Alterations in Gut Microbiota of Patients With COVID-19 During Time of Hospitalization.

BACKGROUND & AIMS: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects gastrointestinal tissues, little is known about the roles of gut commensal microbes in susceptibility to and severity of infection. We investigated changes in fecal microbiomes of patients with SARS-CoV-2 infection during hospitalization and associations with severity and fecal shedding of virus. METHODS: We performed shotgun metagenomic sequencing analyses of fecal samples from 15 patients with Coronavirus Disease 2019 (COVID-19) in Hong Kong, from February 5 through March 17, 2020. Fecal samples were collected 2 or 3 times per week from time of hospitalization until discharge; disease was categorized as mild (no radiographic evidence of pneumonia), moderate (pneumonia was present), severe (respiratory rate ≥30/min, or oxygen saturation ≤93% when breathing ambient air), or critical (respiratory failure requiring mechanical ventilation, shock, or organ failure requiring intensive care). We compared microbiome data with those from 6 subjects with community-acquired pneumonia and 15 healthy individuals (controls). We assessed gut microbiome profiles in association with disease severity and changes in fecal shedding of SARS-CoV-2. RESULTS: Patients with COVID-19 had significant alterations in fecal microbiomes compared with controls, characterized by enrichment of opportunistic pathogens and depletion of beneficial commensals, at time of hospitalization and at all timepoints during hospitalization. Depleted symbionts and gut dysbiosis persisted even after clearance of SARS-CoV-2 (determined from throat swabs) and resolution of respiratory symptoms. The baseline abundance of Coprobacillus, Clostridium ramosum, and Clostridium hathewayi correlated with COVID-19 severity; there was an inverse correlation between abundance of Faecalibacterium prausnitzii (an anti-inflammatory bacterium) and disease severity. Over the course of hospitalization, Bacteroides dorei, Bacteroides thetaiotaomicron, Bacteroides massiliensis, and Bacteroides ovatus, which downregulate expression of angiotensin-converting enzyme 2 (ACE2) in murine gut, correlated inversely with SARS-CoV-2 load in fecal samples from patients. CONCLUSIONS: In a pilot study of 15 patients with COVID-19, we found persistent alterations in the fecal microbiome during the time of hospitalization, compared with controls. Fecal microbiota alterations were associated with fecal levels of SARS-CoV-2 and COVID-19 severity. Strategies to alter the intestinal microbiota might reduce disease severity.

Persistent Organic Pollutants as Risk Factors for Obesity and Diabetes.

PURPOSE OF REVIEW: The rising prevalence of obesity and diabetes cannot be fully explained by known risk factors, such as unhealthy diet, a sedentary lifestyle, and family history. This review summarizes the available studies linking persistent organic pollutants (POPs) to obesity and diabetes and discusses plausible underlying mechanisms. RECENT FINDINGS: Increasing evidence suggest that POPs may act as obesogens and diabetogens to promote the development of obesity and diabetes and induce metabolic dysfunction. POPs are synthesized chemicals and are used widely in our daily life. These chemicals are resistant to degradation in chemical or biological processes, which enable them to exist in the environment persistently and to be bio-accumulated in animal and human tissue through the food chain. Increasingly, epidemiologic studies suggest a positive association between POPs and risk of developing diabetes. Understanding the relationship of POPs with obesity and diabetes may shed light on preventive strategies for obesity and diabetes.

Identification of novel long noncoding RNAs associated with TGF-ß/Smad3-mediated renal inflammation and fibrosis by RNA sequencing.

We have previously shown that transforming growth factor-ß/Smad3-dependent miRNAs play a critical role in renal inflammation and fibrosis. However, off-target effects of miRNAs limit their therapeutic application. Recently, emerging roles of long noncoding RNAs (lncRNAs) in diseases have been recognized. In this study, we used high-throughput RNA sequencing to identify the Smad3-dependent lncRNAs related to renal inflammation and fibrosis in Smad3 knockout mouse models of unilateral ureteral obstructive nephropathy and immunologically induced anti-glomerular basement membrane glomerulonephritis. Compared with wild-type mice, 151 lncRNAs in the unilateral ureteral obstructive nephropathy kidney and 413 lncRNAs in kidneys with anti-glomerular basement membrane glomerulonephritis were significantly altered in Smad3 knockout mice. Among them, 21 common lncRNAs were up-regulated in wild-type, but down-regulated in Smad3 knockout, kidneys in both disease models in which progressive renal inflammation and fibrosis were abolished when the Smad3 gene was deleted or suppressed. Real-time PCR confirmed these findings and revealed the functional link between Smad3-dependent lncRNAs np_5318/np_17856 and progressive kidney injury. Results demonstrate that the identification and characterization of functional lncRNAs associated with kidney disease may represent a promising research direction into renal disorder and may lead to the development of new lncRNA therapies for kidney diseases.

microRNAs in Diabetic Kidney Disease.

Diabetes and diabetic kidney diseases have continually exerted a great burden on our society. Although the recent advances in medical research have led to a much better understanding of diabetic kidney diseases, there is still no successful strategy for effective treatments for diabetic kidney diseases. Recently, treatment of diabetic kidney diseases relies either on drugs that reduce the progression of renal injury or on renal replacement therapies, such as dialysis and kidney transplantation. On the other hand, searching for biomarkers for early diagnosis and effective therapy is also urgent. Discovery of microRNAs has opened to a novel field for posttranscriptional regulation of gene expression. Results from cell culture experiments, experimental animal models, and patients under diabetic conditions reveal the critical role of microRNAs during the progression of diabetic kidney diseases. Functional studies demonstrate not only the capability of microRNAs to regulate expression of target genes, but also their therapeutic potential to diabetic kidney diseases. The existence of microRNAs in plasma, serum, and urine suggests their possibility to be biomarkers in diabetic kidney diseases. Thus, identification of the functional role of microRNAs provides an essentially clinical impact in terms of prevention and treatment of progression in diabetic kidney diseases as it enables us to develop novel, specific therapies and diagnostic tools for diabetic kidney diseases.

Smad7 suppresses renal fibrosis via altering expression of TGF-ß/Smad3-regulated microRNAs.

Blockade of transforming growth factor-ß (TGF-ß) signaling by Smad7 gene therapy is known to prevent experimental renal fibrosis. This study investigated whether Smad7 suppresses renal fibrosis via altering the renal expression of fibrosis-related microRNAs. Application of gene therapy into diseased kidneys of obstructive nephropathy and kidney cells by overexpressing Smad7 restored miR-29b but inhibited the expression of miR-192 and miR-21, resulting in blockade of renal fibrosis. Furthermore, Smad7 overexpression also suppressed advanced glycated end products- and angiotensin II-regulated expression of these microRNAs. In contrast, disruption of Smad7 gene in mice demonstrated opposite results by enhancing the loss of miR-29b and upregulation of miR-192 and miR-21, resulting in promotion of renal fibrosis in ligated kidneys of a model of obstructive nephropathy. More importantly, treatment with anti-miR-29b, miR-21 and miR-192 mimics in Smad7 overexpressing tubular epithelial cells abrogated the suppressive function of Smad7 on renal fibrosis, suggesting that these microRNAs act downstream of Smad7 to override the Smad7 function. In conclusion, Smad7 protects kidneys from fibrosis by regulating TGF-ß/Smad3-mediated renal expression of miR-21, miR-192, and miR-29b. Restored renal miR-29b but suppressed miR-192 and miR-21 may be a mechanism by which gene therapy with Smad7 inhibits renal fibrosis.

The microRNA miR-433 promotes renal fibrosis by amplifying the TGF-ß/Smad3-Azin1 pathway.

The TGF-ß/Smad3 pathway plays a major role in tissue fibrosis, but the precise mechanisms are not fully understood. Here we identified microRNA miR-433 as an important component of TGF-ß/Smad3-driven renal fibrosis. The miR-433 was upregulated following unilateral ureteral obstruction, a model of aggressive renal fibrosis. In vitro, overexpression of miR-433 enhanced TGF-ß1-induced fibrosis, whereas knockdown of miR-433 suppressed this response. Furthermore, Smad3, but not Smad2, bound to the miR-433 promoter to induce its expression. Delivery of an miR-433 knockdown plasmid to the kidney by ultrasound microbubble-mediated gene transfer suppressed the induction and progression of fibrosis in the obstruction model. The antizyme inhibitor Azin1, an important regulator of polyamine synthesis, was identified as a target of miR-433. Overexpression of miR-433 suppressed Azin1 expression, while, in turn, Azin1 overexpression suppressed TGF-ß signaling and the fibrotic response. Thus, miR-433 is an important component of TGF-ß/Smad3-induced renal fibrosis through the induction of a positive feedback loop to amplify TGF-ß/Smad3 signaling, and may be a potential therapeutic target in tissue fibrosis.

Role of the TGF-ß/BMP-7/Smad pathways in renal diseases.

TGF-ß (transforming growth factor-ß) and BMP-7 (bone morphogenetic protein-7), two key members in the TGF-ß superfamily, play important but diverse roles in CKDs (chronic kidney diseases). Both TGF-ß and BMP-7 share similar downstream Smad signalling pathways, but counter-regulate each other to maintain the balance of their biological activities. During renal injury in CKDs, this balance is significantly altered because TGF-ß signalling is up-regulated by inducing TGF-ß1 and activating Smad3, whereas BMP-7 and its downstream Smad1/5/8 are down-regulated. In the context of renal fibrosis, Smad3 is pathogenic, whereas Smad2 and Smad7 are renoprotective. However, this counter-balancing mechanism is also altered because TGF-ß1 induces Smurf2, a ubiquitin E3-ligase, to target Smad7 as well as Smad2 for degradation. Thus overexpression of renal Smad7 restores the balance of TGF-ß/Smad signalling and has therapeutic effect on CKDs. Recent studies also found that Smad3 mediated renal fibrosis by up-regulating miR-21 (where miR represents microRNA) and miR-192, but down-regulating miR-29 and miR-200 families. Therefore restoring miR-29/miR-200 or suppressing miR-21/miR-192 is able to treat progressive renal fibrosis. Furthermore, activation of TGF-ß/Smad signalling inhibits renal BMP-7 expression and BMP/Smad signalling. On the other hand, overexpression of renal BMP-7 is capable of inhibiting TGF-ß/Smad3 signalling and protects the kidney from TGF-ß-mediated renal injury. This counter-regulation not only expands our understanding of the causes of renal injury, but also suggests the therapeutic potential by targeting TGF-ß/Smad signalling or restoring BMP-7 in CKDs. Taken together, the current understanding of the distinct roles and mechanisms of TGF-ß and BMP-7 in CKDs implies that targeting the TGF-ß/Smad pathway or restoring BMP-7 signalling may represent novel and effective therapies for CKDs.

Diverse roles of TGF-ß receptor II in renal fibrosis and inflammation in vivo and in vitro.

TGF-ß1 binds receptor II (TßRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TßRII may function to initiate the downstream TGF-ß signalling and determine the diverse role of TGF-ß1 in kidney injury. The hypothesis was examined in a model of unilateral ureteral obstructive (UUO) nephropathy and in kidney fibroblasts and tubular epithelial cells in which the TßRII was deleted conditionally. We found that disruption of TßRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF-ß/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TßRII enhanced NF-κB signalling and renal inflammation including up-regulation of Il-1ß and Tnfα in the UUO kidney. Similarly, in vitro disruption of TßRII from kidney fibroblasts or tubular epithelial cells inhibited TGF-ß1-induced Smad signalling and fibrosis but impaired the anti-inflammatory effect of TGF-ß1 on IL-1ß-stimulated NF-κB activation and pro-inflammatory cytokine expression. In conclusion, TßRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF-ß/Smad3, but not the non-canonical TGF-ß signalling pathway, may be a key mechanism by which disruption of TßRII protects against renal fibrosis. In addition, deletion of TßRII also enhances NF-κB signalling along with up-regulation of renal pro-inflammatory cytokines, which may be associated with the impairment of anti-inflammatory properties of TGF-ß1.

Disruption of Smad4 impairs TGF-ß/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro.

The mechanism by which TGF-ß regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-ß signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1ß, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1ß-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-ß1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-ß1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

Differential recruitment of methyl CpG-binding domain factors and DNA methyltransferases by the orphan receptor germ cell nuclear factor initiates the repression and silencing of Oct4.

The pluripotency gene Oct4 encodes a key transcription factor that maintains self-renewal of embryonic stem cell (ESC) and is downregulated upon differentiation of ESCs and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, mediates Oct4 gene expression. Here, we show that the orphan nuclear receptor germ cell nuclear factor (GCNF) initiates Oct4 repression and DNA methylation by the differential recruitment of methyl-CpG binding domain (MBD) and DNA methyltransferases (Dnmts) to the Oct4 promoter. When compared with wild-type ESCs and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in retinoic acid (RA)-differentiated GCNF(-/-) ESCs and GCNF(-/-) embryos. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified MBD3, MBD2, and de novo Dnmts as GCNF interacting factors. Upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2 as well as Dnmt3A. In differentiated GCNF(-/-) ESCs, recruitment of MBD3 and MBD2 as well as Dnmt3A to Oct4 promoter is lost and subsequently Oct4 repression and DNA methylation failed to occur. Hypomethylation of the Oct4 promoter is also observed in RA-differentiated MBD3(-/-) and Dnmt3A(-/-) ESCs, but not in MBD2(-/-) and Dnmt3B(-/-) ESCs. Thus, recruitment of MBD3, MBD2, and Dnmt3A by GCNF links two events: gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ESC differentiation.

Chemokines in renal injury.

The main function of chemokines is to guide inflammatory cells in their migration to sites of inflammation. During the last 2 decades, an expanding number of chemokines and their receptors have driven broad inquiry into how inflammatory cells are recruited in a variety of diseases. Although this review focuses on chemokines and their receptors in renal injury, proinflammatory IL-17, TGFß, and TWEAK signaling pathways also play a critical role in their expression. Recent studies in transgenic mice as well as blockade of chemokine signaling by neutralizing ligands or receptor antagonists now allow direct interrogation of chemokine action. The emerging role of regulatory T cells and Th17 cells during renal injury also forges tight relationships between chemokines and T cell infiltration in the development of kidney disease. As chemokine receptor blockade inches toward clinical use, the field remains an attractive area with potential for unexpected opportunity in the future.

Smad3-mediated upregulation of miR-21 promotes renal fibrosis.

TGF-ß/Smad signaling plays a role in fibrogenesis, but therapies targeting TGF-ß are ineffective in treating renal fibrosis. Here, we explored the therapeutic potential of targeting TGF-ß-induced microRNA in the progression of renal fibrosis. Microarray analysis and real-time PCR revealed upregulation of miR-21 in tubular epithelial cells (TECs) in response to TGF-ß. Lack of Smad3, but not lack of Smad2, prevented cells from upregulating miR-21 in response to TGF-ß. In addition, Smad3-deficient mice were protected from upregulation of miR-21 and fibrosis in the unilateral ureteral obstruction model. In contrast, conditional knockout of Smad2 enhanced miR-21 expression and renal fibrosis. Furthermore, ultrasound-microbubble-mediated gene transfer of a miR-21-knockdown plasmid halted the progression of renal fibrosis in established obstructive nephropathy. In conclusion, these data demonstrate that Smad3, but not Smad2, signaling increases expression of miR-21, which promotes renal fibrosis. Inhibition of miR-21 may be a therapeutic approach to suppress renal fibrosis.

TGF-ß/Smad3 signaling promotes renal fibrosis by inhibiting miR-29.

TGF-ß/Smad3 signaling promotes fibrosis, but the development of therapeutic interventions involving this pathway will require the identification and ultimate targeting of downstream fibrosis-specific genes. In this study, using a microRNA microarray and real-time PCR, wild-type mice had reduced expression of miR-29 along with the development of progressive renal fibrosis in obstructive nephropathy. In contrast, Smad3 knockout mice had increased expression of miR-29 along with the absence of renal fibrosis in the same model of obstruction. In cultured fibroblasts and tubular epithelial cells, Smad3 mediated TGF-ß(1)-induced downregulation of miR-29 by binding to the promoter of miR-29. Furthermore, miR-29 acted as a downstream inhibitor and therapeutic microRNA for TGF-ß/Smad3-mediated fibrosis. In vitro, overexpression of miR-29b inhibited, but knockdown of miR-29 enhanced, TGF-ß(1)-induced expression of collagens I and III by renal tubular cells. Ultrasound-mediated gene delivery of miR-29b either before or after established obstructive nephropathy blocked progressive renal fibrosis. In conclusion, miR-29 is a downstream inhibitor of TGF-ß/Smad3-mediated fibrosis and may have therapeutic potential for diseases involving fibrosis.

Autotaxin signaling facilitates ß cell dedifferentiation and dysfunction induced by Sirtuin 3 deficiency.

OBJECTIVE: ß cell dedifferentiation may underlie the reversible reduction in pancreatic ß cell mass and function in type 2 diabetes (T2D). We previously reported that ß cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic ß cell dedifferentiation in Sirt3-deficient ß cells. METHODS: We applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on ß cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the ß cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. RESULTS: In Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by ß cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced ß cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. CONCLUSIONS: Sirt3 prevents ß cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse ß cell dysfunction associated with glucolipotoxicity.

C-reactive protein promotes acute renal inflammation and fibrosis in unilateral ureteral obstructive nephropathy in mice.

Elevated blood level of C-reactive protein (CRP) is associated with increased risk of chronic kidney disease. However, whether this association reflects functional importance of CRP in the pathogenesis of kidney disease remains unclear. In this study, we examined the biological role of CRP in a well-characterized model of progressive kidney disease, unilateral ureteral obstruction (UUO), in mice that express the human CRP gene (CRPtg). Compared with wild-type (Wt) mice at 3 days after UUO, CRPtg mice developed more severe renal inflammation with a significant increase in tubulointerstitial T cells and macrophages, upregulation of proinflammatory cytokines (IL-1ß and TNF-α), chemokines (MCP-1), and adhesion molecules (ICAM-1). Renal fibrosis was also significantly enhanced in CRPtg mice as demonstrated by increased expression of tubulointerstitial α-smooth muscle actin and collagen types I and III compared with Wt mice. Interestingly, on days 7 and 14 after UUO, an equal severity of renal inflammation and fibrosis were observed in CRPtg and Wt mice. These findings suggested that CRP may have a role in the initiation of renal inflammation and fibrosis. Further study revealed that enhanced early renal inflammation and fibrosis on day 3 in CRPtg mice was associated with a significant upregulation of endogenous mouse CRP and FcγRI mRNA and increased activation of both NF-κB/p65 and TGF-ß/Smad2/3 signaling, while equal severity of progressive renal injury at day 7 and day 14 between CRPtg and Wt mice were attributed to equivalent levels of CRP, FcγRI, phospho-NF-κB/p65, and TGF-ß/Smad2/3 signaling. Based on these findings, we conclude that CRP may not only be a biomarker, but also a mediator in the early development of renal inflammation and fibrosis in a mouse model of UUO. Enhanced activation of both NF-κB and TGF-ß/Smad signaling pathways may be mechanisms by which CRP promotes early renal inflammation and fibrosis.

Essential role for Smad3 in angiotensin II-induced tubular epithelial-mesenchymal transition.

Angiotensin II (Ang II) is a key mediator of chronic kidney disease, in which epithelial-mesenchymal transition (EMT) is a critical process mediated by the TGFbeta/Smad signalling pathway. The present study examined the specific role of Smads in Ang II-induced EMT in vitro and in vivo. We found that Ang II signalled through the receptor of AT1, not AT2, to activate Smad2/3 and induce EMT in a normal rat tubular epithelial cell line (NRK52E). Activation of Smads by Ang II was attributed to degradation of an inhibitory Smad7, which was mediated by the AT1-Smurf2-dependent ubiquitin degradation mechanism because blockade of AT1 receptor or knockdown of Smurf2 inhibited Smad7 loss, thereby reducing Smad2/3 activation and EMT in response to Ang II. In contrast, over-expression of Smad7 inhibited Ang II-induced Smad2/3 activation and EMT in NRK52E cells and in a rat model of remnant kidney disease. Moreover, knockdown of Smad3, not Smad2, attenuated Ang II-induced EMT. In conclusion, Ang II activates Smad signalling to induce EMT, which is mediated by a loss of Smad7 through the AT1-Smurf2-dependent ubiquitin degradation pathway. Smad3, but not Smad2, may be a mediator of EMT, while Smad7 may play a protective role in EMT in response to Ang II.

miR-192 mediates TGF-beta/Smad3-driven renal fibrosis.

TGF-beta/Smad3 promotes renal fibrosis, but the mechanisms that regulate profibrotic genes remain unclear. We hypothesized that miR-192, a microRNA expressed in the kidney may mediate renal fibrosis in a Smad3-dependent manner. Microarray and real-time PCR demonstrated a tight association between upregulation of miR-192 in the fibrotic kidney and activation of TGF-beta/Smad signaling. Deletion of Smad7 promoted miR-192 expression and enhanced Smad signaling and fibrosis in obstructive kidney disease. In contrast, overexpression of Smad7 to block TGF-beta/Smad signaling inhibited miR-192 expression and renal fibrosis in the rat 5/6 nephrectomy model; in vitro, overexpression of Smad7 in tubular epithelial cells abolished TGF-beta1-induced miR-192 expression. Furthermore, Smad3 but not Smad2 mediated TGF-beta1-induced miR-192 expression by binding to the miR-192 promoter. Last, overexpression of a miR-192 mimic promoted and addition of a miR-192 inhibitor blocked TGF-beta1-induced collagen matrix expression. Taken together, miR-192 may be a critical downstream mediator of TGF-beta/Smad3 signaling in the development of renal fibrosis.

Advanced glycation end-products induce tubular CTGF via TGF-beta-independent Smad3 signaling.

Advanced glycation end-products (AGEs) can induce expression of connective tissue growth factor (CTGF), which seems to promote the development of diabetic nephropathy, but the exact signaling mechanisms that mediate this induction are unknown. Here, AGEs induced CTGF expression in tubular epithelial cells (TECs) that either lacked the TGF-beta1 gene or expressed dominant TGF-beta receptor II, demonstrating independence of TGF-beta. Furthermore, conditional knockout of the gene encoding TGF-beta receptor II from the kidney did not prevent AGE-induced renal expression of CTGF and collagen I. More specific, AGEs induced CTGF expression via the receptor for AGEs-extracellular signal-regulated kinase (RAGE-ERK)/p38 mitogen-activated protein kinase-Smad cross-talk pathway because inhibition of this pathway by several methods (anti-RAGE antibody, specific inhibitors, or dominant negative adenovirus to ERK1/2 and p38) blocked this induction. Overexpressing Smad7 abolished AGE-induced Smad3 phosphorylation and CTGF expression, demonstrating the necessity for activation of Smad signaling in this process. More important, knockdown of either Smad3 or Smad2 demonstrated that Smad3 but not Smad2 is essential for CTGF induction in response to AGEs. In conclusion, AGEs induce tubular CTGF expression via the TGF-beta-independent RAGE-ERK/p38-Smad3 cross-talk pathway. These data suggest that overexpression of Smad7 or targeting Smad3 may have therapeutic potential for diabetic nephropathy.

Smad2 protects against TGF-beta/Smad3-mediated renal fibrosis.

Smad2 and Smad3 interact and mediate TGF-beta signaling. Although Smad3 promotes fibrosis, the role of Smad2 in fibrogenesis is largely unknown. In this study, conditional deletion of Smad2 from the kidney tubular epithelial cells markedly enhanced fibrosis in response to unilateral ureteral obstruction. In vitro, Smad2 knockdown in tubular epithelial cells increased expression of collagen I, collagen III, and TIMP-1 and decreased expression of the matrix-degrading enzyme MMP-2 in response to TGF-beta1 compared with similarly treated wild-type cells. We obtained similar results in Smad2-knockout fibroblasts. Mechanistically, Smad2 deletion promoted fibrosis through enhanced TGF-beta/Smad3 signaling, evidenced by greater Smad3 phosphorylation, nuclear translocation, promoter activity, and binding of Smad3 to a collagen promoter (COL1A2). Moreover, deletion of Smad2 increased autoinduction of TGF-beta1. Conversely, overexpression of Smad2 attenuated TGF-beta1-induced Smad3 phosphorylation and collagen I matrix expression in tubular epithelial cells. In conclusion, in contrast to Smad3, Smad2 protects against TGF-beta-mediated fibrosis by counteracting TGF-beta/Smad3 signaling.

Pancreatic Sirtuin 3 Deficiency Promotes Hepatic Steatosis by Enhancing 5-Hydroxytryptamine Synthesis in Mice With Diet-Induced Obesity.

Sirtuin 3 (SIRT3) is a protein deacetylase regulating ß-cell function through inhibiting oxidative stress in obese and diabetic mice, but the detailed mechanism and potential effect of ß-cell-specific SIRT3 on metabolic homeostasis, and its potential effect on other metabolic organs, are unknown. We found that glucose tolerance and glucose-stimulated insulin secretion were impaired in high-fat diet (HFD)-fed ß-cell-selective Sirt3 knockout (Sirt3 f/f;Cre/+) mice. In addition, Sirt3 f/f;Cre/+ mice had more severe hepatic steatosis than Sirt3 f/f mice upon HFD feeding. RNA sequencing of islets suggested that Sirt3 deficiency overactivated 5-hydroxytryptamine (5-HT) synthesis as evidenced by upregulation of tryptophan hydroxylase 1 (TPH1). 5-HT concentration was increased in both islets and serum of Sirt3 f/f;Cre/+ mice. 5-HT also facilitated the effect of palmitate to increase lipid deposition. Treatment with TPH1 inhibitor ameliorated hepatic steatosis and reduced weight gain in HFD-fed Sirt3 f/f;Cre/+ mice. These data suggested that under HFD feeding, SIRT3 deficiency in ß-cells not only regulates insulin secretion but also modulates hepatic lipid metabolism via the release of 5-HT.