Role In Kidney Fibrosis Research Articles

Collagen formation, function and role in kidney disease.

Highly abundant in mammals,collagens define the organization of tissues and participate in cell signalling. Most of the 28 vertebrate collagens, with the exception of collagens VI, VII, XXVI and XXVIII, can be categorized into five subgroups: fibrillar collagens, network-forming collagens, fibril-associated collagens with interrupted triple helices, membrane-associated collagens with interrupted triple helices and multiple triple-helix domains with interruptions. Collagen peptides are synthesized from the ribosome and enter the rough endoplasmic reticulum, where they undergo numerous post-translational modifications. The collagen chains form triple helices that can be secreted to form a diverse array of supramolecular structures in the extracellular matrix. Collagens are ubiquitously expressed and have been linked to a broad spectrum of disorders, including genetic disorders with kidney phenotypes. They also have an important role in kidney fibrosis and mass spectrometry-based proteomic studies have improved understanding of the composition of fibrosis in kidney disease. A wide range of therapeutics are in development for collagen and kidneydisorders, including genetic approaches, chaperone therapies, proteindegradation strategies and anti-fibrotic therapies. Improved understanding of collagens and their role in disease is needed to facilitate the development of more specific treatments for collagen and kidney disorders.

Modulators of Alpha-2 Macroglobulin Upregulation by High Glucose in Glomerular Mesangial Cells

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the −405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD.

Nuclear translocation of SIRT4 mediates deacetylation of U2AF2 to modulate renal fibrosis through alternative splicing-mediated upregulation of CCN2.

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.

Nuclear translocation of SIRT4 mediates deacetylation of U2AF2 to modulate renal fibrosis through alternative splicing-mediated upregulation of CCN2

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.

Lactate: a missing link between metabolism and inflammation in CKD progression?

Persistent enhancement of glycolysis in kidney tubular epithelial cells has been linked to the progression of chronic kidney disease, although the underlying mechanisms are largely unknown. In this issue of Kidney International, Wang etal. report that the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 plays a crucial role in kidney fibrosis by enhancing histone H4 lysine 12 lactylation through lactate accumulation. This increases the transcription of nuclear factor-κB-related genes and promotes inflammation and fibrosis. Inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 reduces these effects, indicating therapeutic potential for kidney fibrosis.

RIPK3 causes mitochondrial dysfunction and albuminuria in diabetic podocytopathy through PGAM5-Drp1 signaling

BackgroundReceptor-interacting protein kinase (RIPK)3 is an essential molecule for necroptosis and its role in kidney fibrosis has been investigated using various kidney injury models. However, the relevance and the underlying mechanisms of RIPK3 to podocyte injury in albuminuric diabetic kidney disease (DKD) remain unclear. Here, we investigated the role of RIPK3 in glomerular injury of DKD. MethodsWe analyzed RIPK3 expression levels in the kidneys of patients with biopsy-proven DKD and animal models of DKD. Additionally, to confirm the clinical significance of circulating RIPK3, RIPK3 was measured by ELISA in plasma obtained from a prospective observational cohort of patients with type 2 diabetes, and estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), which are indicators of renal function, were followed up during the observation period. To investigate the role of RIPK3 in glomerular damage in DKD, we induced a DKD model using a high-fat diet in Ripk3 knockout and wild-type mice. To assess whether mitochondrial dysfunction and albuminuria in DKD take a Ripk3-dependent pathway, we used single-cell RNA sequencing of kidney cortex and immortalized podocytes treated with high glucose or overexpressing RIPK3. ResultsRIPK3 expression was increased in podocytes of diabetic glomeruli with increased albuminuria and decreased podocyte numbers. Plasma RIPK3 levels were significantly elevated in albuminuric diabetic patients than in non-diabetic controls (p = 0.002) and non-albuminuric diabetic patients (p = 0.046). The participants in the highest tertile of plasma RIPK3 had a higher incidence of renal progression (hazard ratio [HR] 2.29 [1.05–4.98]) and incident chronic kidney disease (HR 4.08 [1.10–15.13]). Ripk3 knockout improved albuminuria, podocyte loss, and renal ultrastructure in DKD mice. Increased mitochondrial fragmentation, upregulated mitochondrial fission-related proteins such as phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp1), and mitochondrial ROS were decreased in podocytes of Ripk3 knockout DKD mice. In cultured podocytes, RIPK3 inhibition attenuated mitochondrial fission and mitochondrial dysfunction by decreasing p-mixed lineage kinase domain-like protein (MLKL), PGAM5, and p-Drp1 S616 and mitochondrial translocation of Drp1. ConclusionsThe study demonstrates that RIPK3 reflects deterioration of renal function of DKD. In addition, RIPK3 induces diabetic podocytopathy by regulating mitochondrial fission via PGAM5-Drp1 signaling through MLKL. Inhibition of RIPK3 might be a promising therapeutic option for treating DKD.

#2394 Inhibition of ACSS2-mediated H3K9 crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Abstract Background and Aims Histone lysine crotonylation (Kcr), a novel posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. Method Human renal tissue, fixed in formaldehyde, and embedded in paraffin, was selected from the files of the Service of Pathology of West China Hospital, Sichuan University.ACSS2 knockout mice and tubular specific knock out of ACSS2 mice in C57BL/6N background and wild-type littermates (WT) were used to induce the FAN and UUO model or as control groups (n = 6 each). In the parallel study, C57BL/6N mice were randomly divided into three groups: control, FAN/UUO and FAN/UUO+ACSS2 inhibitor (n = 6 each). At day 7, mice were sacrificed and the serum creatinine, BUN and urinary albumin/creatinine ratio (ACR) were tested. Renal histological injury was measured by HE and Masson's trichrome staining. SA-β-gal staining was used to check cellular senescence. Renal tissues of the mice were analyzed by western blot and qPCR assay for the expression of ACSS2, H3K9cr, fibrosis and cellular senescence related proteins and genes. RNA-sequencing and ChIP-sequencing combined with in vitro cell experiments were used to figure out the underlying mechanism. Results In this study, we reported that histone Kcr of tubular epithelial cells was abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) was found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys revealed that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppressed H3K9cr-mediated IL-1β expression, which thereby alleviated IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Conclusion Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

#2660 WWP2-induced pericytes to myofibroblast transition contributes to the progression of fibrosis in CKD in diabetes

Abstract Background and Aims CKD in diabetes can be characterized by different clinical and histological phenotypes, associated to different molecular pathways specifically involved in the progression of kidney disease. CKD in diabetes includes different phenotypes, commonly classified according to Mazzucco et al (PMID:11920336) as diabetic Nephropathy (DN), non-diabetic renal disease (NDRD) and mixed forms (DN+NDRD). Among these, DN represents the primary cause of end stage renal disease (ESRD) and our group demonstrated that renal damage in DN is characterized by a specific molecular feature, represented by the activation of lysine63 (K63)-ubiquitination (Ub) pathway. In particular, we demonstrated that accumulation of K63-Ub proteins is involved in the progression of renal fibrosis (PMID: 27881486). WWP2 is an E3 ubiquitin-protein ligase that modulates myofibroblast pro-fibrotic activation in cardiac fibrosis (PMID: 31399586), however its specific role in kidney fibrosis in diabetes and its subsequent involvement in K63-Ub pathway has not been described so far. Thus aim of this study was to evaluate the involvement of WWP2 in the progression of kidney fibrosis in diabetes and its involvement in K63-Ub pathway. Method WWP2, K-63Ub, alpha-sma and PDGFbeta-receptor expression were evaluated in: i) 22 kidney biopsies from patients with a biopsy proven diagnosis of DN (n = 11), NDRD (n = 6) and CKD without diabetes (n = 5), by immunohistochemistry and immunofluorescence; ii) streptozotocin (STZ)-treated DBA/2J mice, a model of human DN, in the presence or absence of a specific inhibitor of K63Ub (NSC697923) by immunohistochemistry (n = 3 for each group); iii) in HK2 tubular cells, EAHY926 endothelial cells and in human-derived pericytes under hyperglycemic conditions by western blotting and qPCR. Results Immunohistochemistry showed that WWP2 was expressed both in CKD and in DN patients’ biopsies when compared to patients with NDRD (p < 0.05 and p < 0.01 respectively), both at tubular, glomerular and in the vascular compartment. Immunofluorescence confirmed that WWP2 increased expression in DN patients compared to NDRD, was associated to accumulation of K63Ub proteins in the tubular compartment as well as in the vascular compartment in particular in pericytes, cells that envelop the surface of vessels and have been described as the major source of scar-forming myofibroblasts in CKD (PMID: 19008372). The involvement of WWP2 in the K63-Ub pathway was confirmed in vivo in DBA2J diabetic mice in which the specific inhibition by NSC697923 of the K63Ub pathway, significantly reduced WWP2 expression in kidney tissues from diabetic mice (p < 0.05). These results were confirmed in vitro where specific inhibition of K63Ub pathway by NSC697923 significantly reduced hyperglycaemia-induced WWP2 expression in HK2 cells by western blotting and qPCR (p < 0.01). Hyperglycaemic conditions did not influence WWP2 expression in EAHY926 endothelial cells (p = n.s.). Conversely, using qPCR, in pericytes we observed an hyperglycemia-induced expression of both WWP2 levels (RR>2) and alpha-sma levels (RR>3) at different time points. Moreover, western blotting showed that hyperglycemia also induced a decrease of PDGF-beta-receptor marker of pericytes, thus suggesting their transition to myofibroblasts. Conclusion These findings demonstrate the influence of WWP2 on the k63-Ub pathway thus driving fibrogenesis in DN patients, in particular through pericyte to myofibroblast transition. WWP2 could represent a potential novel target for therapeutic intervention in the treatment of chronic kidney disease in diabetes.

Inhibition of ACSS2-mediated histone crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Histone lysine crotonylation (Kcr), as a posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. In this study, we report that histone Kcr of tubular epithelial cells is abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) is found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys reveal that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppress H3K9cr-mediated IL-1β expression, which thereby alleviate IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

PDGF-D Is Dispensable for the Development and Progression of Murine Alport Syndrome

Alport syndrome is an inherited kidney disease, which can lead to glomerulosclerosis and fibrosis, as well as end-stage kidney disease in children and adults. Platelet-derived growth factor-D (PDGF-D) mediates glomerulosclerosis and interstitial fibrosis in various models of kidney disease, prompting investigation of its role in a murine model of Alport syndrome. Invitro, PDGF-D induced proliferation and profibrotic activation of conditionally immortalized human parietal epithelial cells. In Col4a3-/- mice, a model of Alport syndrome, PDGF-D mRNA and protein were significantly up-regulated compared with non-diseased wild-type mice. To analyze the therapeutic potential of PDGF-D inhibition, Col4a3-/- mice were treated with a PDGF-D neutralizing antibody. Surprisingly, PDGF-D antibody treatment had no effect on renal function, glomerulosclerosis, fibrosis, or other indices of kidney injury compared with control treatment with unspecific IgG. To characterize the role of PDGF-D in disease development, Col4a3-/- mice with a constitutive genetic deletion of Pdgfd were generated and analyzed. No difference in pathologic features or kidney function was observed in Col4a3-/-Pdgfd-/- mice compared with Col4a3-/-Pdgfd+/+ littermates, confirming the antibody treatment data. Mechanistically, lack of proteolytic PDGF-D activation in Col4a3-/- mice might explain the lack of effects invivo. In conclusion, despite its established role in kidney fibrosis, PDGF-D, without further activation, does not mediate the development and progression of Alport syndrome in mice.

Myeloid PFKFB3-mediated glycolysis promotes kidney fibrosis.

Excessive renal fibrosis is a common pathology in progressive chronic kidney diseases. Inflammatory injury and aberrant repair processes contribute to the development of kidney fibrosis. Myeloid cells, particularly monocytes/macrophages, play a crucial role in kidney fibrosis by releasing their proinflammatory cytokines and extracellular matrix components such as collagen and fibronectin into the microenvironment of the injured kidney. Numerous signaling pathways have been identified in relation to these activities. However, the involvement of metabolic pathways in myeloid cell functions during the development of renal fibrosis remains understudied. In our study, we initially reanalyzed single-cell RNA sequencing data of renal myeloid cells from Dr. Denby's group and observed an increased gene expression in glycolytic pathway in myeloid cells that are critical for renal inflammation and fibrosis. To investigate the role of myeloid glycolysis in renal fibrosis, we utilized a model of unilateral ureteral obstruction in mice deficient of Pfkfb3, an activator of glycolysis, in myeloid cells (Pfkfb3 ΔMϕ ) and their wild type littermates (Pfkfb3 WT). We observed a significant reduction in fibrosis in the obstructive kidneys of Pfkfb3 ΔMϕ mice compared to Pfkfb3 WT mice. This was accompanied by a substantial decrease in macrophage infiltration, as well as a decrease of M1 and M2 macrophages and a suppression of macrophage to obtain myofibroblast phenotype in the obstructive kidneys of Pfkfb3 ΔMϕ mice. Mechanistic studies indicate that glycolytic metabolites stabilize HIF1α, leading to alterations in macrophage phenotype that contribute to renal fibrosis. In conclusion, our study implicates that targeting myeloid glycolysis represents a novel approach to inhibit renal fibrosis.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-β (TGF-β)-dependent and -independent signaling. TIF1γ hindered TGF-β signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-β signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-β signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

G9a and DNMT1 inhibition modulates CDKN1A promoter methylation and the cell cycle leading to improvement in kidney fibrosis

BackgroundEpigenetic mechanisms, including histone and DNA methylation, play a key role in kidney fibrosis, but the precise mechanism remains unclear. Concerted action between histone and DNA-methyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We investigated the role of G9a and DNMT1 in kidney fibrosis pathogenesis and aimed to elucidate key G9a and DNMT1 targets contributing to kidney fibrosis maintenance. MethodsG9a and DNMT1 were detected in human fibrotic kidneys, UUO mouse kidneys, and TGFβ1-induced HK-2 cells. G9a and DNMT1 expression was knocked down by siRNA or inhibited with CM272 in HK-2 and UUO mouse, and transcriptomic responses to CM272 were examined. Antifibrogenic activity and safety of CM272 were studied in UUO mouse. Cell cycle were analyzed with flow cytometry. Gene expression regulation was analyzed by chromatin immunoprecipitation and methylation-specific PCR. ResultsG9a and DNMT1 were overexpressed in human fibrotic kidneys, UUO mouse kidneys, and TGFβ1-induced HK-2 cells. G9a/DNMT1 inhibition potently alleviated fibrosis in vitro and vivo. G9a/DNMT1 inhibition reduced the expression of E2F targets and altered the methylation status of CDKN1A leading to the attenuated cell-cycle arrest. TGFβ1-induced overexpression of G9a or DNMT1 resulted in the enrichment of H3K9me2 and 5-methylcytosine at CDKN1A promoter. ConclusionsOur data link G9a and DNMT1 to CDKN1A regulatory function and kidney fibrosis. Combined targeting G9a and DNMT1 could be a promising strategy for the treatment of kidney fibrosis.

#5526 COMPLEMENT C3 EXERTS DIRECT PRO-FIBROTIC EFFECTS ON KIDNEY TUBULES

Abstract Background and Aims Renal complement expression has been observed in both experimental and human kidney diseases. We have previously reported massive tubular C3 expression in fibrotic TGF-beta1 transgenic mice (Nephrol Dial Transpl 2015, 30; S3:FP301). Still, little is known whether this intra-renal complement is only an inflammatory marker of renal fibrosis or also exerts direct pro-fibrotic effects in the pathogenesis. In the present study we aimed to investigate the effect of C3a receptor activation in murine primary tubular epithelial cells. Method Primary tubular epithelial cells (TEC) were isolated from kidneys of four weeks-old male C57Bl6 mouse. The cells were grown in DMEM/12 medium supplemented with 2% FBS and recombinant 5 ng/ml EGF, and characterized for epithelial and mesenchymal markers. Verified TEC between P4 and P10 passages were used for the experiments. TEC cells were seeded on 6-well plates and treated for 24 hours with PBS (CTL, n=3), 100 nM C3a receptor agonist peptide (C3aR, n=3) or 10 ng/ml recombinant TGF-beta1 (TGFb, n=3). Then, mRNA and protein expressions were assessed and evaluated using Kruskal-Wallis test (p<0.05). Results TGF-beta induced a 2-fold Col1a1 and 1,6-fold Tgfb1 mRNA expression, accompanied by 4-fold Egr2 but did not affect Ccl2 or C3 mRNA expression. In contrast, C3aR group depicted 1,6-fold C3 and 1,8-fold Ccl2 mRNA expression, an only 1,6-fold Egr2 but a 2-fold Col1a1 and 1,5-fold Tgfb1 mRNA expression. Despite the similar Tgfb1 mRNA overexpression in TGFb and C3aR groups, marked TGFB1 protein overexpression (15-fold) was only observed in TGF-beta treated cells but not in the C3aR group. Conclusion Our study data indicate that renal complement C3 production can exert autocrine / paracrine direct pro-fibrotic effects on tubular epithelial cells unrelated to TGF-beta protein levels. Therefore, local C3 overexpression might play a significant pathogenetic role in kidney fibrosis.

Iron therapy mitigates chronic kidney disease progression by regulating intracellular iron status of kidney macrophages.

Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and proinflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of proinflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron markedly decreased TGF-β expression and suppressed TGF-β-driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, validating the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.

Type IV Collagen and SOX9 Are Molecular Targets of BET Inhibition in Experimental Glomerulosclerosis.

Progressive glomerulonephritis (GN) is characterized by an excessive accumulation of extracellular (ECM) proteins, mainly type IV collagen (COLIV), in the glomerulus leading to glomerulosclerosis. The current therapeutic approach to GN is suboptimal. Epigenetic drugs could be novel therapeutic options for human disease. Among these drugs, bromodomain and extra-terminal domain (BET) inhibitors (iBETs) have shown beneficial effects in experimental kidney disease and fibrotic disorders. Sex-determining region Y-box 9 (SOX9) is a transcription factor involved in regulating proliferation, migration, and regeneration, but its role in kidney fibrosis is still unclear. We investigated whether iBETs could regulate ECM accumulation in experimental GN and evaluated the role of SOX9 in this process. For this purpose, we tested the iBET JQ1 in mice with anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS). In NTS-injected mice, JQ1 treatment reduced glomerular ECM deposition, mainly by inhibiting glomerular COLIV accumulation and Col4a3 gene overexpression. Moreover, chromatin immunoprecipitation assays demonstrated that JQ1 inhibited the recruitment and binding of BRD4 to the Col4a3 promoter and reduced its transcription. Active SOX9 was found in the nuclei of glomerular cells of NTS-injured kidneys, mainly in COLIV-stained regions. JQ1 treatment blocked SOX9 nuclear translocation in injured kidneys. Moreover, in vitro JQ1 blocked TGF-β1-induced SOX9 activation and ECM production in cultured mesangial cells. Additionally, SOX9 gene silencing inhibited ECM production, including COLIV production. Our results demonstrated that JQ1 inhibited SOX9/COLIV, to reduce experimental glomerulosclerosis, supporting further research of iBET as a potential therapeutic option in progressive glomerulosclerosis.

Gambogenic acid alleviates kidney fibrosis via epigenetic inhibition of EZH2 to regulate Smad7-dependent mechanism

BackgroundEpigenetics regulating gene expression plays important role in kidney fibrosis. Natural products originating from diverse sources including plants and microorganisms are capable to influence epigenetic modifications. Gambogenic acid (GNA) is a caged xanthone extracted from gamboge resin, exudation of Garcinia hanburyi Hook.f., and the effect of GNA on kidney fibrosis with its underlying mechanism on epigenetics remains unknown. PurposeThis study aimed to explore the role of GNA against kidney fibrogenesis by histone methylation mediating gene expression. MethodsTwo experimental mice of unilateral ureteral obstruction (UUO) and folic acid (FA) were given two dosages of GNA (3 and 6 mg/kg/d). TGF-β1 was used to stimulate mouse tubular epithelial (TCMK-1) cells and siRNAs were transfected to verify the underlying mechanisms of GNA. Histological changes were evaluated by HE, MASSON stainings, immunohistochemistry and immunofluorescence. Western blot and qPCR were used to measure protein/gene transcription levels. ResultsGNA dose-dependently alleviated UUO-induced kidney fibrosis and FA-induced kidney early fibrosis, indicated by the pathology and fibrotic factor changes (α-SMA, collagen I, collagen VI, and fibronectin). Mechanically, GNA reduced enhancer of zeste homolog 2 (EZH2) and H3K27me3, promoted Smad7 transcription, and inhibited TGF-β/Smad3 fibrotic signaling in injured kidneys. Moreover, with TGF-β1-induced EZH2 increasing, GNA suppressed α-SMA, fibronectin and collagen levels in tubular epithelial TCMK-1 cells. Although partially decreasing EZH2, GNA did not influence fibrotic signaling in Smad7 siRNA-transfected TCMK-1 cells. ConclusionEpigenetic inhibition of EZH2 by GNA ameliorated kidney fibrogenesis via regulating Smad7-meidated TGF-β/Smad3 signaling.

POS-551 MONOACYLGLYCEROL LIPASE MEDIATES RENAL FIBROSIS THROUGH ENDOGENOUS CANNABINOID SYSTEM

Chronic kidney disease (CKD) has become one of the most severe diseases that threats human life. The rate of adult chronic kidney disease in China is as high as 10.8%, and death rate is ranked firstly among chronic diseases. Kidney fibrosis is the endstage of CKD. However, there is currently no effective treatment for CKD to reverse the development of renal fibrosis. Researchers are committed to explore the specific mechanisms of kidney fibrosis. Several studies have proved that the endogenous cannabinoid system (ECS) plays an important role in kidney fibrosis and senescence.

A novel role of BK potassium channel activity in preventing the development of kidney fibrosis

Kidney fibrosis is a common characteristic of chronic kidney disease and while the large conductance voltage and calcium-activated potassium channel (BK) is widely expressed in kidneys, its role in kidney fibrosis is unknown. To evaluate this, we found that BK protein expression was decreased in the fibrotic kidneys. Accompanying this was increased fibrotic marker protein expression of fibronectin, vimentin and α-smooth muscle actin and increased mRNA expressions of fibronectin, α-smooth muscle actin, collagen III and collagen I. These changes occurred in the unilateral ureteral obstruction and folic acid models of fibrosis and were more pronounced in BK knockout than in wild-type mice. Activation of BK activity by chemical NS1619 or BMS191011 channel openers attenuated kidney fibrosis in these two models while protecting kidney function in wild-type mice. BK deficiency up-regulated transforming growth factor-β (TGF-β)/transcription factor Smad2/3 signaling in the fibrotic kidney, whereas activation of BK activity inhibited this signaling pathway both invivo and invitro. BK channel activation increased the degradation of TGF-β receptors induced by TGF-β1 invivo and invitro. Furthermore, in cell lines HK-2, NRK49, and NRK-52E, BK channel activation by NS1619 led to increased caveolae formation and facilitated localization of TGF-β receptors in the microdomains of lipid rafts. Thus, our data demonstrated that BK activation has an anti-fibrotic effect on kidney fibrosis by inhibiting the TGF-β signaling pathway through accelerating TGF-β receptor degradation via the caveolae route. Hence, our study provides innovative insight into BK as a potential therapeutic target for the treatment of kidney fibrosis.

PP2Ac\u03b1 promotes macrophage accumulation and activation to exacerbate tubular cell death and kidney fibrosis through activating Rap1 and TNF\u03b1 production

Macrophage accumulation and activation play an essential role in kidney fibrosis; however, the underlying mechanisms remain to be explored. By analyzing the kidney tissues from patients and animal models with kidney fibrosis, we found a large induction of PP2Acα in macrophages. We then generated a mouse model with inducible macrophage ablation of PP2Acα. The knockouts developed less renal fibrosis, macrophage accumulation, or tubular cell death after unilateral ureter obstruction or ischemic reperfusion injury compared to control littermates. In cultured macrophages, PP2Acα deficiency resulted in decreased cell motility by inhibiting Rap1 activity. Moreover, co-culture of PP2Acα−/− macrophages with tubular cells resulted in less tubular cell death attributed to downregulated Stat6-mediated tumor necrosis factor α (TNFα) production in macrophages. Together, this study demonstrates that PP2Acα promotes macrophage accumulation and activation, hence accelerates tubular cell death and kidney fibrosis through regulating Rap1 activation and TNFα production.