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Clopidogrel, a P2Y12 inhibitor, is a novel anti-fibrosis agent for chronic kidney disease (CKD), but its mechanisms remain unclear, which we investigated by silencing P2Y12 or treating unilateral ureteral obstruction (UUO) in LysM-Cre/Rosa Tomato mice with clopidogrel in vivo and in vitro. We found that P2Y12 was significantly increased and correlated with progressive renal fibrosis in CKD patients and UUO mice. Phenotypically, up to 82% of P2Y12-expressing cells within the fibrosing kidney were of macrophage origin, identified by co-expressing CD68/F4/80 antigens or a macrophage-lineage-tracing marker Tomato. Unexpectedly, more than 90% of P2Y12-expressing macrophages were undergoing macrophage-to-myofibroblast transition (MMT) by co-expressing alpha smooth muscle actin (α-SMA), which was also confirmed by single-cell RNA sequencing. Functionally, clopidogrel improved the decline rate of the estimated glomerular filtration rate (eGFR) in patients with CKD and significantly inhibited renal fibrosis in UUO mice. Mechanistically, P2Y12 expression was induced by transforming growth factor β1 (TGF-β1) and promoted MMT via the Smad3-dependent mechanism. Thus, silencing or pharmacological inhibition of P2Y12 was capable of inhibiting TGF-β/Smad3-mediated MMT and progressive renal fibrosis in vivo and in vitro. In conclusion, P2Y12 is highly expressed by macrophages in fibrosing kidneys and mediates renal fibrosis by promoting MMT via TGF-β/Smad3 signaling. Thus, P2Y12 inhibitor maybe a novel and effective anti-fibrosis agent for CKD.

Rationale: Poor β cell proliferation is one of the detrimental factors hindering islet cell replacement therapy for patients with diabetes. Smad3 is an important transcriptional factor of TGF-β signaling and has been shown to promote diabetes by inhibiting β cell proliferation. Therefore, we hypothesize that Smad3-deficient islets may be a novel cell replacement therapy for diabetes. Methods: We examined this hypothesis in streptozocin-induced type-1 diabetic mice and type-2 diabetic db/db mice by transplanting Smad3 knockout (KO) and wild type (WT) islets under the renal capsule, respectively. The effects of Smad3KO versus WT islet replacement therapy on diabetes and diabetic kidney injury were examined. In addition, RNA-seq was applied to identify the downstream target gene underlying Smad3-regulated β cell proliferation in Smad3KO-db/db versus Smad3WT-db/db mouse islets. Results: Compared to Smad3WT islet therapy, treatment with Smad3KO islets produced a much better therapeutic effect on both type-1 and type-2 diabetes by significantly lowering serum levels of blood glucose and HbA1c and protected against diabetic kidney injuries by preventing an increase in serum creatinine and the development of proteinuria, mesangial matrix expansion, and fibrosis. These were associated with a significant increase in grafted β cell proliferation and blood insulin levels, resulting in improved glucose intolerance. Mechanistically, RNA-seq revealed that compared with Smad3WT-db/db mouse islets, deletion of Smad3 from db/db mouse islets markedly upregulated E2F3, a pivotal regulator of cell cycle G1/S entry. Further studies found that Smad3 could bind to the promoter of E2F3, and thus inhibit β cell proliferation via an E2F3-dependent mechanism as silencing E2F3 abrogated the proliferative effect on Smad3KO β cells. Conclusion: Smad3-deficient islet replacement therapy can significantly improve both type-1 and type-2 diabetes and protect against diabetic kidney injury, which is mediated by a novel mechanism of E2F3-dependent β cell proliferation.

Tumor innervation is a common phenomenon with unknown mechanism. Here, we discovered a direct mechanism of tumor-associated macrophage (TAM) for promoting de novo neurogenesis via a subset showing neuronal phenotypes and pain receptor expression associated with cancer-driven nocifensive behaviors. This subset is rich in lung adenocarcinoma associated with poorer prognosis. By elucidating the transcriptome dynamics of TAM with single-cell resolution, we discovered a phenomenon “macrophage to neuron-like cell transition” (MNT) for directly promoting tumoral neurogenesis, evidenced by macrophage depletion and fate-mapping study in lung carcinoma models. Encouragingly, we detected neuronal phenotypes and activities of the bone marrow–derived MNT cells (MNTs) in vitro. Adoptive transfer of MNTs into NOD/SCID mice markedly enhanced their cancer-associated nocifensive behaviors. We identified macrophage-specific Smad3 as a pivotal regulator for promoting MNT at the genomic level; its disruption effectively blocked the tumor innervation and cancer-dependent nocifensive behaviors in vivo. Thus, MNT may represent a precision therapeutic target for cancer pain.

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism. Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E). Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80+ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling. Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, but treatment remains ineffective. C-reactive protein (CRP) is pathogenic in DN, which significantly correlated with dipeptidyl peptidase-4 (DPP4) expression in diabetic patients with unknown reason. Here, using our unique CRP(tg)-db/db mice, we observed human CRP markedly induced renal DPP4 associated with enhanced kidney injury compared with db/db mice. Interestingly, linagliptin, a US Food and Drug Administration (FDA)-approved specific DPP4 inhibitor, effectively blocked this CRP-driven DN in the CRP(tg)-db/db mice. Mechanistically, CRP evoked DPP4 in cultured renal tubular epithelial cells, where CD32b/nuclear factor κB (NF-κB) signaling markedly enriched p65 binding on the DPP4 promoter region to increase its transcription. Unexpectedly, we further discovered that CRP triggers dimerization of DPP4 with CD32b at protein level, forming a novel DPP4/CD32b/NF-κB signaling circuit for promoting CRP-mediated DN. More importantly, linagliptin effectively blocked the circuit, thereby inhibiting the CRP/CD32b/NF-κB-driven renal inflammation and fibrosis. Thus, DPP4 may represent a precise druggable target for CRP-driven DN.

Transforming growth factor β (TGF-β) is a well-known key mediator for the progression and metastasis of lung carcinoma. However, cost-effective anti-TGF-β therapeutics for lung cancer remain to be explored. Specifically, the low efficacy in drug delivery greatly limits the clinical application of small molecular inhibitors of TGF-β. In the present study, specific inhibitor of Smad3 (SIS3) is developed into a self-carried nanodrug (SCND-SIS3) using the reprecipitation method, which largely improves its solubility and bioavailability while reduces its nephrotoxicity. Compared to unmodified-SIS3, SCND-SIS3 demonstrates better anti-cancer effects through inducing tumor cell apoptosis, inhibiting angiogenesis, and boosting NK cell-mediated immune responses in syngeneic Lewis Lung Cancer (LLC) mouse model. Better still, it could achieve comparable anti-cancer effect with just one-fifth the dose of unmodified-SIS3. Mechanistically, RNA-sequencing analysis and cytokine array results unveil a TGF-β/Smad3-dependent immunoregulatory landscape in NK cells. In particular, SCND-SIS3 promotes NK cell cytotoxicity by ameliorating Smad3-mediated transcriptional inhibition of Ndrg1. Furthermore, improved NK cell cytotoxicity by SCND-SIS3 is associated with higher expression of activation receptor Nkp46, and suppressed levels of Trib3 and TSP1 as compared with unmodified-SIS3. Taken together, SCND-SIS3 possesses superior anti-cancer effects with enhanced bioavailability and biocompatibility, therefore representing as a novel therapeutic strategy for lung carcinoma with promising clinical potential.

Smad3 deficiency prevents the development of type 2 diabetic nephropathy; however, the underlying molecular mechanisms remain unknown. In this study, we aimed to identify Smad3‐related genes involved in the pathogenesis of diabetic kidney disease. High‐throughput RNA sequencing was performed to profile the whole transcriptome in the diabetic kidney of Smad3 WT‐db/db, Smad3 KO‐db/db, Smad3+/− db/db and their littermate control db/m mice at 20 weeks. The gene ontology, pathways and alternative splicing of differentially expressed protein‐coding genes and long non‐coding RNAs related to Smad3 in diabetic kidney were analysed. Compared to Smad3 WT‐db/db mice, Smad3 KO‐db/db mice exhibited an alteration of genes associated with RNA splicing and metabolism, whereas heterozygosity deletion of Smad3 (Smad3+/− db/db mice) significantly altered genes related to cell division and cell cycle. Notably, three protein‐coding genes (Upk1b, Psca and Gdf15) and two lncRNAs (NONMMUG023520.2 and NONMMUG032975.2) were identified to be Smad3‐dependent and to be associated with the development of diabetic nephropathy. By using whole transcriptome RNA sequencing, we identified novel Smad3 transcripts related to the development of diabetic nephropathy. Thus, targeting these transcripts may represent a novel and effective therapy for diabetic nephropathy.

Angiotensin‐converting enzyme‐2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1‐7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II‐induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild‐type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7‐28 days following a chronic Ang II infusion (P

Diabetic nephropathy (DN) is a major cause of end-stage kidney disease, where TGF-β1/Smad signaling plays an important role in the disease progression. Our previous studies demonstrated a combination of Traditional Chinese Medicine derived Smad7 agonist Asiatic Acid (AA) and Smad3 inhibitor Naringenin (NG), AANG, effectively suppressed the progression of renal fibrosis

Cancer‐associated fibroblasts (CAFs) are important in tumor microenvironment (TME) driven cancer progression. However, CAFs are heterogeneous and still largely underdefined, better understanding their origins will identify new therapeutic strategies for cancer. Here, the authors discovered a new role of macrophage‐myofibroblast transition (MMT) in cancer for de novo generating protumoral CAFs by resolving the transcriptome dynamics of tumor‐associated macrophages (TAM) with single‐cell resolution. MMT cells (MMTs) are observed in non‐small‐cell lung carcinoma (NSCLC) associated with CAF abundance and patient mortality. By fate‐mapping study, RNA velocity, and pseudotime analysis, existence of novel macrophage‐lineage‐derived CAF subset in the TME of Lewis lung carcinoma (LLC) model is confirmed, which is directly transited via MMT from M2‐TAM in vivo and bone‐marrow‐derived macrophages (BMDM) in vitro. Adoptive transfer of BMDM‐derived MMTs markedly promote CAF formation in LLC‐bearing mice. Mechanistically, a Smad3‐centric regulatory network is upregulated in the MMTs of NSCLC, where chromatin immunoprecipitation sequencing(ChIP‐seq) detects a significant enrichment of Smad3 binding on fibroblast differentiation genes in the macrophage‐lineage cells in LLC‐tumor. More importantly, macrophage‐specific deletion and pharmaceutical inhibition of Smad3 effectively block MMT, therefore, suppressing the CAF formation and cancer progression in vivo. Thus, MMT may represent a novel therapeutic target of CAF for cancer immunotherapy., Here, the authors discovered a direct mechanism of tumor‐associated macrophages for de novo generating protumoral cancer‐associated fibroblasts (CAF) via macrophage‐myofibroblast transition (MMT) by resolving their transcriptome dynamics at single‐cell resolution. Encouragingly, targeting the MMT key regulator Smad3 effectively blocked the production and protumoral actions of CAF, evidencing its translational potential as a novel therapeutic target in the tumour microenvironment for cancer.

Diabetic cardiomyopathy (DCM) is a common diabetic complication characterized by diastolic relaxation abnormalities, myocardial fibrosis and chronic heart failure. Although TGF‐β/Smad3 signalling has been shown to play a critical role in chronic heart disease, the role and mechanisms of Smad3 in DCM remain unclear. We reported here the potential role of Smad3 in the development of DCM by genetically deleting the Smad3 gene from db/db mice. At the age of 32 weeks, Smad3WT‐db/db mice developed moderate to severe DCM as demonstrated by a marked increase in the left ventricular (LV) mass, a significant fall in the LV ejection fraction (EF) and LV fractional shortening (FS), and progressive myocardial fibrosis and inflammation. In contrast, db/db mice lacking Smad3 (Smad3KO‐db/db) were protected against the development of DCM with normal cardiac function and undetectable myocardial inflammation and fibrosis. Interestingly, db/db mice with deleting one copy of Smad3 (Smad3 ± db/db) did not show any cardioprotective effects. Mechanistically, we found that deletion of Smad3 from db/db mice largely protected cardiac Smad7 from Smurf2‐mediated ubiquitin proteasome degradation, thereby inducing IBα to suppress NF‐kB‐driven cardiac inflammation. In addition, deletion of Smad3 also altered Smad3‐dependent miRNAs by up‐regulating cardiac miR‐29b while suppressing miR‐21 to exhibit the cardioprotective effect on Smad3KO‐db/db mice. In conclusion, results from this study reveal that Smad3 is a key mediator in the pathogenesis of DCM. Targeting Smad3 may be a novel therapy for DCM.

Plasma levels of neuropeptide Y (NPY) are elevated in patients with acute myocardial infarction (AMI), but its role in AMI remains unclear, which was examined here in NPY wild-type/knockout (WT/KO) mice treated with/without exogenous NPY and its Y1 receptor antagonist (Y1Ra) BIBP 3226. We found that AMI mice lacking NPY developed more severe AMI than WT mice with worse cardiac dysfunction, progressive cardiac inflammation and fibrosis, and excessive apoptosis but impairing angiogenesis. All of these changes were reversed when the NPY KO mice were treated with exogenous NPY in a dose-dependent manner. Interestingly, treatment with NPY also dose dependently attenuated AMI in WT mice, which was blocked by BIBP 3226. Phenotypically, cardiac NPY was de novo expressed by infiltrating macrophages during the repairing or fibrosing process in heart-failure patients and AMI mice. Mechanistically, NPY was induced by transforming growth factor (TGF)-β1 in bone marrow-derived macrophages and signaled through its Y1R to exert its pathophysiological activities by inhibiting p38/nuclear factor κB (NF-κB)-mediated M1 macrophage activation while promoting the reparative M2 phenotype in vivo and in vitro. In conclusion, NPY can attenuate AMI in mice. Inhibition of cardiac inflammation and fibrosis while enhancing angiogenesis but reducing apoptosis may be the underlying mechanisms through which NPY attenuates cardiac remodeling and deterioration of function following AMI.

Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage–myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, α-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGF-β1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-β1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.

Rationale: Transforming Growth Factor-beta (TGF-β) /Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases, but its role in beta cell function and type 2 diabetes is unknown. Methods: The role of Smad3 in beta cell function under type 2 diabetes condition was investigated by genetically deleting Smad3 from db/db mice. Phenotypic changes of pancreatic islets and beta cell function were compared between Smad3 knockout db/db (Smad3KO-db/db) mice and Smad3 wild-type db/db (Smad3WT-db/db) mice, and other littermate controls. Islet-specific RNA-sequencing was performed to identify Smad3-dependent differentially expressed genes associated with type 2 diabetes. In vitro beta cell proliferation assay and insulin secretion assay were carried out to validate the mechanism by which Smad3 regulates beta cell proliferation and function. Results: The results showed that Smad3 deficiency completely protected against diabetes-associated beta cell loss and dysfunction in db/db mice. By islet-specific RNA-sequencing, we identified 8160 Smad3-dependent differentially expressed genes associated with type 2 diabetes, where Smad3 deficiency markedly prevented the down-regulation of those genes. Mechanistically, Smad3 deficiency preserved the expression of beta cell development mediator Pax6 in islet, thereby enhancing beta cell proliferation and function in db/db mice in vivo and in Min6 cells in vitro. Conclusions: Taken together, we discovered a pathogenic role of Smad3 in beta cell loss and dysfunction via targeting the protective Pax6. Thus, Smad3 may represent as a novel therapeutic target for type 2 diabetes prevention and treatment.

Transforming growth factor β1 (TGF-β1) plays a promoting role in tumor growth via a mechanism associated with hyperactive Smad3 and suppressed Smad7 signaling in the tumor microenvironment. We report that retrieving the balance between Smad3 and Smad7 signaling with asiatic acid (AA, a Smad7 inducer) and naringenin (NG, a Smad3 inhibitor) effectively inhibited tumor progression in mouse models of invasive melanoma (B16F10) and lung carcinoma (LLC) by promoting natural killer (NK) cell development and cytotoxicity against cancer. Mechanistically, we found that Smad3 physically bound Id2 and IRF2 to suppress NK cell production and NK cell-mediated cytotoxicity against cancer. Treatment with AA and NG greatly inhibited Smad3 translation and phosphorylation while it restored Smad7 expression, and, therefore, it largely promoted NK cell differentiation, maturation, and cytotoxicity against cancer via Id2/IRF2-associated mechanisms. In contrast, silencing Id2 or IRF2 blunted the protective effects of AA and NG on NK cell-dependent anti-cancer activities. Thus, treatment with AA and NG produced an additive effect on inactivating TGF-β1/Smad3 signaling, and, therefore, it suppressed melanoma and lung carcinoma growth by promoting NK cell immunity against cancer via a mechanism associated with Id2 and IRF2.

Tumor-associated macrophages (TAM) have important roles in cancer progression, but the signaling behind the formation of protumoral TAM remains understudied. Here, by single-cell RNA sequencing, we revealed that the pattern recognition receptor Mincle was highly expressed in TAM and significantly associated with mortality in patients with non–small cell lung cancer. Cancer cells markedly induced Mincle expression in bone marrow–derived macrophages (BMDM), thus promoting cancer progression in invasive lung carcinoma LLC and melanoma B16F10 in vivo and in vitro. Mincle was predominately expressed in the M2-like TAM in non–small cell lung carcinoma and LLC tumors, and silencing of Mincle unexpectedly promoted M1-like phenotypes in vitro. Mechanistically, we discovered a novel Mincle/Syk/NF-κB signaling pathway in TAM needed for executing their TLR4-independent protumoral activities. Adoptive transfer of Mincle-silenced BMDM significantly suppressed TAM-driven cancer progression in the LLC-bearing NOD/SCID mice. By modifying our well-established ultrasound microbubble–mediated gene transfer protocol, we demonstrated that tumor-specific silencing of Mincle effectively blocked Mincle/Syk/NF-κB signaling, therefore inhibiting the TAM-driven cancer progression in the syngeneic mouse cancer models. Thus, our findings highlight the function of Mincle as a novel immunotherapeutic target for cancer via blocking the Mincle/Syk/NF-κB circuit in TAM.

Background Transforming growth factor (TGF)-β/Smad3 signaling is highly activated in kidneys of patients with type 2 diabetic nephropathy (T2DN), however, the precise role of Smad3 in the pathogenesis of diabetic nephropathy remains unclear. Methods Smad3 knockout (KO)-db/db mice were generated by intercrossing of male and female double-heterozygous Smad3+/− db/m mice. Renal functions including urinary albumin excretion and serum creatinine were determined. Renal histological injury including renal fibrosis and inflammation were examined by periodic acid Schiff (PAS), periodic acid-silver methenamine (PASM), and immunohistochemistry (IHC) staining. Results Smad3 knockout (KO)-db/db mice were protected from the development of diabetic kidney injury, characterized by the normal levels of urinary albumin excretion and serum creatinine without any evidence for renal fibrosis and inflammation. In contrast, Smad3 wild-type (WT) db/db and Smad3+/− db/db mice developed progressively decline in renal function over the 12 to 32-week time course, including increased microalbuminuria and elevated levels of serum creatinine. Pathologically, Smad3 WT db/db and Smad3+/− db/db mice exhibited a marked deposition of collagen-I (col I), collagen-IV(col-IV), and an increased infiltration of F4/80+ macrophages in kidney. Mechanistically, Smad3 deficiency decreased the lncRNA Erbb4-IR transcription, while increased miR-29b transcription and therefore protected the kidney from progressive renal injury in db/db mice. Conclusion Results from this study imply that Smad3 may represent as a novel and effective therapeutic target for T2DN.

Transforming growth factor-β/Smad3 signaling plays an important role in diabetic nephropathy, but its underlying working mechanism remains largely unexplored. The current study uncovered the pathogenic role and underlying mechanism of a novel Smad3-dependent long noncoding RNA (lncRNA) (LRNA9884) in type 2 diabetic nephropathy (T2DN). We found that LRNA9884 was significantly upregulated in the diabetic kidney of

Macrophage migration inhibitory factor (MIF) is elevated in patients with acute kidney injury (AKI) and is suggested as a potential predictor for renal replacement therapy in AKI. In this study, we found that MIF also plays a pathogenic role and is a therapeutic target for AKI. In a cisplatin-induced AKI mouse model, elevated plasma MIF correlated with increased serum creatinine and the severity of renal inflammation and tubular necrosis, whereas deletion of MIF protected the kidney from cisplatin-induced AKI by largely improving renal functional and histological injury, and suppressing renal inflammation including upregulation of cytokines such as interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), IL-6, inducible nitric oxide synthase (iNOS), MCP-1, IL-8, and infiltration of macrophages, neutrophils, and T cells. We next developed a novel therapeutic strategy for AKI by blocking the endogenous MIF with an MIF inhibitor, ribosomal protein S19 (RPS19). Similar to the MIF-knockout mice, treatment with RPS19, but not the mutant RPS19, suppressed cisplatin-induced AKI. Mechanistically, we found that both genetic knockout and pharmacological inhibition of MIF protected against AKI by inactivating the CD74-nuclear factor κB (NF-κB) signaling. In conclusion, MIF is pathogenic in cisplatin-induced AKI. Targeting MIF with an MIF inhibitor RPS19 could be a promising therapeutic potential for AKI.

Tubular cell necrosis is a key histological feature of acute kidney injury (AKI). Necroptosis is a type of programed necrosis, which is executed by mixed lineage kinase domain-like protein (MLKL) upon its binding to the plasma membrane. Emerging evidence indicates that necroptosis plays a critical role in the development of AKI. However, it is unclear whether renal tubular cells undergo necroptosis in vivo and how the necroptotic pathway is regulated during AKI. Repulsive guidance molecule (RGM)-b is a member of the RGM family. Our previous study demonstrated that RGMb is highly expressed in kidney tubular epithelial cells, but its biological role in the kidney has not been well characterized. In the present study, we found that RGMb reduced membrane-associated MLKL levels and inhibited necroptosis in cultured cells. During ischemia/reperfusion injury (IRI) or oxalate nephropathy, MLKL was induced to express on the apical membrane of proximal tubular (PT) cells. Specific knockout of Rgmb in tubular cells (Rgmb cKO) increased MLKL expression at the apical membrane of PT cells and induced more tubular cell death and more severe renal dysfunction compared with wild-type mice. Treatment with the necroptosis inhibitor Necrostatin-1 or GSK'963 reduced MLKL expression on the apical membrane of PT cells and ameliorated renal function impairment after IRI in both wild-type and Rgmb cKO mice. Taken together, our results suggest that proximal tubular cell necroptosis plays an important role in AKI, and that RGMb protects against AKI by inhibiting MLKL membrane association and necroptosis in proximal tubular cells.

We recently showed that imbalance of TGF-β/Smad signaling with over-activation of Smad3 but lower levels of Smad7 is a central mechanism of tissue fibrosis. In the present study, we report here that inhibition of Smad3 with naringenin (NG) and upregulation of Smad7 with asiatic acid (AA) produced an additive effect on inhibition of renal fibrosis in a mouse model of obstructive nephropathy. We found that AA, a triterpene from Centella Asiatica, functioned as a Smad7 agonist and suppressed TGF-β/Smad3-mediated renal fibrosis by inducing Smad7. Whereas, NG, a flavonoid from grapefruits and citrus fruits, was a Smad3 inhibitor that inhibited renal fibrosis by blocking Smad3 phosphorylation and transcription. The combination of AA and NG produced an additive effect on inhibition of renal fibrosis by blocking Smad3 while upregulating Smad7. Thus, rebalancing the disorder of TGF-β/Smad signaling by treatment with AA and NG may represent as a novel and effective therapy for chronic kidney disease associated with fibrosis.

T cells play a critical role in acute allograft rejection. TGF-β/Smad3 signaling is a key pathway in regulating T cell development. We report here that Smad3 is a key transcriptional factor of TGF-β signaling that differentially regulates T cell immune responses in a mouse model of cardiac allograft rejection in which donor hearts from BALB/c mice were transplanted into Smad3 knockout (KO) and wild type (WT) mice. Results showed that the cardiac allograft survival was prolonged in Smad3 KO recipients. This allograft protection was associated with a significant inhibition of proinflammatory cytokines (IL-1β, TNF-α, and MCP-1) and infiltration of neutrophils, CD3+ T cells, and F4/80+ macrophages. Importantly, deletion of Smad3 markedly suppressed T-bet and IFN-γ while enhancing GATA3 and IL-4 expression, resulting in a shift from the Th1 to Th2 immune responses. Furthermore, mice lacking Smad3 were also protected from the Th17-mediated cardiac injury, although the regulatory T cell (Treg) response was also suppressed. In conclusion, Smad3 is an immune regulator in T cell-mediated cardiac allograft rejection. Loss of Smad3 results in a shift from Th1 to Th2 but suppressing Th17 immune responses. Thus, modulation of TGF-β/Smad3 signaling may be a novel therapy for acute allograft rejection.

// Xiao-Yu Dai 1,2,* , Li Zhou 1,2,* , Xiao-Ru Huang 1 , Ping Fu 2 and Hui-Yao Lan 1 1 Department of Medicine and Therapeutics, and Li Ka Shing Institute of Health Sciences, and Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China 2 Division of Nephrology, West China Hospital of Sichuan University, Chengdu, China * These authors have contributed equally to this work Correspondence to: Hui Y. Lan, email: // Ping Fu, email: // Keywords : chronic aristolochic acid nephropathy, Smad7, renal fibrosis, renal inflammation Received : January 26, 2015 Accepted : March 05, 2015 Published : March 30, 2015 Abstract Chronic Aristolochic Acid Nephropathy (AAN) is a progressive chronic kidney disease related to herb medicine. However, treatment for chronic AAN remains ineffective. We report here that Smad7 is protective and has therapeutic potential for chronic AAN. In a mouse model of chronic AAN, progressive renal injury was associated with a loss of renal Smad7 and disruption of Smad7 largely aggravated the severity of chronic AAN as demonstrated by a significant increase in levels of 24-hour urinary protein excretion, serum creatinine, and progressive renal fibrosis and inflammation. In contrast, restored Smad7 locally in the kidneys of Smad7 knockout mice prevented the progression of chronic AAN. Further studies revealed that worsen chronic AAN in Smad7 knockout mice was associated with enhanced activation of TGF-β/Smad3 and NF-κB signaling pathways, which was reversed when renal Smad7 was restored. Importantly, we also found that overexpression of Smad7 locally in the kidneys with established chronic AAN was capable of attenuating progressive chronic AAN by inactivating TGF-β/Smad3-medated renal fibrosis and NF-κB-driven renal inflammation. In conclusion, Smad7 plays a protective role in the pathogenesis of chronic AAN and overexpression of Smad7 may represent a novel therapeutic potential for chronic AAN.

Transforming growth factor β (TGF-β)/Smad3 signaling plays a role in tissue fibrosis. We report here that Erbb4-IR is a novel long non-coding RNA (lncRNA) responsible for TGF-β/Smad3-mediated renal fibrosis and is a specific therapeutic target for chronic kidney disease. Erbb4-IR was induced by TGF-β1 via a Smad3-dependent mechanism and was highly upregulated in the fibrotic kidney of mouse unilateral ureteral obstructive nephropathy (UUO). Silencing Erbb4-IR blocked TGF-β1-induced collagen I and alpha-smooth muscle actin (α-SMA) expressions in vitro and effectively attenuated renal fibrosis in the UUO kidney by blocking TGF-β/Smad3 signaling. Mechanistic studies revealed that Smad7, a downstream negative regulator of TGF-β/Smad signaling, is a target gene of Erbb4-IR because a binding site of Erbb4-IR was found on the 3′ UTR of Smad7 gene. Mutation of this binding site prevented the suppressive effect of Erbb4-IR on the Smad7 reporter activity; in contrast, overexpression of Erbb4-IR largely inhibited Smad7 but increased collagen I and α-SMA transcriptions. Thus, kidney-specific silencing of Erbb4-IR upregulated renal Smad7 and thus blocked TGF-β/Smad3-mediated renal fibrosis in vivo and in vitro. In conclusion, the present study identified that Erbb4-IR is a novel lncRNA responsible for TGF-β/Smad3-mediated renal fibrosis by downregulating Smad7. Targeting Erbb4-IR may represent a precise therapeutic strategy for progressive renal fibrosis.

Regulatory T cells (Tregs) have been shown to play a protective role in glomerulonephritis (GN) and Foxp3 is a master transcription factor in Treg development. In this study, we examined the functional role and mechanisms of Foxp3 in a mouse model of accelerated anti-glomerular basement membrane (anti-GBM) GN induced in antigen-primed Foxp3 transgenic (Tg) mice. Compared with littermate of wildtype (WT) mice in which induced severe crescentic GN developed with progressive renal dysfunction, Foxp3 Tg mice had reduced crescent formation, urinary protein excretion, plasma creatinine and decline in creatinine clearance. The protective role of Foxp3 in crescentic GN was associated with a markedly suppressed expression of proinflammatory interleukin-1 beta (IL-1β), tumour necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1), and diminished infiltration of the kidneys by CD3+ T cells and F4/80+ macrophages. Moreover, overexpression of Foxp3 resulted in a significant increase in CD4+ Foxp3+ Tregs systemically and in the diseased kidneys, thereby blunting Th1, Th2, and Th17 responses systemically and in the kidneys. In conclusion, Foxp3 protects against kidney injury in crescentic GN through enhancement of Treg numbers and function, and suppression of Th1, Th2 and Th17 immune responses at the systemic and local tissue levels.

Angiotensin-converting enzyme-2 (ACE2) is downregulated in hypertensive nephropathy. The present study investigated the mechanisms whereby loss of ACE2 promoted angiotensin II–induced hypertensive nephropathy in ACE2 gene knockout mice. We found that compared with wild-type animals, mice lacking ACE2 developed much more severe hypertensive nephropathy in response to chronic angiotensin II infusion, including higher levels of blood pressure, urinary protein excretion, serum creatinine, and progressive renal fibrosis and inflammation. Mechanistic studies revealed that worsening kidney injury in ACE2 knockout mice was associated with an increase in Smurf2 (Smad-specific E3 ubiquitin protein ligase 2), a decrease in renal Smad7, and marked activation of TGF-β (transforming growth factor β)/Smad3 and NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling, suggesting that Smurf2-dependent Smad7 ubiquitin degradation may be a key mechanism whereby loss of ACE2 promotes angiotensin II–induced TGF-β/Smad3 and NF-κB–mediated hypertensive nephropathy. This was validated by restoring Smad7 locally in the kidneys of ACE2 knockout mice to block angiotensin II–induced TGF-β/Smad3-mediated renal fibrosis and NF-κB–driven renal inflammation. Moreover, we found that angiotensin II could induce microRNA-21 in the mouse kidney and in cultured mesangial cells via a Smad3-dependent mechanism, which was enhanced by deleting ACE2 but inhibited by overexpressing renal Smad7. In conclusion, loss of ACE2 promotes angiotensin II–induced renal injury by targeting Smad7 for degradation via a Smurf2-dependent mechanism. Overexpression of renal Smad7 protects against hypertensive nephropathy by inactivating angiotensin II–induced TGF-β/Smad3 and NF-κB pathways and by targeting the Smad3-dependent microRNA-21 axis.

Transforming growth factor (TGF)-β1 signals through downstream Smad-dependent and -independent pathways to exert its biological actions. It has been reported that overexpression of TGF-β1 results in the development of psoriasis-like lesions in a mouse model of K5.TGF-β(WT) transgenic mice. However, the signalling mechanisms by which TGF-β1 mediates the development of psoriasis-like lesions remain unknown. The aim of the present study was to investigate the hypothesis that TGF-β1 mediates the development of psoriasis-like lesions via a Smad3-dependent mechanism. This was tested in a mouse model of K5.TGF-β(WT) transgenic mice by blocking TGF-β signalling with a specific Smad3 inhibitor. Topical treatment with a Smad3 inhibitor markedly blocked TGF-β/Smad3 signalling and progressive psoriasis-like lesions in K5.TGF-β(WT) transgenic mice, as evidenced by decreased skin severity scores, double skin fold thickness (DSFT) scores, infiltration of CD3(+) T cells and F4/80(+) macrophages and the degree of fibrosis in the dermis. This was associated with a marked reduction in TGF-β1, interleukin (IL)-6, IL-23 and IL-17A both locally in skin plaque lesions and systemically in the plasma, resulting in inhibition of both the T helper (Th) 17 cell transcription factor RORγt and accumulation of CD4(+) IL-17A(+) cells within the skin plaque lesions. In conclusion, TGF-β1 mediates the development of psoriasis-like lesions via a Smad3-dependent, Th17-mediated mechanism. Targeting TGF-β/Smad3 signalling with a Smad3 inhibitor may represent a novel and effective therapy for psoriasis.

Smad7 is well demonstrated as a negative regulator of TGF-β signaling. Its alteration in expression often results in diseases such as cancer and fibrosis. However, the exact role of Smad7 in regulating bone remodeling during mammalian development has not been properly delineated. In this study we performed experiments to clarify the involvement of Smad7 in regulating osteogenesis and osteoclastogenesis both invivo and invitro. Genetically engineered Smad7(ΔE1) (KO) mice were used, whereby partial functional of Smad7 is lost by deleting exon I of the Smad7 gene and the truncated proteins cause a hypomorphic allele. Analysis with μCT imagery and bone histomorphometry showed that the KO mice had lower TbN, TbTh, higher TbSp in the metaphysic region of the femurs at 6, 12, 24weeks from birth, as well as decreased MAR and increased osteoclast surface compared with the WT mice. In vitro BM-MSC multi-lineage differentiation evaluation showed that the KO group had reduced osteogenic potential, fewer mineralized nodules, lower ALP activity, and reduced gene expression of Col1A1, Runx2 and OCN. The adipogenic potential was elevated in the KO group with more formation of lipid droplets, and increased gene expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was elevate, with emergence of more osteoclasts, larger resorptive areas, and increased gene expression of TRAP and CTR. Our results indicate that partial loss of Smad7 function in mice leads to compromised bone formation and enhanced bone resorption. Thus, Smad7 is acknowledged as a novel key regulator between osteogenesis and osteoclastogenesis.

Peritoneal fibrosis is a major cause of ultrafiltration failure in patients receiving continuous ambulatory peritoneal dialysis. Transforming growth factor (TGF)-β1 is an important mediator in this process; however, its signaling mechanisms had not been explored. Thus, we examined TGF-β1/Smad signaling in human peritoneal biopsy specimens associated with continuous ambulatory peritoneal dialysis. We found that TGF-β/Smad2/3 signaling was highly activated in patients with increased collagen deposition and thickening of the peritoneal membrane who were receiving continuous ambulatory peritoneal dialysis. Long-term exposure of wild-type mice to 4.25% peritoneal dialysis solution for 30 days induced significant peritoneal fibrosis with impaired peritoneal equilibrium, which was prevented in Smad3 knockout mice. In contrast, conditional Smad2 gene deletion in the peritoneum exacerbated peritoneal fibrosis and dysfunction. The contrasting roles of Smad2 and Smad3 in peritoneal fibrosis were also examined in vitro . Cultured mesothelial cells from Smad3 knockout mice were resistant to TGF-β1–induced collagen I production and the transition toward a myofibroblast phenotype as seen in wild-type cells, whereas Smad2 deficiency in mesothelial cells failed to modulate the profibrotic response to TGF-β1. In conclusion, this study found activation of TGF-β/Smad signaling in peritoneal fibrosis in patients receiving continuous ambulatory peritoneal dialysis and identifies opposing roles for Smad2 and Smad3 in peritoneal dialysis–associated peritoneal fibrosis. These findings provide a mechanistic basis for future therapies targeting TGF-β/Smad signaling in peritoneal fibrosis.

Loss of miR-29 is associated with cardiac fibrosis. This study examined the role and therapeutic potential of miR-29 in mouse model of hypertension induced by angiotensin II (AngII). By using microRNA microarray, in situ hybridization, and real-time polymerase chain reaction, we found that AngII-induced cardiac fibrosis in the hypertensive heart and in cultured cardiac fibroblasts were associated with downregulation of miR-29a-c via a Smad3-dependent mechanism. In vitro knockdown of miR-29b enhanced but overexpression of miR-29b inhibited AngII-induced fibrosis, revealing a protective role of miR-29b in cardiac fibrosis in response to AngII. This was further demonstrated in vivo by the ability of overexpressing miR-29b in the mouse heart to prevent AngII-mediated cardiac fibrosis and cardiac dysfunction. Importantly, we also found that restored miR-29b in the established hypertensive heart was capable of blocking progressive cardiac fibrosis and improving cardiac dysfunction, demonstrating a therapeutic potential of miR-29b for chronic heart disease. Further studies revealed that targeting the transforming growth factor (TGF)-β1 coding sequence region, thereby inhibiting TGF-β/Smad3 signaling, could be a new mechanism by which miR-29b inhibited AngII-induced cardiac fibrosis. In conclusion, miR-29b plays a protective role in AngII-mediated cardiac remodeling and may be a therapeutic agent for cardiac fibrosis by targeting the TGF-β/Smad3 pathway.

Aims Smad7 plays a negative regulatory role in many inflammatory diseases, but its effect on hypertensive disease remains unknown. The present study tested the hypothesis that overexpression of Smad7 may have therapeutic potential for angiotensin II (Ang II)-mediated hypertensive cardiac remodelling. Methods and results Hypertensive heart disease was induced in mice by subcutaneous infusion of Ang II for 28 days and treated with Smad7 by a non-invasive ultrasound-microbubble-mediated inducible Smad7 gene transfer. We found that cardiac Smad7 was largely reduced in the hypertensive heart and overexpression of cardiac Smad7 protected against the fall in the left ventricular (LV) ejection fraction (EF), an increase in LV mass, and cardiac inflammation and fibrosis such as up-regulation of pro-inflammatory cytokines (IL-1β, TNF-α) and fibrotic markers (collagen I, α-SMA), and infiltration of CD3+ T cells and F4/80+ macrophages. Further studies revealed that inactivation of the Sp1-TGF-β/Smad3-NF-κB (NF-κB, nuclear factor κB) pathways and prevention of cardiac miR-29 loss were mechanisms by which overexpression of Smad7 inhibited Ang II-mediated cardiac remodelling. Importantly, we also found that treatment with Smad7 when hypertensive cardiopathy established at day 14 halted the progression of cardiac injury by blunting the fall of EF and an increase in LV mass, and blocking TGF-β/Smad3-mediated cardiac fibrosis and NF-κB-driven inflammation. Conclusion Smad7 plays a protective role in Ang II-induced cardiac remodelling via mechanisms involving the Sp1-TGF-β/Smad-NF-κB-miR-29 regulatory network. Thus, Smad7 may be a novel therapeutic agent for hypertensive cardiovascular diseases.

Podocyte injury and the appearance of proteinuria are features of minimal-change disease (MCD). Cyclosporin A (CsA) and tacrolimus (FK506) has been reported to reduce proteinuria in patients with nephrotic syndrome, but mechanisms remain unknown. We, therefore, investigated the protective mechanisms of CsA and FK506 on proteinuria in a rat model of MCD induced by puromycin aminonucleoside (PAN) and in vitro cultured mouse podocytes. Our results showed that CsA and FK506 treatment decreased proteinuria via a mechanism associated to a reduction in the foot-process fusion and desmin, and a recovery of synaptopodin and podocin. In PAN-treated mouse podocytes, pre-incubation with CsA and FK506 restored the distribution of the actin cytoskeleton, increased the expression of synaptopodin and podocin, improved podocyte viability, and reduced the migrating activities of podocytes. Treatment with CsA and FK506 also inhibited PAN-induced podocytes apoptosis, which was associated with the induction of Bcl-xL and inhibition of Bax, cleaved caspase 3, and cleaved PARP expression. Further studies revealed that CsA and FK506 inhibited PAN-induced p38 and JNK signaling, thereby protecting podocytes from PAN-induced injury. In conclusion, CsA and FK506 inhibit proteinuria by protecting against PAN-induced podocyte injury, which may be associated with inhibition of the MAPK signaling pathway.

Mincle (macrophage-inducible C-type lectin, Clec4e) is a transmembrane pattern recognition receptor involving the innate immunity, but its role in kidney disease is still unexplored. In the obstructed kidney of the unilateral ureteral obstruction model of renal injury, Mincle was specifically detected in the infiltrating M1 macrophages (CD68 + iNOS + cells) on day one but was rapidly reduced following reduction of M1 macrophages during the progression of kidney injury. Interestingly, Mincle-expressing macrophages were progressively increased in the cisplatin-induced acute kidney injury model, where iNOS expression was detected in the CD68 + cells following Mincle induction. Adaptive transfer of Mincle + M1 macrophages largely promoted cisplatin-induced renal inflammation, which was prevented by the knockdown of Mincle in the transferred cells. Mincle was tightly regulated by TLR4/NF-κB signaling as evidenced by the binding of NF-κB/p65 to the promoter region of Mincle in LPS-primed macrophages. Blocking TLR4 or NF-κB suppressed LPS-induced Mincle expression on macrophages. Importantly, Mincle was found to be essential for maintaining the inflammatory phenotypes of M1 macrophages through the Syk signaling pathway since knockdown of Mincle or inhibition of Syk suppressed LPS-induced IL-1β, MCP-1, and iNOS expression. Thus, Mincle is induced specifically on M1 macrophages, where Mincle-Syk signaling promotes and maintains inflammatory phenotypes of M1 macrophages in acute renal inflammation. Hence, targeting Mincle may be a novel therapy for acute kidney injury associated with M1 macrophages.

This study aimed to evaluate the validation of the leptin receptor-deficient mice model for secondary osteoporosis associated with type 2 diabetes mellitus (T2DM) at bone micro-architectural level. Thirty three 36-week old male mice were divided into four groups: normal control (db/m) (n = 7), leptin receptor-deficient T2DM (db/db) (n = 8), human C-reactive protein (CRP) transgenic normal control (crp/db/m) (n = 7) and human CRP transgenic T2DM (crp/db/db) (n = 11). Lumber vertebrae (L5) and bilateral lower limbs were scanned by micro-CT to analyze trabecular and cortical bone quality. Right femora were used for three-point bending to analyze the mechanical properties. Trabecular bone quality at L5 was better in db/db or crp/db/db group in terms of bone mineral density (BMD), bone volume fraction, connectivity density, trabecular number and separation (all p crp/db/db group was significantly shorter than db/m group (p db/db and crp/db/db groups (p > 0.05). Maximum loading and energy yield in mechanical test were similar among groups while the elastic modulus in db/db and crp/db/db significantly lower than db/m. The leptin-receptor mice is not a proper model for secondary osteoporosis associated with T2DM.

C-reactive protein (CRP) is associated with progressive diabetic nephropathy in patients with type-2 diabetes (T2DN). However, role of CRP in T2DN remains unclear. We report here that CRP is pathogenic in T2DN in db/db mice that express human CRP (CRPtg-db/db). Compared to the littermate db/db mice, CRPtg-db/db developed more severe T2DN, showing higher levels of fasting blood glucose and microalbuminuria and more progressive renal inflammation and fibrosis. Enhanced T2DN in CRPtg-db/db mice were associated with over-activation of CRP-CD32b, NF-κB, TGF-β/Smad3 and mTOR signaling. Further studies in vitro defined that CRP activated Smad3 directly at 15 mins via the CD32b- ERK/p38 MAP kinase crosstalk pathway and indirectly at 24 hours through a TGF-β1-dependent mechanism. Importantly, CRP also activated mTOR signaling at 30 mins via a Smad3-dependent mechanism as Smad3 bound mTOR physically and CRP-induced mTOR signaling was abolished by a neutralizing CD32b antibody and a specific Smad3 inhibitor. Finally, we also found that CRP induced renal fibrosis through a CD32b-Smad3-mTOR pathway because blocking mTOR signaling with rapamycin inhibited CRP-induced CTGF and collagen I expression. Thus, CRP is pathogenic in T2DN. CRP may promote CD32b- NF-κB signaling to mediate renal inflammation; whereas, CRP may enhance renal fibrosis in T2DN via CD32b-Smad3-mTOR signaling.

The TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes.We delivered Smad7 plasmids into the kidney of db/db mice using kidney-targeting, ultrasound-mediated, microbubble-inducible gene transfer. The histopathology, ultrastructural pathology and pathways of TGF-β/SMAD2/3-mediated fibrosis and NF-κB-dependent inflammation were evaluated.In this mouse model of type 2 diabetes, Smad7 gene therapy significantly inhibited diabetic kidney injury, compared with mice treated with empty vectors. Symptoms inhibited included: (1) proteinuria and renal function impairment; (2) renal fibrosis such as glomerular sclerosis, tubulo-interstitial collagen matrix abundance and renal inflammation, including Inos (also known as Nos2), Il1b and Mcp1 (also known as Ccl2) upregulation, as well as macrophage infiltration; and (3) podocyte and endothelial cell injury as demonstrated by immunohistochemistry and/or electron microscopy. Further study demonstrated that the improvement of type 2 diabetic kidney injury by overexpression of Smad7 was associated with significantly inhibited local activation of the TGF-β/SMAD and NF-κB signalling pathways in the kidney.Our results clearly demonstrate that kidney-targeting Smad7 gene transfer may be an effective therapy for type 2 diabetic nephropathy, acting via simultaneous modulation of the TGF-β/SMAD and NF-κB signalling pathways.

Although C-reactive protein (CRP) has been implicated as a risk factor in diabetes, its pathogenic importance in diabetic kidney disease (DKD) remains unclear. The present study investigated the potential role of CRP in DKD.Diabetes was induced by streptozotocin in human CRP transgenic and wild-type mice for assessment of kidney injury at 24 weeks by real-time PCR, immunohistochemistry and western blot analysis. In vitro, the pathogenic effect of CRP was investigated using human kidney tubular epithelial cells cultured with high glucose and/or CRP.We found that CRP transgenic mice developed much more severe diabetic kidney injury than wild-type mice, as indicated by a significant increase in urinary albumin excretion and kidney injury molecule-1 abundance, enhanced infiltration of macrophages and T cells, and upregulation of pro-inflammatory cytokines (IL-1β, TNFα) and extracellular matrix (collagen I, III and IV). Enhanced renal inflammation and fibrosis in CRP transgenic mice was associated with upregulation of CRP receptor, CD32a, and over-activation of the TGF-β/SMAD and nuclear factor κB signalling pathways. In vitro, CRP significantly upregulated pro-inflammatory cytokines (IL-1β, TNFα, monocyte chemoattractant protein-1 [MCP-1]) and pro-fibrotic growth factors (TGF-β1, connective tissue growth factor [CTGF]) via CD32a/64. CRP was induced by high glucose, which synergistically promoted high glucose-mediated renal inflammation and fibrosis.CRP is not only a biomarker, but also a mediator in DKD. Enhanced activation of TGF-β/SMAD and nuclear factor κB signalling pathways may be the mechanisms by which CRP promotes renal inflammation and fibrosis under diabetic conditions.

OBJECTIVE - Although Smad3 has been considered as a downstream mediator of transforming growth factor-β (TGF-β) signaling in diabetes complications, the role of Smad7 in diabetes remains largely unclear. The current study tests the hypothesis that Smad7 may play a protective role and has therapeutic potential for diabetic kidney disease. RESEARCH DESIGN AND METHODS - Protective role of Smad7 in diabetic kidney disease was examined in streptozotocin-induced diabetic mice that have Smad7 gene knockout (KO) and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. RESULTS - We found that mice deficient for Smad7 developed more severe diabetic kidney injury than wild-type mice as evidenced by a significant increase in microalbuminuria, renal fibrosis (collagen I, IV, and fibronectin), and renal inflammation (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], monocyte chemoattractant protein-1 [MCP-1], intracellular adhesion molecule-1 [ICAM-1], and macrophages). Further studies revealed that enhanced renal fibrosis and inflammation in Smad7 KO mice with diabetes were associated with increased activation of both TGF-β/Smad2/3 and nuclear factor-κB (NF-κB) signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney in diabetic rats by an ultrasound-microbubble-mediated technique. Although overexpression of renal Smad7 had no effect on levels of blood glucose, it significantly attenuated the development of microalbuminuria, TGF-β/Smad3-mediated renal fibrosis such as collagen I and IV and fibronectin accumulation and NF-κB/p65-driven renal inflammation including IL-1β, TNF-α, MCP-1, and ICAM-1 expression and macrophage infiltration in diabetic rats. CONCLUSIONS-Smad7 plays a protective role in diabetic renal injury. Overexpression of Smad7 may represent a novel therapy for the diabetic kidney complication. © 2011 by the American Diabetes Association., link_to_subscribed_fulltext

Proliferative glomerulonephritis in humans is characterized by the presence of leukocytes in glomeruli. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) can potentially stimulate or affect T cell, macrophage, and neutrophil function. To define the roles of GM-CSF and G-CSF in leukocyte-mediated glomerulonephritis, glomerular injury was studied in mice genetically deficient in either GM-CSF (GM-CSF -/- mice) or G-CSF (G-CSF -/- mice). Two models of glomerulonephritis were studied: neutrophil-mediated heterologous-phase anti-glomerular basement membrane (GBM) glomerulonephritis and T cell/macrophage-mediated crescentic autologous-phase anti-GBM glomerulonephritis. Both GM-CSF -/- and G-CSF -/- mice were protected from heterologous-phase anti-GBM glomerulonephritis compared with genetically normal (CSF WT) mice, with reduced proteinuria and glomerular neutrophil numbers. However, only GM-CSF -/- mice were protected from crescentic glomerular injury in the autologous phase, whereas G-CSF -/- mice were not protected and in fact had increased numbers of T cells in glomeruli. Humoral responses to the nephritogenic antigen were unaltered by deficiency of either GM-CSF or G-CSF, but glomerular T cell and macrophage numbers, as well as dermal delayed-type hypersensitivity to the nephritogenic antigen, were reduced in GM-CSF -/- mice. These studies demonstrate that endogenous GM-CSF plays a role in experimental glomerulonephritis in both the autologous and heterologous phases of injury.

TGF-β/Smad3 signaling plays a pivotal role in the pathogenesis of peritoneal fibrosis associated with peritoneal dialysis (PD). MicroRNA-29 (miR-29) is known as a potent downstream inhibitor of TGF-β/Smad3 in renal fibrosis. In this study, we examined the therapeutic potential for miR-29b on PD-related peritoneal fibrosis in a mouse model of PD induced by daily infusion of 4.25% dextrose-containing PD fluid (PDF). MiR-29b-expressing plasmid was delivered into the peritoneum via an ultrasound-microbubble-mediated system before and at day 14 after PDF. We found that mice on PD developed peritoneal fibrosis with impaired peritoneal function, which was associated with a loss of miR-29b. In contrast, overexpression of miR-29b before the PDF infusion showed a protective effect on peritoneal fibrosis including EMT and prevented peritoneal dysfunction. Moreover, delayed miR-29b treatment until peritoneal fibrosis was established at day 14 also halted the progression of peritoneal fibrosis at day 28. Further studies identified that blockade of the Sp1-TGF-β/Smad3 pathway may be a mechanism by which miR-29b inhibited peritoneal fibrosis. In conclusion, treatment with miR-29b may represent a novel and effective therapy for PD-associated peritoneal fibrosis.

Interleukin-4 deficiency enhances Th1 responses and crescentic glomerulonephritis in mice. Evidence suggests that crescentic glomerulonephritis (GN) is due to T helper cell 1 (Th1) directed delayed-type hypersensitivity (DTH)-like injury. As endogenous interleukin (IL)-4, (the pivotal cytokine in Th2 responses) may attenuate Th1 responses in this disease, we compared the development of crescentic GN, induced by a planted antigen, in mice genetically deficient in IL-4 (IL-4−/−) with disease in normal mice (IL-4+/+). IL-4−/− mice developed more severe GN with increased renal impairment (CCr 35 ± 7 μl/min vs. 133 ± 14 μl/min, P < 0.002) and crescent formation (55.7 ± 8.4% vs. 4.9 ± 1.2%, P < 0.002). This was associated with increased glomerular fibrin deposition, glomerular CD4+ T cell infiltration and macrophage recruitment. Systemically, IL-4−/− mice showed an increased antigen specific Th1 response indicated by increased skin DTH, and increased IgG3 and IgG2b. Decreased IgG1 levels indicated a reduced Th2 response. These results demonstrate a protective role for endogenous IL-4 in crescentic GN. They show that IL-4 deficiency promotes crescentic glomerular injury and amplifies local and systemic Th1 responses. They support the hypothesis that crescent formation results from Th1 immune responses to antigens in the glomerulus.

Smad7 has been shown to negatively regulate fibrosis and inflammation, but its role in angiotensin II (Ang II)-induced hypertensive cardiac remodeling remains unknown. Therefore, the present study investigated the role of Smad7 in hypertensive cardiopathy induced by angiotensin II infusion. Hypertensive cardiac disease was induced in Smad7 gene knockout (KO) and wild-type (WT) mice by subcutaneous infusion of Ang II (1.46 mg/kg/day) for 28 days. Although equal levels of high blood pressure were developed in both Smad7 KO and WT mice, Smad7 KO mice developed more severe cardiac injury as demonstrated by impairing cardiac function including a significant increase in left ventricular (LV) mass (P, published_or_final_version

Smad7 is an inhibitory Smad and plays a protective role in obstructive and diabetic kidney disease. However, the role and mechanisms of Smad7 in hypertensive nephropathy remains unexplored. Thus, the aim of this study was to investigate the role and regulatory mechanisms of Smad7 in ANG II-induced hypertensive nephropathy. Smad7 gene knockout (KO) and wild-type (WT) mice received a subcutaneous infusion of ANG II or control saline for 4 weeks via osmotic mini-pumps. ANG II infusion produced equivalent hypertension in Smad7 KO and WT mice; however, Smad7 KO mice exhibited more severe renal functional injury as shown by increased proteinuria and reduced renal function (both p, published_or_final_version

It is known that angiotensin (Ang)-converting enzyme (ACE) 2 catalyzes Ang II to Ang 1-7 to prevent the detrimental effect of Ang II on blood pressure, renal fibrosis, and inflammation. However, mechanisms of renoprotective role of Ace2 remain largely unclear. The present study tested the hypothesis that deficiency of Ace2 may accelerate intrarenal Ang II-mediated fibrosis and inflammation independent of blood pressure in a model of unilateral ureteral obstructive (UUO) nephropathy induced in Ace2(+/y) and Ace2(-/y) mice. Results showed that both Ace2(+/y) and Ace2(-/y) mice had normal levels of blood pressure and plasma Ang II/Ang 1-7. In contrast, deletion of ACE2 resulted in a fourfold increase in the ratio of intrarenal Ang II/Ang 1-7 in the UUO nephropathy. These changes were associated with the development of more intensive tubulointerstitial fibrosis (α-SMA, collagen I) and inflammation (TNF-α, IL-1β, MCP-1, F4/80(+) cells, and CD3(+)T cells) in Ace2(-/y) mice at day 3 (all P

TGF-β1 binds receptor II (TβRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TβRII may function to initiate the downstream TGF-β signalling and determine the diverse role of TGF-β1 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 TβRII was deleted conditionally. We found that disruption of TβRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF-β/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TβRII enhanced NF-κB signalling and renal inflammation including up-regulation of Il-1β and Tnfα in the UUO kidney. Similarly, in vitro disruption of TβRII from kidney fibroblasts or tubular epithelial cells inhibited TGF-β1-induced Smad signalling and fibrosis but impaired the anti-inflammatory effect of TGF-β1 on IL-1β-stimulated NF-κB activation and pro-inflammatory cytokine expression. In conclusion, TβRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF-β/Smad3, but not the non-canonical TGF-β signalling pathway, may be a key mechanism by which disruption of TβRII protects against renal fibrosis. In addition, deletion of TβRII 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-β1.

Background: Although blockade of Rho kinase with pharmacologic inhibitors ameliorates renal fibrosis and diabetic kidney disease (DKD), the underlined mechanisms remain largely unclear. The present study tested the hypothesis that ROCK1 may regulate the early development of albuminuria via the megalin/cubilin-dependent mechanism. Methods: A DKD model was induced in ROCK1 knockout and wild-type mice by streptozotocin (STZ). The effect of deleted ROCK1 on urinary albumin excretion and the expression of megalin/cubilin were examined. In addition, the effect of blocking ROCK activities with an inhibitor (Y-27632) on tubular albumin reabsorption was tested in a normal rat tubular epithelial cell line (NRK52E) under high-glucose conditions. Expression of transforming growth factor (TGF)-β1, interleukin-1β and collagen-1 was also been examined. Results: Urinary albumin excretion was significantly increased in ROCK1 WT mice at 8 weeks after STZ injection. In contrast, mice lacking ROCK1 gene were protected against the development of albuminuria. This was associated with the protection against the loss of megalin/cubilin and an increase in TGF-β 1, IL-1β, and fibrosis in the kidney. In vitro, we also found that blockade of Rho kinase with inhibitor Y-27632 prevented high-glucose-induced loss of megalin expression and an increase of TGF-β 1, thereby increasing the absorption rate of FITC-labeled albumin by tubular epithelial cells. Conclusion: ROCK1 may play a role in the development of albuminuria in DKD by downregulating the endocytosis receptors complex-megalin/cubilin. Copyright © 2011 S. Karger AG, Basel., link_to_subscribed_fulltext

Autoimmune crescentic glomerulonephritis is characterized by severe immune response with glomerular crescentic formation and fibrosis in the kidney. Recent studies indicate that overexpression of renal Smad7 attenuates both renal fibrosis and inflammation in rat remnant kidney. However, little attention has been paid to the potential role of TGF-beta/Smad signaling in autoimmune kidney disease. This study tested the hypothesis that blocking TGF-beta signaling by overexpression of Smad7 may have a therapeutic effect in a mouse model of autoimmune crescentic glomerulonephritis that was induced in C57BL/6 x DBA/2J F1 hybrid mice by giving DBA/2J donor lymphocytes. Smad7 gene was transfected into the kidney using the ultrasound-microbubble-mediated system. Results showed that overexpression of Smad7 blocked both renal fibrosis and inflammatory pathways in terms of Smad2/3 and NF-kappaB activation (P < 0.01), thereby inhibiting alpha-smooth muscle actin; collagen I, III, and IV accumulation; and expression of inflammatory cytokines (IL-1beta and IL-6), adhesion molecule/chemokine (intercellular adhesion molecule-1, monocyte chemoattractant protein-1), and inducible nitric oxide synthase (all P < 0.01). Leukocyte infiltration (CD4(+) cells and macrophages) was also suppressed (P < 0.005). Severe histologic damage (glomerular crescent formation and tubulointerstitial injury) and functional injury including proteinuria were significantly improved (all P < 0.05). This study provides important evidence that overexpression of Smad7 may have therapeutic potential for autoimmune kidney disease.

Crescentic glomerulonephritis is characterized by glomerular fibrin deposition, and experimental crescentic glomerulonephritis has been shown to be fibrin-dependent. Net fibrin deposition is a balance between activation of the coagulation system causing glomerular fibrin deposition and fibrin removal by the plasminogen-plasmin (fibrinolytic) system. Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis by inhibiting plasminogen activators and has effects on leukocyte recruitment and matrix deposition. To test the hypothesis that the presence of PAI-1 and its levels were a determinant of injury in crescentic glomerulonephritis, accelerated anti-glomerular basement membrane glomerulonephritis was induced in mice genetically deficient in PAI-1 (PAI-1 -/-), PAI-1 heterozygotes (PAI-1 +/-), and mice engineered to overexpress PAI-1 (PAI-1 tg). Compared with strain-matched genetically normal animals, PAI-1 -/- mice with glomerulonephritis developed fewer glomerular crescents, less glomerular fibrin deposition, fewer infiltrating leukocytes, and less renal collagen accumulation at day 14 of disease. The reduction in disease persisted at day 28, when injury had become more established. In contrast, mice overexpressing the PAI-1 gene (PAI-1 tg), that have basal plasma and renal PAI-1 levels several times, normal developed increased glomerular crescent formation, more glomerular fibrin deposition, increased numbers of infiltrating leukocytes, and more renal collagen at both time points. These studies demonstrate that PAI-1 is a determinant of glomerular fibrin deposition and renal injury in crescentic glomerulonephritis.

Th1 responsiveness to nephritogenic antigens determines susceptibility to crescentic glomerulonephritis in mice. The pattern of glomerulonephritis (GN) developing in response to a planted antigen (sheep anti-mouse GBM globulin) was compared in two strains of mice which demonstrated either a predominant Th1 (C57BL/6) or Th2 (BALB/c) response to this antigen. GN was induced with a subnephritogenic i.v. dose of sheep anti-mouse GBM globulin in mice presensitized to sheep globulin. Sensitized C57BL/6 mice showed pronounced cutaneous delayed-type hypersensitivity (DTH) following the challenge with sheep globulin, low titers of circulating anti-sheep globulin antibody and high interferon γ (IFN γ ) and low interleukin 4 (IL-4) production by splenic T cells, consistent with a predominant Th1 pattern of immune response. Sensitized BALB/c mice did not develop DTH following cutaneous challenge with sheep globulin, had higher circulating anti-sheep globulin antibody titers, and showed high IL-4 and low IFN γ production by splenic T cells compared with C57BL/6 mice, consistent with a predominant Th2 response. In C57BL/6 mice, GN developing in response to sheep globulin exhibited a severe crescentic pattern with prominent glomerular T cell and macrophage influx and fibrin deposition. In vivo depletion with a monoclonal anti-CD4 antibody demonstrated that this injury was T helper cell dependent. Treatment with monoclonal anti-mouse IFN γ antibody significantly reduced glomerular injury and crescent formation and attenuated the cutaneous DTH response. GN induced by the same protocol in BALB/c mice exhibited pronounced glomerular IgG and complement deposition. Crescent formation, fibrin deposition, and glomerular T cell and macrophage infiltration were significantly less than observed in C57BL/6 mice, and injury was not T cell dependent in the effector phase. These data suggest that the pattern of glomerular injury induced by a planted antigen can be determined by the balance of T helper cell subset activation. A Th1 response induces a severe crescentic pattern of GN, which like cutaneous DTH, is T helper cell and IFN γ dependent.