How to inhibit transforming growth factor beta safely in diabetic kidney disease.

PURPOSE OF REVIEW: Diabetic kidney disease (DKD) is a leading cause of mortality and morbidity in diabetes. This review aims to discuss the major features of DKD, to identify the difficult barrier encountered in developing a therapeutic strategy and to provide a potentially superior novel approach to retard DKD. RECENT FINDINGS: Renal inflammation and fibrosis are prominent features of DKD. Transforming growth factor beta (TGFß) with its activity enhanced in DKD plays a key pathological profibrotic role in promoting renal fibrosis. However, TGFß is a difficult drug target because it has multiple important physiological functions, such as immunomodulation. These physiological functions of TGFß can be interrupted as a result of complete blockade of the TGFß pathway if TGFß is directly targeted, leading to catastrophic side-effects, such as fulminant inflammation. Cell division autoantigen 1 (CDA1) is recently identified as an enhancer of profibrotic TGFß signaling and inhibitor of anti-inflammatory SIRT1. Renal CDA1 expression is elevated in human DKD as well as in rodent models of DKD. Targeting CDA1, by either genetic approach or pharmacological approach in mice, leads to concurrent attenuation of renal fibrosis and inflammation without any deleterious effects observed. SUMMARY: Targeting CDA1, instead of directly targeting TGFß, represents a superior approach to retard DKD.

Genetic deletion of cell division autoantigen 1 retards diabetes-associated renal injury.

Cell division autoantigen 1 (CDA1) enhances TGF-ß signaling in renal and vascular cells, and renal expression of CDA1 is elevated in animal models of diabetes. In this study, we investigated the genetic deletion of Tspyl2, the gene encoding CDA1, in C57BL6 and ApoE knockout mice. The increased renal expression of TGF-ß1, TGF-ß type I and II receptors, and phosphorylated Smad3 associated with diabetes in wild-type mice was attenuated in diabetic CDA1 knockout mice. Notably, CDA1 deletion significantly reduced diabetes-associated renal matrix accumulation and immunohistochemical staining for collagens III and IV and attenuated glomerular and tubulointerstitial injury indices, despite the presence of persistent hyperglycemia, polyuria, renal hypertrophy, and hyperfiltration. Furthermore, CDA1 deletion reduced gene expression of TGF-ß1 receptors in the kidney, resulting in a functionally attenuated response to exogenous TGF-ß, including reduced levels of phosphorylated Smad3 and ERK1/2, in primary kidney cells from CDA1 knockout animals. Taken together, these data suggest that CDA1 deletion reduces but does not block renal TGF-ß signaling. Because direct antagonism of TGF-ß or its receptors has unwanted effects, CDA1 may be a potential therapeutic target for retarding DN and perhaps, other kidney diseases associated with TGF-ß-mediated fibrogenesis.

Cell division autoantigen 1 enhances signaling and the profibrotic effects of transforming growth factor-ß in diabetic nephropathy.

Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-ß (TGF-ß) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-ß signaling, modulating the expression of TGF-ß, TGF-ß receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-ß together were synergistic in stimulating TGF-ß signaling and target gene expression. CDA1 knockdown effectively blocked TGF-ß-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-ß type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-ß signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-ß signaling in the kidney.

Ultrasound combined with microbubbles enhances the renoprotective effects of methylprednisolone in rats with adriamycin-induced nephropathy.

The purpose of this study was to investigate the effect of ultrasound combined with microbbules (SonoVueTM) on the potency of methylprednisolone in attenuating the renal injury induced by adriamycin in rats. Animal model was established by two intravenous injections of 4 mg/kg adriamycin with a 2-week interval in rats. One week later, the adriamycin injected rats were randomly divided into 7 groups, receiving various treatments daily for 2 weeks. Two doses of methylprednisolone (20 or 40 mg/kg) were administrated alone or 20 mg/kg methylprednisolone and 100 µL SonoVueTM microbbules (1-5 × 108 bubbles/mL; mean diameter of bubbles: 2.5 µm) was co-administrated by intravenous injections from the tail vein. The ultrasound was applied at a frequency of 0.8 MHz and a spatial average temporal average intensity of 2.79 W/cm2 for 5 min at a 50% duty cycle (1 s on 1 s off) on the back skin of the anatomic position of the kidney in rats of two groups combined with ultrasound. Renal injury were analyzed using immunohistochemical staining, real-time PCR, light and transmission electron microcopies. The kidney function related biochemical indexes were measured by automatic biochemistry analyzer. The results showed that adriamycin induced a typical renal injury and 40 mg/kg methylprednisolone injection significantly ameliorated the abnormality of key parameters such as proteinuria, renal mRNA and protein expression levels of nephrin, collagens III and IV as well as podocyte impairment, glomerulosclerosis and tubulointerstitial injury indexes. However, a sub-dose of methylprednisolone at 20 mg/kg was ineffective when administered intravenously, but its potency at this dosage was enhanced by co-administration with 100 µL SonoVueTM microbubbles plus ultrasound irradiation. In conclusion, ultrasound combined with microbubbles can significantly increase local renal drug delivery leading to enhanced therapeutic effect of low dose methylprednisolone in ameliorating adriamycin-induced nephropathy in rats.

Targeting the CDA1/CDA1BP1 Axis Retards Renal Fibrosis in Experimental Diabetic Nephropathy.

Targeting cell division autoantigen 1 (CDA1) is postulated to attenuate the profibrotic actions of transforming growth factor-ß in diabetic nephropathy. This study has identified a regulatory protein for CDA1 and has then used genetic and pharmacological approaches to test in vivo whether strategies to target this pathway would lead to reduced renal injury. A novel protein, named CDA1BP1 (CDA1 binding protein 1), was identified as critical in regulating the profibrotic activity of CDA1. Genetic deletion of CDA1BP1 attenuated key parameters of renal fibrosis in diabetic mice. Furthermore, a series of short synthetic CDA1BP1 peptides competitively inhibited CDA1-CDA1BP1 binding in vitro with a hybrid peptide, CHA-050, containing a 12mer CDA1BP1 peptide and a previously known "cell-penetrating peptide," dose-dependently reducing expression of collagens I and III in HK-2 cells. In vivo, a d-amino acid retro-inverso peptide, CHA-061, significantly attenuated diabetes-associated increases in the renal expression of genes involved in fibrotic and proinflammatory pathways. In a delayed intervention study, CHA-061 treatment reversed diabetes-associated molecular and pathological changes within the kidney. Specifically, CHA-061 significantly attenuated renal extracellular matrix accumulation and glomerular injury. Taken together, targeting the CDA1/CDA1BP1 axis is a safe, efficacious, and feasible approach to retard experimental diabetic nephropathy.

Diabetes Reduces Severity of Aortic Aneurysms Depending on the Presence of Cell Division Autoantigen 1 (CDA1).

Diabetes is a negative risk factor for aortic aneurysm, but the underlying explanation for this phenomenon is unknown. We have previously demonstrated that cell division autoantigen 1 (CDA1), which enhances transforming growth factor-ß signaling, is upregulated in diabetes. We hypothesized that CDA1 plays a key role in conferring the protective effect of diabetes against aortic aneurysms. Male wild-type, CDA1 knockout (KO), apolipoprotein E (ApoE) KO, and CDA1/ApoE double-KO (dKO) mice were rendered diabetic. Whereas aneurysms were not observed in diabetic ApoE KO and wild-type mice, 40% of diabetic dKO mice developed aortic aneurysms. These aneurysms were associated with attenuated aortic transforming growth factor-ß signaling, reduced expression of various collagens, and increased aortic macrophage infiltration and matrix metalloproteinase 12 expression. In the well-characterized model of angiotensin II-induced aneurysm formation, concomitant diabetes reduced fatal aortic rupture and attenuated suprarenal aortic expansion, changes not seen in dKO mice. Furthermore, aortic CDA1 expression was downregulated ∼70% within biopsies from human abdominal aortic aneurysms. The identification that diabetes is associated with upregulation of vascular CDA1 and that CDA1 deletion in diabetic mice promotes aneurysm formation provides evidence that CDA1 plays a role in diabetes to reduce susceptibility to aneurysm formation.

MSP8 is a non-essential merozoite surface protein in Plasmodium falciparum.

MSP8 is a recently identified merozoite surface protein that shares similar structural features with the leading vaccine candidate MSP1. Both proteins contain two C-terminal epidermal growth factor (EGF)-like domains, a glycosylphosphatidylinositol (GPI) anchor attachment sequence and undergo proteolytic processing. By double recombination, we have disrupted the MSP8 gene in P. falciparum 3D7 parasites, and confirmed integration by southern hybridisation and PCR. Western blot analysis of lysates from asynchronous cultures and isolated merozoites demonstrated the absence of MSP8 in two cloned knockout lines. There was no significant difference in growth rate observed between 3D7 and the cloned DeltaMSP8 lines. Thus, unlike MSP1, MSP8 is not required for asexual stage parasite growth and replication in vitro. Further analysis of the cloned lines showed that loss of MSP8 had no effect on the levels of expression of other merozoite surface proteins including MSP1-5, 7 and 10. Stage-specific immunoblots showed that MSP8 expression commences in late rings and extends throughout the rest of the erythrocytic life cycle in the 3D7 parent line, but is absent from all stages in the DeltaMSP8 transfectants.

Apical location of a novel EGF-like domain-containing protein of Plasmodium falciparum.

Using bioinformatics analyses of the unfinished malaria genome sequence, we have identified a novel protein of Plasmodium falciparum that contains two epidermal growth factor (EGF)-like domains near the C-terminus of the protein. The sequence contains a single open reading frame of 1572bp with the potential to encode a protein of 524 residues containing hydrophobic regions at the extreme N- and C-termini which appear to represent signal peptide and glycosylphosphatidylinositol (GPI)-attachment sites, respectively. RT-PCR analysis has confirmed that the novel gene is transcribed in asexual stages of P. falciparum. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4, MSP5 or MSP8 expressed as GST fusion proteins. Antisera to the C-terminal fragments react with two bands of 80 and 36kDa in P. falciparum parasite lysates whereas antisera to the most N-terminal fusion protein only recognises the 80kDa band, suggesting that the novel protein may undergo processing in a similar way to MSP1 and MSP8, but with fewer cleavage events. Immunoblot analysis of stage-specific parasite samples reveals that the protein is present in trophozoites, schizonts and in isolated merozoites. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localised to the surfaces of trophozoites, schizonts and free merozoites in an apical distribution. Based on the accepted nomenclature in the field we now designate this protein MSP10. We have shown that the MSP10 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited sequence diversity in an approximately lkb region of MSP10, encompassing the two EGF-like domains. A sequence similar to MSP10 can be identified in the available P. yoelii genomic sequence, offering the possibility of ascertaining whether this novel protein can induce host protective responses in an in vivo model.

[Investigation and analysis of the defense mechanism and domestic violence of male patients with somatization disorder].

OBJECTIVE: To evaluate the relationship between the psychological impediment of the patients with somatization disorder (SD) and domestic violence, and to enhance peoples recognition of this kind of patients' harmful action to others so as to attach more importance to the patients and to the improvement of mental health service of the community. METHODS: Questionnaire scoring was conducted among the wives of the SD group and the control group concerning domestic violence. It was also conducted in the SD group, the control group and the wives of the two groups concerning defense mechanism. RESULTS: The average score on demestic violence of the SD group was significantly higher than that of the normal control. Immature and intermediate defenses were more usual in the SD group than in the normal control (P < 0.01). CONCLUSION: Psychological impediment of SD patients is an important cause of domestic violence.

Antiproliferative autoantigen CDA1 transcriptionally up-regulates p21(Waf1/Cip1) by activating p53 and MEK/ERK1/2 MAPK pathways.

We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells arrests cell growth and inhibits DNA synthesis at S-phase. Here we show that CDA1-induced arrest of cell growth is accompanied by increases in protein and mRNA levels of the cyclin-dependent kinase (Cdk) inhibitor protein, p21(Waf1/Cip1) (p21). Both p21 induction and cell growth arrest are reversed when CDA1 expression is inhibited. CDA1 also increases p53 protein, but not its mRNA, in a time- and dose-dependent manner. MDM2, a ubiquitin ligase regulating p53 degradation, is inactivated by CDA1, suggesting that p53 protein accumulation is due to decreased protein degradation. Knockdown of p53, using siRNA targeting two sites of p53 mRNA, abrogates transcriptional induction of p21 by CDA1. Deletion of the p53 responsive element in the distal region of p21 promoter attenuates promoter activity in response to CDA1. DNA damage caused by camptothecin treatment increases mRNA and protein levels of CDA1, accompanied by induction of p53. The DNA damage-induced p53 induction is markedly attenuated by CDA1 knockdown. CDA1 induces phosphorylation of ERK1/2(p44/42), an activity blocked by PD98059 and U0126, inhibitors of the upstream kinase MEK1/2. The MEK inhibitors also block induction of p21 mRNA and abrogate p21 promoter activity stimulated by CDA1. Cell cycle kinases, Cdk1, -2, -4, and -6 are inhibited by CDA1 overexpression. We conclude that CDA1 induces p53- and MEK/ERK1/2 MAPK-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.