCOS) is a disorder with endocrinal and metabolic problems in reproductive aged women. Evidence shows that PCOS is in a high prone trend to develop kidney diseases. In this study, we investigated the mediators responsible for PCOS-related kidney injury. We found that tumor necrosis factor (TNF-α) levels were significantly increased in serum and primary cultured granulosa cells (GCs) from PCOS patients. Serum TNF-α levels were positively correlated with serum testosterone and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, suggesting its positive role in the severity of PCOS. Serum TNF-α levels were also positively correlated with the levels of urinary KapU, LamU, α1-MU and ß2-MU, the markers for renal tubular cell-derived proteinuria. We established a PCOS mouse model by resection of the right kidney, followed by daily administration of dihydrotestosterone (DHT, 27.5 µg, i.p.) from D7 for 90 days. We found that TNF-α levels were significantly increased in the ovary and serum of the mice, accompanied by increased renal tubular cell apoptosis, inflammation and fibrosis in kidneys. Furthermore, the receptor of TNF-α, tumor necrosis factor receptor 1 (TNFR1), was significantly upregulated in renal tubular cells. We treated human ovarian granulosa-like tumor cells (KGN) with DHT (1 µg/ml) in vitro, the conditioned medium derived from the granulosa cell culture greatly accelerated apoptotic injury in human proximal tubular epithelial cells (HKC-8), which was blocked after knockdown of TNF-α in KGN cells. Furthermore, knockdown of TNFR1 in renal tubular epithelial cells greatly ameliorated cell injury induced by granulosa cell-derived conditioned medium. These results suggest that serum TNF-α plays a key role in mediating inflammation and apoptosis in renal tubular cells associated with PCOS-related kidney injury.

Cannabinoid receptor type 2 promotes kidney fibrosis through orchestrating ß-catenin signaling.

The endocannabinoid system has multiple effects. Through interacting with cannabinoid receptor type 1 and type 2, this system can greatly affect disease progression. Previously, we showed that activated cannabinoid receptor type 2 (CB2) mediated kidney fibrosis. However, the underlying mechanisms remain underdetermined. Here, we report that CB2 was upregulated predominantly in kidney tubular epithelial cells in unilateral urinary obstruction and ischemia-reperfusion injury models in mice, and in patients with a variety of kidney diseases. CB2 expression was closely correlated with the progression of kidney fibrosis and accompanied by the activation of ß-catenin. Furthermore, CB2 induced the formation of a ß-arrestin 1/Src/ß-catenin complex, which further triggered the nuclear translocation of ß-catenin and caused fibrotic injury. Incubation with XL-001, an inverse agonist to CB2, or knockdown of ß-arrestin 1 inhibited CB2-triggered activation of ß-catenin and fibrotic injury. Notably, CB2 potentiated Wnt1-induced ß-arrestin 1/ß-catenin activation and augmented the pathogenesis of kidney fibrosis in mice with unilateral ischemia-reperfusion injury or folic acid-induced nephropathy. Knockdown of ß-arrestin 1 inhibited the CB2 agonist AM1241-induced ß-catenin activation and kidney fibrosis. By promoter sequence analysis, putative transcription factor binding sites for T-cell factor/lymphoid enhancer factor were found in the promoter regions of the CB2 gene regardless of the species. Overexpression of ß-catenin induced the binding of T-cell factor/lymphoid enhancer factor-1 to these sites, promoted the expression of CB2, ß-arrestin 1, and the proto-oncogene Src, and triggered their accumulation. Thus, the CB2/ß-catenin pathway appears to create a reciprocal activation feedback loop that plays a central role in the pathogenesis of kidney fibrosis.

C-X-C motif chemokine receptor 4 aggravates renal fibrosis through activating JAK/STAT/GSK3ß/ß-catenin pathway.

Chronic kidney disease (CKD) has a high prevalence worldwide. Renal fibrosis is the common pathological feature in various types of CKD. However, the underlying mechanisms are not determined. Here, we adopted different CKD mouse models and cultured human proximal tubular cell line (HKC-8) to examine the expression of C-X-C motif chemokine receptor 4 (CXCR4) and ß-catenin signalling, as well as their relationship in renal fibrosis. In CKD mice and humans with a variety of nephropathies, CXCR4 was dramatically up-regulated in tubules, with a concomitant activation of ß-catenin. CXCR4 expression level was positively correlated with the expression of ß-catenin target MMP-7. AMD3100, a CXCR4 receptor blocker, and gene knockdown of CXCR4 significantly inhibited the activation of JAK/STAT and ß-catenin signalling, protected against tubular injury and renal fibrosis. CXCR4-induced renal fibrosis was inhibited by treatment with ICG-001, an inhibitor of ß-catenin signalling. In HKC-8 cells, overexpression of CXCR4 induced activation of ß-catenin and deteriorated cell injury. These effects were inhibited by ICG-001. Stromal cell-derived factor (SDF)-1α, the ligand of CXCR4, stimulated the activation of JAK2/STAT3 and JAK3/STAT6 signalling in HKC-8 cells. Overexpression of STAT3 or STAT6 decreased the abundance of GSK3ß mRNA. Silencing of STAT3 or STAT6 significantly blocked SDF-1α-induced activation of ß-catenin and fibrotic lesions. These results uncover a novel mechanistic linkage between CXCR4 and ß-catenin activation in renal fibrosis in association with JAK/STAT/GSK3ß pathway. Our studies also suggest that targeted inhibition of CXCR4 may provide better therapeutic effects on renal fibrosis by inhibiting multiple downstream signalling cascades.

Tubule-derived exosomes play a central role in fibroblast activation and kidney fibrosis.

Extracellular vesicles such as exosomes are involved in mediating cell-cell communication by shuttling an assortment of proteins and genetic information. Here, we tested whether renal tubule-derived exosomes play a central role in mediating kidney fibrosis. The production of exosomes was found to be increased in the early stage of unilateral ureteral obstruction, ischemia reperfusion injury or 5/6 nephrectomy models of kidney disease. Exosome production occurred primarily in renal proximal tubular epithelium and was accompanied by induction of sonic hedgehog (Shh). In vitro, upon stimulation with transforming growth factor-ß1, kidney proximal tubular cells (HKC-8) increased exosome production. Purified exosomes from these cells were able to induce renal interstitial fibroblast (NRK-49F) activation. Conversely, pharmacologic inhibition of exosome secretion with dimethyl amiloride, depletion of exosome from the conditioned media or knockdown of Shh expression abolished the ability of transforming growth factor-ß1-treated HKC-8 cells to induce NRK-49F activation. In vivo, injections of tubular cell-derived exosomes aggravated kidney injury and fibrosis, which was negated by an Shh signaling inhibitor. Blockade of exosome secretion in vivo ameliorated renal fibrosis after either ischemic or obstructive injury. Furthermore, knockdown of Rab27a, a protein that is essential for exosome formation, also preserved kidney function and attenuated renal fibrotic lesions in mice. Thus, our results suggest that tubule-derived exosomes play an essential role in renal fibrogenesis through shuttling Shh ligand. Hence, strategies targeting exosomes could be a new avenue in developing therapeutics against renal fibrosis.

Wnt/ß-catenin signaling mediates both heart and kidney injury in type 2 cardiorenal syndrome.

In type 2 cardiorenal syndrome, chronic heart failure is thought to cause or promote chronic kidney disease; however, the underlying mechanisms remain poorly understood. We investigated the role of Wnt signaling in heart and kidney injury in a mouse model of cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC). At 8 weeks after TAC, cardiac hypertrophy, inflammation, and fibrosis were prominent, and echocardiography confirmed impaired cardiac function. The cardiac lesions were accompanied by upregulation of multiple Wnt ligands and activation of ß-catenin, as well as activation of the renin-angiotensin system (RAS). Wnt3a induced multiple components of the RAS in primary cardiomyocytes and cardiac fibroblasts in vitro. TAC also caused proteinuria and kidney fibrosis, accompanied by klotho depletion and ß-catenin activation in the kidney. Pharmacologic blockade of ß-catenin with a small molecule inhibitor or the RAS with losartan ameliorated cardiac injury, restored heart function, and mitigated the renal lesions. Serum from TAC mice was sufficient to activate ß-catenin and trigger tubular cell injury in vitro, indicating a role for circulating factors. Multiple inflammatory cytokines were upregulated in the circulation of TAC mice, and tumor necrosis factor-α was able to inhibit klotho, induce ß-catenin activation, and cause tubular cell injury in vitro. These studies identify Wnt/ß-catenin signaling as a common pathogenic mediator of heart and kidney injury in type 2 cardiorenal syndrome after TAC. Targeting this pathway could be a promising therapeutic strategy to protect both organs in cardiorenal syndrome.

Tenascin-C protects against acute kidney injury by recruiting Wnt ligands.

The development of acute kidney injury (AKI) is a complex process involving tubular, inflammatory, and vascular components, but less is known about the role of the interstitial microenvironment. We have previously shown that the extracellular matrix glycoprotein tenascin-C (TNC) is induced in fibrotic kidneys. In mouse models of AKI induced by ischemia-reperfusion injury (IRI) or cisplatin, TNC was induced de novo in the injured sites and localized to the renal interstitium. The circulating level of TNC protein was also elevated in AKI patients after cardiac surgery. Knockdown of TNC by shRNA in vivo aggravated AKI after ischemic or toxic injury. This effect was associated with reduced renal ß-catenin expression, suggesting an impact on Wnt signaling. In vitro, TNC protected tubular epithelial cells against apoptosis and augmented Wnt1-mediated ß-catenin activation. Co-immunoprecipitation revealed that TNC physically interacts with Wnt ligands. Furthermore, a TNC-enriched kidney tissue scaffold prepared from IRI mice was able to recruit and concentrate Wnt ligands from the surrounding milieu ex vivo. The ability to recruit Wnt ligands in this ex vivo model diminished after TNC depletion. These studies indicate that TNC is specifically induced at sites of injury and recruits Wnt ligands, thereby creating a favorable microenvironment for tubular repair and regeneration after AKI.

Wnt/ß-catenin links oxidative stress to podocyte injury and proteinuria.

Podocyte injury is the major cause of proteinuria in primary glomerular diseases. Oxidative stress has long been thought to play a role in triggering podocyte damage; however, the underlying mechanism remains poorly understood. Here we show that the Wnt/ß-catenin pathway is involved in mediating oxidative stress-induced podocyte dysfunction. Advanced oxidation protein products, a marker and trigger of oxidative stress, were increased in the serum of patients with chronic kidney disease and correlated with impaired glomerular filtration, proteinuria, and circulating level of Wnt1. Both serum from patients with chronic kidney disease and exogenous advanced oxidation protein products induced Wnt1 and Wnt7a expression, activated ß-catenin, and reduced expression of podocyte-specific markers in vitro and in vivo. Blockade of Wnt signaling by Klotho or knockdown of ß-catenin by shRNA in podocytes abolished ß-catenin activation and the upregulation of fibronectin, desmin, matrix metalloproteinase-9, and Snail1 triggered by advanced oxidation protein products. Furthermore, conditional knockout mice with podocyte-specific ablation of ß-catenin were protected against podocyte injury and albuminuria after treatment with advanced oxidation protein products. The action of Wnt/ß-catenin was dependent on the receptor of advanced glycation end products (RAGE)-mediated NADPH oxidase induction, reactive oxygen species generation, and nuclear factor-κB activation. These studies uncover a novel mechanistic linkage of oxidative stress, Wnt/ß-catenin activation, and podocyte dysfunction.

(Pro)renin Receptor Is an Amplifier of Wnt/ß-Catenin Signaling in Kidney Injury and Fibrosis.

The (pro)renin receptor (PRR) is a transmembrane protein with multiple functions. However, its regulation and role in the pathogenesis of CKD remain poorly defined. Here, we report that PRR is a downstream target and an essential component of Wnt/ß-catenin signaling. In mouse models, induction of CKD by ischemia-reperfusion injury (IRI), adriamycin, or angiotensin II infusion upregulated PRR expression in kidney tubular epithelium. Immunohistochemical staining of kidney biopsy specimens also revealed induction of renal PRR in human CKD. Overexpression of either Wnt1 or ß-catenin induced PRR mRNA and protein expression in vitro Notably, forced expression of PRR potentiated Wnt1-mediated ß-catenin activation and augmented the expression of downstream targets such as fibronectin, plasminogen activator inhibitor 1, and α-smooth muscle actin (α-SMA). Conversely, knockdown of PRR by siRNA abolished ß-catenin activation. PRR potentiation of Wnt/ß-catenin signaling did not require renin, but required vacuolar H+ ATPase activity. In the mouse model of IRI, transfection with PRR or Wnt1 expression vectors promoted ß-catenin activation, aggravated kidney dysfunction, and worsened renal inflammation and fibrotic lesions. Coexpression of PRR and Wnt1 had a synergistic effect. In contrast, knockdown of PRR expression ameliorated kidney injury and fibrosis after IRI. These results indicate that PRR is both a downstream target and a crucial element in Wnt signal transmission. We conclude that PRR can promote kidney injury and fibrosis by amplifying Wnt/ß-catenin signaling.

Klotho Ameliorates Kidney Injury and Fibrosis and Normalizes Blood Pressure by Targeting the Renin-Angiotensin System.

Loss of Klotho and activation of the renin-angiotensin system (RAS) are common pathological findings in chronic kidney diseases. However, whether these two events are intricately connected is poorly understood. We hypothesized that Klotho might protect kidneys by targeted inhibition of RAS activation in diseased kidneys. To test this hypothesis, mouse models of remnant kidney, as well as adriamycin nephropathy and unilateral ureteral obstruction, were utilized. At 6 weeks after 5/6 nephrectomy, kidney injury was evident, characterized by elevated albuminuria and serum creatinine levels, and excessive deposition of interstitial matrix proteins. These lesions were accompanied by loss of renal Klotho expression, up-regulation of RAS components, and development of hypertension. In vivo expression of exogenous Klotho through hydrodynamic-based gene delivery abolished the induction of multiple RAS proteins, including angiotensinogen, renin, angiotensin-converting enzyme, and angiotensin II type 1 receptor, and normalized blood pressure. Klotho also inhibited ß-catenin activation and ameliorated renal fibrotic lesions. Similar results were obtained in mouse models of adriamycin and obstructive nephropathy. In cultured kidney tubular epithelial cells, Klotho dose-dependently blocked Wnt1-triggered RAS activation. Taken together, these results demonstrate that Klotho exerts its renal protection by targeted inhibition of RAS, a pathogenic pathway known to play a key role in the evolution and progression of hypertension and chronic kidney disorders.

The CXCR4-AT1 axis plays a vital role in glomerular injury via mediating the crosstalk between podocyte and mesangial cell.

Glomeruli stand at the center of nephrons to accomplish filtration and albumin interception. Podocytes and mesangial cells are the major constituents in the glomeruli. However, their interdependency in glomerular injury has rarely been reported. Herein, we investigated the role of C-X-C chemokine receptor type 4 (CXCR4) in mediating the crosstalk between podocytes and mesangial cells. We found CXCR4 and angiotensin II (AngII) increased primarily in injured podocytes. However, type-1 receptor of angiotensin II (AT1) and stromal cell-derived factor 1α (SDF-1α), a ligand of CXCR4, were evidently upregulated in mesangial cells following the progression of podocyte injury. Ectopic expression of CXCR4 in 5/6 nephrectomy mice increased the decline of renal function and glomerular injury, accelerated podocyte injury and mesangial cell activation, and initiated CXCR4-AT1 axis signals. Additionally, treatment with losartan, an AT1 blocker, interrupted the cycle of podocyte injury and mesangial matrix deposition triggered by CXCR4. Podocyte-specific ablation of CXCR4 gene blocked podocyte injury and mesangial cell activation. In vitro, CXCR4 overexpression induced oxidative stress and renin angiotensin system (RAS) activation in podocytes, and triggered the communication between podocytes and mesangial cells. In cultured mesangial cells, AngII treatment induced the expression of SDF-1α, which was secreted into the supernatant to further promote oxidative stress and cell injury in podocytes. Collectively, these results demonstrate that the CXCR4-AT1 axis plays a vital role in glomerular injury via mediating pathologic crosstalk between podocytes and mesangial cells. Our findings uncover a novel pathogenic mechanism by which the CXCR4-AT1 axis promotes glomerular injury.

FAM3A plays a key role in protecting against tubular cell pyroptosis and acute kidney injury.

Acute kidney injury (AKI) is in high prevalence worldwide but with no therapeutic strategies. Programmed cell death in tubular epithelial cells has been reported to accelerate a variety of AKI, but the major pathways and underlying mechanisms are not defined. Herein, we identified that pyroptosis was responsible for AKI progression and related to ATP depletion in renal tubular cells. We found that FAM3A, a mitochondrial protein that assists ATP synthesis, was decreased and negatively correlated with tubular cell injury and pyroptosis in both mice and patients with AKI. Knockout of FAM3A worsened kidney function decline, increased macrophage and neutrophil cell infiltration, and facilitated tubular cell pyroptosis in ischemia/reperfusion injury model. Conversely, FAM3A overexpression alleviated tubular cell pyroptosis, and inhibited kidney injury in ischemic AKI. Mechanistically, FAM3A promoted PI3K/AKT/NRF2 signaling, thus blocking mitochondrial reactive oxygen species (mt-ROS) accumulation. NLRP3 inflammasome sensed the overload of mt-ROS and then activated Caspase-1, which cleaved GSDMD, pro-IL-1ß, and pro-IL-18 into their mature forms to mediate pyroptosis. Of interest, NRF2 activator alleviated the pro-pyroptotic effects of FAM3A depletion, whereas the deletion of NRF2 blocked the anti-pyroptotic function of FAM3A. Thus, our study provides new mechanisms for AKI progression and demonstrates that FAM3A is a potential therapeutic target for treating AKI.

MAGL protects against renal fibrosis through inhibiting tubular cell lipotoxicity.

Rationale: Renal fibrosis, with no therapeutic approaches, is a common pathological feature in various chronic kidney diseases (CKD). Tubular cell injury plays a pivotal role in renal fibrosis. Commonly, injured tubular cells exhibit significant lipid accumulation. However, the underlying mechanisms remain poorly understood. Methods: 2-arachidonoylglycerol (2-AG) levels in CKD patients and CKD model specimens were measured using mass spectrometry. 2-AG-loaded nanoparticles were infused into unilateral ureteral obstruction (UUO) mice. Lipid accumulation and renal fibrosis were tested. Furthermore, monoacylglycerol lipase (MAGL), the hydrolyzing enzyme of 2-AG, was assessed in CKD patients and models. Tubular cell-specific MAGL knock-in mice were generated. Moreover, MAGL recombination protein was also administered to unilateral ischemia reperfusion injury (UIRI) mice. Besides, a series of methods including RNA sequencing, metabolomics, primary cell culture, lipid staining, etc. were used. Results: 2-AG was increased in the serum or kidneys from CKD patients and models. Supplement of 2-AG further induced lipid accumulation and fibrogenesis through cannabinoid receptor type 2 (CB2)/ß-catenin signaling. ß-catenin knockout blocked 2-AG/CB2-induced fatty acid ß-oxidation (FAO) deficiency and lipid accumulation. Remarkably, MAGL significantly decreased in CKD, aligning with lipid accumulation and fibrosis. Specific transgene of MAGL in tubular cells significantly preserved FAO, inhibited lipid-mediated toxicity in tubular cells, and finally retarded fibrogenesis. Additionally, supplementation of MAGL in UIRI mice also preserved FAO function, inhibited lipid accumulation, and protected against renal fibrosis. Conclusion: MAGL is a potential diagnostic marker for kidney function decline, and also serves as a new therapeutic target for renal fibrosis through ameliorating lipotoxicity.

Kidney tubular epithelial cells control interstitial fibroblast fate by releasing TNFAIP8-encapsulated exosomes.

Kidney fibrosis, characterized by the activation and expansion of the matrix-producing fibroblasts, is the common outcome of chronic kidney disease (CKD). While fibroblast proliferation is well studied in CKD, little is known about the regulation and mechanism of fibroblast depletion. Here, we show that exosomes derived from stressed/injured tubules play a pivotal role in dictating fibroblast apoptosis and fate. When human kidney tubular cells (HK-2) were stimulated with TGF-ß1, they produced and released increased amounts of exosomes (TGFß-Exo), which prevented renal interstitial fibroblasts from apoptosis. In vivo, injections of TGFß-Exo promoted renal fibroblast survival, whereas blockade of exosome secretion accelerated fibroblast apoptosis in obstructive nephropathy. Proteomics profiling identified the tumor necrosis factor-α-induced protein 8 (TNFAIP8) as a key component enriched in TGFß-Exo. TNFAIP8 was induced in renal tubular epithelium and enriched in the exosomes from fibrotic kidneys. Knockdown of TNFAIP8 in tubular cells abolished the ability of TGFß-Exo to prevent fibroblast apoptosis. In vivo, gain- or loss- of TNFAIP8 prevented or aggravated renal fibroblast apoptosis after obstructive injury. Mechanistically, exosomal-TNFAIP8 promoted p53 ubiquitination leading to its degradation, thereby inhibiting fibroblasts apoptosis and inducing their proliferation. Collectively, these results indicate that tubule-derived exosomes play a critical role in controlling the size of fibroblast population during renal fibrogenesis through shuttling TNFAIP8 to block p53 signaling. Strategies to target exosomes may be effective strategies for the therapy of fibrotic CKD.

Pentraxin 3 plays a key role in tubular cell senescence and renal fibrosis through inducing ß-catenin signaling.

Renal fibrosis is the common pathological feature of various chronic kidney diseases (CKD). Tubular cell senescence plays a key role in the progression of renal fibrosis. However, the underlying mechanisms are still in mystery. In this study, we identified, Pentraxin 3 (PTX3), belonging to the Pentraxin family, is a new fibrogenic factor. PTX3 was increased in various CKD models. PTX3 was primarily localized in tubular epithelial cells and upregulated, accompanied by mitochondrial dysfunction and cellular senescence. Overexpression of PTX3 aggravated mitochondrial damage and accelerated cell senescence in tubular cells, leading to more severe fibrogenesis in kidneys. However, knockout of PTX3 significantly preserved mitochondrial homeostasis, and blocked cellular senescence in primary cultured tubular cells. Furthermore, KYA1797K, a destabilizer of ß-catenin, greatly inhibited PTX3-induced mitochondrial dysfunction, tubular cell senescence, and renal fibrosis. Overexpression of PTX3 triggered nuclear translocation of ß-catenin, an activating form of ß-catenin. PTX3-induced mitochondrial dysfunction and tubular cell senescence were also significantly inhibited by knockdown of p16INK4A, a senescence-related protein. In a clinical cohort, we found PTX3 was increased in urine and serum in patients with CKD. Urinary PTX3 negatively correlated with eGFR. PTX3 also increased gradually following the severity of diseases, triggering the fibrogenesis. Taken together, our results provide strong evidences that PTX3 is a new fibrogenic factor in the development of renal fibrosis through ß-catenin-induced mitochondrial dysfunction and cell senescence. This study further suggests PTX3 is a new diagnostic factor to renal fibrosis and provides a new therapeutic target against renal fibrosis.

Sirtuin 6 is a key contributor to gender differences in acute kidney injury.

Acute kidney injury (AKI) is rapidly increasing nowadays and at a high risk to progress into chronic kidney disease (CKD). Of note, men are more susceptive to AKI, suggesting gender differences in AKI patients. However, the underlying mechanisms remain largely unclear. To test it, we adopted two experimental models of AKI, including ischemia/reperfusion injury and rhabdomyolysis, which were constructed in age-matched male and female mice. We found severe damages of tubular apoptosis, mitochondrial dysfunction, and loss of renal function showing in male mice, while female mice only had very mild injury. We further tested the expression of Sirtuins, and found that female mice could preserve more Sirtuin members' expression in case of kidney damage. Among Sirtuin family, Sirtuin 6 was maximally preserved in injured kidney in female mice, suggesting its important role involved in the gender differences of AKI pathogenesis. We then found that knockdown of androgen receptor (AR) attenuated tubular damage, mitochondrial dysfunction and retarded the loss of renal function. Overexpression of Sirtuin 6 also showed similar results. Furthermore, in cultured tubular cells, dihydrotestosterone (DHT) decreased Sirtuin 6 expression and exacerbated cell apoptosis. Ectopic expression of Sirtuin 6 sufficiently inhibited DHT-induced cell apoptosis. Mechanically, we found AR inhibited Sirtuin 6, leading to the repression of binding of Sirtuin 6 with PGC-1α. This resulted in acetylation of PGC-1α and inhibition of its activity, further triggered the loss of mitochondrial homeostasis. Our results provided new insights to the underlying mechanisms of gender differences in AKI, suggesting Sirtuin 6 maybe a new therapeutic target for preventing AKI in male patients.

CXC Chemokine Receptor 2 Accelerates Tubular Cell Senescence and Renal Fibrosis via ß-Catenin-Induced Mitochondrial Dysfunction.

Renal fibrosis is a common feature of various chronic kidney diseases (CKD). However, its underlying mechanism has not been totally clarified. C-X-C motif chemokine receptor (CXCR) family plays a role in renal fibrosis, however, detailed mechanisms have not been elucidated. Here, we report that CXCR2 has a potential role in tubular cell senescence and renal fibrosis, and is associated with ß-catenin-activated mitochondrial dysfunction. CXCR2 is one of most increased members among CXCR family in unilateral ureteral obstruction (UUO) mice. CXCR2 was expressed primarily in tubules and co-localized with p16INK4A, a cellular senescence marker, and ß-catenin. Administration of SB225002, a selective CXCR2 antagonist, significantly inhibited the activation of ß-catenin signaling, restored mitochondrial function, protected against tubular cell senescence and renal fibrosis in unilateral ureteral obstruction (UUO) mice. In unilateral ischemia-reperfusion injury (UIRI) mice, treatment with interlukin-8 (IL-8), the ligand of CXCR2, further aggravated ß-catenin activation, mitochondrial dysfunction, tubular cell senescence and renal fibrosis, whereas knockdown of p16INK4A inhibited IL-8-induced these effects. In vitro, SB225002 inhibited mitochondrial dysfunction and tubular cell senescence. Furthermore, ICG-001, a ß-catenin signaling blocker, significantly retarded CXCR2-induced cellular senescence and fibrotic changes. These results suggest that CXCR2 promotes tubular cell senescence and renal fibrosis through inducing ß-catenin-activated mitochondrial dysfunction.

AMPK Activator O304 Protects Against Kidney Aging Through Promoting Energy Metabolism and Autophagy.

Aging is an important risk factor for kidney injury. Energy homeostasis plays a key role in retarding aging, and mitochondria are responsible for energy production. In the kidney, renal tubular cells possess high abundance of mitochondria to meet the high energy consumption. AMPK is an evolutionarily conserved serine/threonine kinase which plays a central role in maintaining energy homeostasis and mitochondrial homeostasis. Besides that, AMPK also commands autophagy, a clearing and recycling process to maintain cellular homeostasis. However, the effect of AMPK activators on kidney aging has not been fully elucidated. To this end, we testified the effects of O304, a novel direct AMPK activator, in naturally aging mice model and D-Galactose (D-Gal)-treated renal tubular cell culture. We identified that O304 beneficially protects against cellular senescence and aged-related fibrosis in kidneys. Also, O304 restored energy metabolism, promoted autophagy and preserved mitochondrial homeostasis. Transcriptomic sequencing also proved that O304 induced fatty acid metabolism, mitochondrial biogenesis and ATP process, and downregulated cell aging, DNA damage response and collagen organization. All these results suggest that O304 has a strong potential to retard aged kidney injury through regulating AMPK-induced multiple pathways. Our results provide an important therapeutic approach to delay kidney aging.

ß-catenin-controlled tubular cell-derived exosomes play a key role in fibroblast activation via the OPN-CD44 axis.

Tubular injury and peripheral fibroblast activation are the hallmarks of chronic kidney disease (CKD), suggesting intimate communication between the two types of cells. However, the underlying mechanisms remain to be determined. Exosomes play a role in shuttling proteins and other materials to recipient cells. In our study, we found that exosomes were aroused by ß-catenin in renal tubular cells. Osteopontin (OPN), especially its N-terminal fragment (N-OPN), was encapsulated in ß-catenin-controlled tubular cell-derived exosome cargo, and subsequently passed to fibroblasts. Through binding with CD44, exosomal OPN promoted fibroblast proliferation and activation. Gene deletion of ß-catenin in tubular cells (Ksp-ß-catenin-/- ) or gene ablation of CD44 (CD44-/- ) greatly ameliorated renal fibrosis. Notably, N-OPN was carried by exosome and secreted into the urine of patients with CKD, and negatively correlated with kidney function. The urinary exosomes from patients with CKD greatly accelerated renal fibrosis, which was blocked by CD44 deletion. These results suggest that exosome-mediated activation of the OPN/CD44 axis plays a key role in renal fibrosis, which is controlled by ß-catenin.

Annexin A2 plays a key role in protecting against cisplatin-induced AKI through ß-catenin/TFEB pathway.

Acute kidney injury (AKI) is in high prevalence in the world. However, the therapeutic strategies for AKI are still in mystery. Studies have shown to improve autophagy and lysosomal function could inhibit AKI. But their modulators need to be explored in detail. Annexin A2 (ANXA2) is a phospholipid-binding protein involving in organelle membrane integrity function, suggesting its important role in autophagy and lysosome homeostasis. It implicates ANXA2 potentially protects against AKI. However, this has not been elucidated. Herein, we found that ANXA2 is increased in renal tubules in cisplatin-induced AKI mice. Ectopic expression of ANXA2 improved lysosomal functions and enhanced autophagic flux, further protecting against renal tubular cell apoptosis and kidney injury. Conversely, knockdown of ANXA2 inhibited lysosomal function and autophagy, which aggravated the progression of AKI. Transcriptome sequencing revealed ß-catenin signaling is highly responsible for this process. In vitro, we found ANXA2 induced ß-catenin activation, further triggering T-cell factor-4 (TCF4)-induced transcription factor EB (TFEB). Furthermore, TFEB promoted lysosome biogenesis to enhance autophagic flux, resulting in the alleviation of AKI. Our new findings underline ANXA2 is a new therapeutic potential for AKI through modulating autophagy and lysosomal function. The underlying mechanism is associated with its inductive effects on ß-catenin/TFEB pathway.