Sumanth Putta

TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN.

Akt kinase is activated by transforming growth factor-β1 (TGF-β) in diabetic kidneys, and has important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in glomerular mesangial cells by inducing the microRNAs (miRNAs) miR-216a and miR-217, both of which target PTEN (phosphatase and tensin homologue), an inhibitor of Akt activation. These miRNAs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and miR-192 through E-box-regulated mechanisms, as shown previously. Akt activation by these miRs led to glomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-β. These studies reveal a mechanism of Akt activation through PTEN downregulation by two miRs, which are regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

Inhibiting MicroRNA-192 Ameliorates Renal Fibrosis in Diabetic Nephropathy

TGF-β1 upregulates microRNA-192 (miR-192) in cultured glomerular mesangial cells and in glomeruli from diabetic mice. miR-192 not only increases collagen expression by targeting the E-box repressors Zeb1/2 but also modulates other renal miRNAs, suggesting that it may be a therapeutic target for diabetic nephropathy. We evaluated the efficacy of a locked nucleic acid (LNA)-modified inhibitor of miR-192, designated LNA-anti-miR-192, in mouse models of diabetic nephropathy. LNA-anti-miR-192 significantly reduced levels of miR-192, but not miR-194, in kidneys of both normal and streptozotocin-induced diabetic mice. In the kidneys of diabetic mice, inhibition of miR-192 significantly increased Zeb1/2 and decreased gene expression of collagen, TGF-β, and fibronectin; immunostaining confirmed the downregulation of these mediators of renal fibrosis. Furthermore, LNA-anti-miR-192 attenuated proteinuria in these diabetic mice. In summary, the specific reduction of renal miR-192 decreases renal fibrosis and improves proteinuria, lending support for the possibility of an anti-miRNA-based translational approach to the treatment of diabetic nephropathy.

Post-transcriptional Up-regulation of Tsc-22 by Ybx1, a Target of miR-216a, Mediates TGF-β-induced Collagen Expression in Kidney Cells

Increased accumulation of extracellular matrix proteins and hypertrophy induced by transforming growth factor-β1 (TGF-β) in renal mesangial cells (MC) are hallmark features of diabetic nephropathy. Although the post-transcriptional regulation of key genes has been implicated in these events, details are not fully understood. Here we show that TGF-β increased microRNA-216a (miR-216a) levels in mouse MC, with parallel down-regulation of Ybx1, a miR-216a target and RNA-binding protein. TGF-β also enhanced protein levels of Tsc-22 (TGF-β-stimulated clone 22) and collagen type I α-2 (Col1a2) expression in MC through far upstream enhancer E-boxes by interaction of Tsc-22 with an E-box regulator, Tfe3. Ybx1 colocalized with processing bodies in MC and formed a ribonucleoprotein complex with Tsc-22 mRNA, and this complex formation was reduced by TGF-β, miR-216a mimics, or Ybx1 shRNA to increase Tsc-22 protein levels but enhanced by miR-216a inhibitor oligonucleotides. Chromatin immunoprecipitation (ChIP) assays revealed that TGF-β could increase the occupancies of Tsc-22 and Tfe3 on enhancer E-boxes of Col1a2. Co-immunoprecipitation assays revealed that TGF-β promoted the interaction of Tsc-22 with Tfe3. These results demonstrate that post-transcriptional regulation of Tsc-22 mediated through Ybx1, a miR-216a target, plays a key role in TGF-β-induced Col1a2 in MC related to the pathogenesis of diabetic nephropathy.

TGF-β Induces Acetylation of Chromatin and of Ets-1 to Alleviate Repression of miR-192 in Diabetic Nephropathy

Acetylation of chromatin and Ets-1 contributes to induction of miR-192, a microRNA involved in kidney pathogenesis. Kidney Fibrosis Through Acetylation Epigenetic changes alter gene expression and can induce pathogenesis. MicroRNA-192 (miR-192) mediates the transcription of genes involved in kidney fibrosis in response to high glucose–induced signaling by TGF-β (transforming growth factor–β). Kato et al. found that in basal conditions, Ets-1 was bound to chromatin upstream of miR-192 and suppressed its expression in murine kidney mesangial cells. After treatment with high glucose or TGF-β, Ets-1 was acetylated in an Akt- and p300-dependent manner and dissociated from miR-192. Although Ets-1 was dispensable for transiently increased expression of miR-192, Ets-1 deficiency in mice or murine mesangial cells prevented the sustained expression of miR-192 in response to TGF-β. Activation of p300 and acetylation of Ets-1 and of histone H3 at lysines 9 and 14 were increased in diabetic db/db mice compared with wild-type mice, suggesting that alleviation of Ets-1 repression may contribute to diabetic nephropathy. MicroRNAs (miRNAs), such as miR-192, mediate the actions of transforming growth factor–β1 (TGF-β) related to the pathogenesis of diabetic kidney diseases. We found that the biphasic induction of miR-192 expression by TGF-β in mouse renal glomerular mesangial cells initially involved the Smad transcription factors, followed by sustained expression that was promoted by acetylation of the transcription factor Ets-1 and of histone H3 by the acetyltransferase p300, which was activated by the serine and threonine kinase Akt. In mesangial cells from Ets-1–deficient mice or in cells in which Ets-1 was knocked down, basal amounts of miR-192 were higher than those in control cells, but sustained induction of miR-192 by TGF-β was attenuated. Furthermore, inhibition of Akt or ectopic expression of dominant-negative histone acetyltransferases decreased p300-mediated acetylation and Ets-1 dissociation from the miR-192 promoter and prevented miR-192 expression in response to TGF-β. Activation of Akt and p300 and acetylation of Ets-1 and histone H3 were increased in glomeruli from diabetic db/db mice compared to nondiabetic db/+ mice, suggesting that this pathway may contribute to diabetic nephropathy. These findings provide insight into the regulation of miRNAs through signaling-mediated changes in transcription factor activity and in epigenetic histone acetylation under normal and disease states.

Regulation of Inflammatory Phenotype in Macrophages by a Diabetes-Induced Long Noncoding RNA

The mechanisms by which macrophages mediate the enhanced inflammation associated with diabetes complications are not completely understood. We used RNA sequencing to profile the transcriptome of bone marrow macrophages isolated from diabetic db/db mice and identified 1,648 differentially expressed genes compared with control db/+ mice. Data analyses revealed that diabetes promoted a proinflammatory, profibrotic, and dysfunctional alternatively activated macrophage phenotype possibly via transcription factors involved in macrophage function. Notably, diabetes altered levels of several long noncoding RNAs (lncRNAs). Because the role of lncRNAs in diabetes complications is unknown, we further characterized the function of lncRNA E330013P06, which was upregulated in macrophages from db/db and diet-induced insulin-resistant type 2 diabetic (T2D) mice, but not from type 1 diabetic mice. It was also upregulated in monocytes from T2D patients. E330013P06 was also increased along with inflammatory genes in mouse macrophages treated with high glucose and palmitic acid. E330013P06 overexpression in macrophages induced inflammatory genes, enhanced responses to inflammatory signals, and increased foam cell formation. In contrast, small interfering RNA–mediated E330013P06 gene silencing inhibited inflammatory genes induced by the diabetic stimuli. These results define the diabetic macrophage transcriptome and novel functional roles for lncRNAs in macrophages that could lead to lncRNA-based therapies for inflammatory diabetes complications.

Effects of cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase on parameters of diabetic nephropathy in a mouse model of type 1 diabetes

We previously showed that the 12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolism is involved in multiple events related to diabetic nephropathy (DN), including glomerular hypertrophy and extracellular matrix deposition (Kang SW, Adler SG, Nast CC, LaPage J, Gu JL, Nadler JL, Natarajan R. Kidney Int 59: 1354-1362, 2001; Kang SW, Natarajan R, Shahed A, Nast CC, LaPage J, Mundel P, Kashtan C, Adler SG. J Am Soc Nephrol 14: 3178-3187, 2003; Kim YS, Lanting L, Adler SG, Natarajan R. Kindney Int 64: 1702-1714, 2003; Reddy MA, Adler SG, Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R. Am J Physiol Renal Physiol 283: F985-F994, 2002). In this study, we investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting 12/15-LO can ameliorate renal injury and DN in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo access and siRNA expression in the kidney, we used double-stranded 12/15-LO siRNA oligonucleotides conjugated with cholesterol. Diabetic DBA/2J mice were injected subcutaneously with either cholesterol-tagged 12/15-LO siRNA, mismatched control siRNA, or vehicle alone, twice weekly for 7 wk. Relative to controls, mice that received 12/15-LO siRNA showed significant reduction in albuminuria, kidney-to-body weight ratios, glomerular mesangial matrix expansion, renal structural damage, and monocyte/macrophage infiltration. These effects were associated with lower renal cortical or glomerular levels of profibrotic markers transforming growth factor-beta, connective tissue growth factor, type I and type IV collagens, plasminogen activator inhibitor 1, and fibronectin. The diabetes-induced increase in glomerular cyclin-dependent kinase inhibitors that are associated with hypertrophy was also prevented by siRNA administration. Our results show for the first time that systemic delivery of cholesterol-tagged siRNAs targeting 12/15-LO has renoprotective effects under diabetic conditions and therefore could be a novel therapeutic approach for DN.

Small RNA sequencing reveals microRNAs that modulate angiotensin II effects in vascular smooth muscle cells

Background: Role of microRNAs in angiotensin II-mediated vascular smooth muscle cell (VSMC) dysfunction is unclear. Results: Angiotensin II up-regulates miR-132 in VSMC. miR-132 induces MCP-1 partly via targeting PTEN, activates CREB, and regulates genes related to cell-cycle and motility. Conclusion: miR-132/212 is a novel modulator of Ang II actions. Significance: miRNAs may serve as new drug targets for Ang II-mediated cardiovascular diseases. Angiotensin II (Ang II)-mediated vascular smooth muscle cell dysfunction plays a critical role in cardiovascular diseases. However, the role of microRNAs (miRNAs) in this process is unclear. We used small RNA deep sequencing to profile Ang II-regulated miRNAs in rat vascular smooth muscle cells (VSMC) and evaluated their role in VSMC dysfunction. Sequencing results revealed several Ang II-responsive miRNAs, and bioinformatics analysis showed that their predicted targets can modulate biological processes relevant to cardiovascular diseases. Further studies with the most highly induced miR-132 and miR-212 cluster (miR-132/212) showed time- and dose-dependent up-regulation of miR-132/212 by Ang II through the Ang II Type 1 receptor. We identified phosphatase and tensin homolog (PTEN) as a novel target of miR-132 and demonstrated that miR-132 induces monocyte chemoattractant protein-1 at least in part via PTEN repression in rat VSMC. Moreover, miR-132 overexpression enhanced cyclic AMP-response element-binding protein (CREB) phosphorylation via RASA1 (p120 Ras GTPase-activating protein 1) down-regulation, whereas miR-132 inhibition attenuated Ang II-induced CREB activation. Furthermore, miR-132 up-regulation by Ang II required CREB activation, demonstrating a positive feedback loop. Notably, aortas from Ang II-infused mice displayed similar up-regulation of miR-132/212 and monocyte chemoattractant protein-1, supporting in vivo relevance. In addition, microarray analysis and reverse transcriptase-quantitative PCR validation revealed additional novel miR-132 targets among Ang II-down-regulated genes implicated in cell cycle, motility, and cardiovascular functions. These results suggest that miR132/212 can serve as a novel cellular node to fine-tune and amplify Ang II actions in VSMC.

The importance of direct and frequent communication

This scenario addresses the standards of veterinary care at an institution with USDA registration for use of covered species and an NIH/OLAW assurance. As described, the institution received a citation for inadequate veterinary care due to lack of follow up and not prescribing or discussing the use of antibiotics after a wound repair on a pig. According to the AAALAC’s position statement, “it is expected that the program of veterinary care will uphold the highest standards of care and ethics.”1 This is required to both maintain animal welfare and achieve valid scientific data. Although it is not uncommon for a principal investigator (PI) to make the decision to euthanize based on their experimental goals and concerns for confounding variables, this situation may have been avoided with appropriate veterinary follow up. Dr. Meyers left instructions to call him if there were any concerns, however, three weeks between visits is not adequate follow up for a sutured wound that may dehisce or become infected. At minimum, Dr. Meyers should have copied the PI on communications post-evaluation and discussed the potential for wound infection and the use of antibiotics as needed. From the given information, it is not clear who was directly responsible for checking and documenting care of the pig. These details should have been clearly stated in the instructions left by Dr. Meyers so that the PI, facility manager, or animal care personnel could have checked on the pig and communicated with Dr. Meyers. The USDA Animal Welfare Act and Animal Welfare Regulations2 states that “daily observation of animals may be accomplished by someone other than the attending veterinarian provided that a mechanism for direct and frequent communication is in place so that timely and accurate information on problems of animal health, behavior, and well-being is conveyed to the attending veterinarian.” Although the Regulations do not specifically require the use of antibiotics, there was a lack of communication and veterinary follow-up. Therefore, the school should accept the citation and reevaluate their medical practices to prevent this problem from recurring. The institute should evaluate the written program of veterinary care description and discuss expectations of the attending veterinarian going forward. The ACLAM Guidelines for Adequate Veterinary Care3 mention that arrangements must be made to assure veterinary services are readily available to meet routine or emergency needs. The attending veterinarian should be prepared to make more frequent visits as necessary. Additionally, the animal care personnel, vivarium manger, and PI should be trained to recognize what is considered abnormal and when Dr. Meyers should be contacted outside of his regularly scheduled visits. This is especially essential to a program that does not have on-site veterinary staff to perform clinical rounds and assess the animal’s recovery daily. Communication must be maintained between the veterinarian, the animal care personnel, and the PI to achieve the common goal: a high standard of animal welfare and good, valid scientific data. Finally, the facility should also reevaluate their current housing conditions to make necessary changes to prevent this from happening in the future4. ❐

Noncompliance in survival surgery technique: report to OLAW.

to Office of Laboratory Animal Welfare (OLAW) depends on whether the study was funded through the Public Health Service, unless Great Eastern’s OLAW Assurance Statement indicates that it will report all animal-related incidents regardless of funding sources1. The scenario does not specify which species was being used, but this incident may be reportable to the United States Department of Agriculture if it involved a species covered by the Animal Welfare Act or the use in biomedical research of an animal species typically used for food or fiber. Great Eastern’s surgica l t raining program seems to have been approved by its IACUC, as it is covered by a protocol. If the approved surgical training protocol defines the parameters of acceptable aseptic surgical technique, it may not be necessary to further specify those accepted and approved practices in an individual protocol or IACUC policy, unless the technique is an exception to that standard of aseptic surgery that must be approved by the IACUC.