Enhancer and super-enhancer landscape in polycystic kidney disease.

Widespread aberrant gene expression is a pathological hallmark of polycystic kidney disease (PKD). Numerous pathogenic signaling cascades, including c-Myc, Fos, and Jun, are transactivated. However, the underlying epigenetic regulators are poorly defined. Here we show that H3K27ac, an acetylated modification of DNA packing protein histone H3 that marks active enhancers, is elevated in mouse and human samples of autosomal dominant PKD. Using comparative H3K27ac ChIP-Seq analysis, we mapped over 16000 active intronic and intergenic enhancer elements in Pkd1-mutant mouse kidneys. We found that the cystic kidney epigenetic landscape resembles that of a developing kidney, and over 90% of upregulated genes in Pkd1-mutant kidneys are co-housed with activated enhancers in the same topologically associated domains. Furthermore, we identified an evolutionarily conserved enhancer cluster downstream of the c-Myc gene and super-enhancers flanking both Jun and Fos loci in mouse and human models of autosomal dominant PKD. Deleting these regulatory elements reduced c-Myc, Jun, or Fos abundance and suppressed proliferation and 3D cyst growth of Pkd1-mutant cells. Finally, inhibiting glycolysis and glutaminolysis or activating Ppara in Pkd1-mutant cells lowerd global H3K27ac levels and its abundance on c-Myc enhancers. Thus, our work suggests that epigenetic rewiring mediates the transcriptomic dysregulation in PKD, and the regulatory elements can be targeted to slow cyst growth.

Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu.

We hypothesized that a functional apolipoprotein LI (APOL1)-miR193a axis (inverse relationship) preserves, but disruption alters, the podocyte molecular phenotype through the modulation of autophagy flux. Podocyte-expressing APOL1G0 (G0-podocytes) showed downregulation but podocyte-expressing APOL1G1 (G1-podocytes) and APOL1G2 (G2-podocytes) displayed enhanced miR193a expression. G0-, G1-, and G2-podocytes showed enhanced expression of light chain (LC) 3-II and beclin-1, but a disparate expression of p62 (low in wild-type but high in risk alleles). G0-podocytes showed enhanced, whereas G1- and G2-podocytes displayed decreased, phosphorylation of Unc-51-like autophagy-activating kinase (ULK)1 and class III phosphatidylinositol 3-kinase (PI3KC3). Podocytes overexpressing miR193a (miR193a-podocytes), G1, and G2 showed decreased transcription of PIK3R3 (PI3KC3's regulatory unit). Since 3-methyladenine (3-MA) enhanced miR193a expression but inhibited PIK3R3 transcription, it appears that 3-MA inhibits autophagy and induces podocyte dedifferentiation via miR193a generation. miR193a-, G1-, and G2-podocytes also showed decreased phosphorylation of mammalian target of rapamycin (mTOR) that could repress lysosome reformation. G1- and G2-podocytes showed enhanced expression of run domain beclin-1-interacting and cysteine-rich domain-containing protein (Rubicon); however, its silencing prevented their dedifferentiation. Docking, protein-protein interaction, and immunoprecipitation studies with antiautophagy-related gene (ATG)14L, anti-UV radiation resistance-associated gene (UVRAG), or Rubicon antibodies suggested the formation of ATG14L complex I and UVRAG complex II in G0-podocytes and the formation of Rubicon complex III in G1- and G2-podocytes. These findings suggest that the APOL1 risk alleles favor podocyte dedifferentiation through blockade of multiple autophagy pathways.

Modulation of apolipoprotein L1-microRNA-193a axis prevents podocyte dedifferentiation in high-glucose milieu.

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms' tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.

Perinatal Outcomes in Premature Placental Calcification and the Association of a Color Doppler Study: Report from a Tertiary Care Hospital in Eastern India.

Introduction: Placental calcification, identified before the 36th week of gestational age, is known as premature placental calcification (PPC). PPC could be a clue for the poor fetal outcome. However, its association with adverse perinatal outcomes is yet to be confirmed. Objective: The primary objective was to determine and compare the perinatal outcomes in pregnancies with and without documented premature placental calcification. Methodology: The present study was a prospective cohort study performed from October 2017 to September 2019. We consecutively enrolled 494 antenatal women who presented to our antenatal OPD after taking consent to participate in our study. Transabdominal sonographies were conducted between 28-36 weeks of gestation to document placental maturity. We compared maternal and fetal outcomes between those who were identified with grade III placental calcification (n = 140) and those without grade III placental calcification (n = 354). Results: The incidence of preeclampsia, at least one abnormal Doppler index, obstetrics cholestasis, placental abruption, and FGR (fetal growth restriction) pregnancies were significantly higher in the group premature placental calcification. We also found a significantly increased incidence of Low APGAR (Appearance, Pulse, Grimace, Activity, and Respiration) scores, NICU (Neonatal Intensive Care Unit) Admission, Abnormal CTG (cardiotocography), meconium-stained liquor, and low birth weight babies in those with grade III placental calcification. Conclusion: Clinicians should be aware of documenting placental grading while performing ultrasonography during 28 to 36 weeks. Ultrasonographically, the absence of PPC can define a subcategory of low-risk pregnant populations which probably need no referral to specialized centers and can be managed in these settings.

Interstitial microRNA miR-214 attenuates inflammation and polycystic kidney disease progression.

Renal cysts are the defining feature of autosomal dominant polycystic kidney disease (ADPKD); however, the substantial interstitial inflammation is an often-overlooked aspect of this disorder. Recent studies suggest that immune cells in the cyst microenvironment affect ADPKD progression. Here we report that microRNAs (miRNAs) are new molecular signals in this crosstalk. We found that miR-214 and its host long noncoding RNA Dnm3os are upregulated in orthologous ADPKD mouse models and cystic kidneys from humans with ADPKD. In situ hybridization revealed that interstitial cells in the cyst microenvironment are the primary source of miR-214. While genetic deletion of miR-214 does not affect kidney development or homeostasis, surprisingly, its inhibition in Pkd2- and Pkd1-mutant mice aggravates cyst growth. Mechanistically, the proinflammatory TLR4/IFN-γ/STAT1 pathways transactivate the miR-214 host gene. miR-214, in turn as a negative feedback loop, directly inhibits Tlr4. Accordingly, miR-214 deletion is associated with increased Tlr4 expression and enhanced pericystic macrophage accumulation. Thus, miR-214 upregulation is a compensatory protective response in the cyst microenvironment that restrains inflammation and cyst growth.

Molecular Docking Studies of a Cyclic Octapeptide-Cyclosaplin from Sandalwood.

Natural products from plants, such as chemopreventive agents, attract huge attention because of their low toxicity and high specificity. The rational drug design in combination with structure-based modeling and rapid screening methods offer significant potential for identifying and developing lead anticancer molecules. Thus, the molecular docking method plays an important role in screening a large set of molecules based on their free binding energies and proposes structural hypotheses of how the molecules can inhibit the target. Several peptide-based therapeutics have been developed to combat several health disorders, including cancers, metabolic disorders, heart-related diseases, and infectious diseases. Despite the discovery of hundreds of such therapeutic peptides however, only few peptide-based drugs have made it to the market. Moreover, the in silico activities of cyclic peptides towards molecular targets, such as protein kinases, proteases, and apoptosis related proteins have not been extensively investigated. In this study, we explored the in silico kinase and protease inhibitor potentials of cyclosaplin, and studied the interactions of cyclosaplin with other apoptosis-related proteins. Previously, the structure of cyclosaplin was elucidated by molecular modeling associated with dynamics that were used in the current study as well. Docking studies showed strong affinity of cyclosaplin towards cancer-related proteins. The binding affinity closer to 10 kcal/mol indicated efficient binding. Cyclosaplin showed strong binding affinities towards protein kinases such as EGFR, VEGFR2, PKB, and p38, indicating its potential role in protein kinase inhibition. Moreover, it displayed strong binding affinity to apoptosis-related proteins and revealed the possible role of cyclosaplin in apoptotic cell death. The protein-ligand interactions using LigPlot displayed some similar interactions between cyclosaplin and peptide-based ligands, especially in case of protein kinases and a few apoptosis related proteins. Thus, the in silico analyses gave the insights of cyclosaplin being a potential apoptosis inducer and protein kinase inhibitor.

Evaluation of Cyclosaplin Efficacy Using a Silk Based 3D Tumor Model.

Development of novel anti-cancer peptides requires a rapid screening process which can be accelerated by using appropriate in vitro tumor models. Breast carcinoma tissue is a three-dimensional (3D) microenvironment, which contains a hypoxic center surrounded by dense proliferative tissue. Biochemical clues provided by such a 3D cell mass cannot be recapitulated in conventional 2D culture systems. In this experiment, we evaluate the efficacy of the sandalwood peptide, cyclosaplin, on an established in vitro 3D silk breast cancer model using the invasive MDA-MB-231 cell line. The anti-proliferative effect of the peptide on the 3D silk tumor model is monitored by alamarBlue assay, with conventional 2D culture as control. The proliferation rate, glucose consumed, lactate dehydrogenase (LDH), and matrix metalloproteinase 9 (MMP-9) activity of human breast cancer cells are higher in 3D constructs compared to 2D. A higher concentration of drug is required to achieve 50% cell death in 3D culture than in 2D culture. The cyclosaplin treated MDA-MB-231 cells showed a significant decrease in MMP-9 activity in 3D constructs. Microscopic analysis revealed the formation of cell clusters evenly distributed in the scaffolds. The drug treated cells were less in number, smaller and showed unusual morphology. Overall, these findings indicate the role of cyclosaplin as a promising anti-cancer therapeutic.

Manipulation of renal gene expression using oligonucleotides.

Oligonucleotides are small molecules 8-50 nucleotides in length that bind via Watson-Crick base pairing to enhance or repress the expression of target RNA. The use of oligonucleotides to manipulate gene expression in the kidney could be a valuable tool to further understand kidney pathophysiology and can serve as an important complement to genetic studies. This chapter serves as a primer on the use of oligonucleotides in the kidney. We provide an overview of the various ways that oligonucleotides can manipulate gene expression. In addition, we describe the advancements in the development of oligonucleotides for laboratory and clinical use. Finally, instruction is provided on the design and implementation of oligonucleotides for in vitro and in vivo laboratory studies.

Grem2 mediates podocyte apoptosis in high glucose milieu.

BACKGROUND: Increased DAN protein (Grem1, Grem2, Grem3, Cerberus, NBL1, SOST, and USAG1) levels are often associated with severe disease-states in adult kidneys. Grem1, SOST, and USAG1 have been demonstrated to be upregulated and play a critical role in the progression of diabetic nephropathy (DN); however, the expression and the role of other DAN family members in DN have not been reported yet. In this study, we investigated the expression and the role of Grem2 in the development of renal lesions in mice with type 2 DN. METHODS: Fourteen-week-old BTBRob/ob (a mouse model of type 2 diabetes mellitus) and control (BTBR, wild type) mice were evaluated for renal functional and structural biomarkers. Urine was collected for protein content assay, and renal tissues were harvested for molecular analysis with real-time PCR, Western blotting, and immunohistochemistry. In vitro studies, human podocytes were transfected with Grem2 plasmid and were evaluated for apoptosis (morphologic assay and Western blotting). To evaluate the Grem2-mediated downstream signaling, the phosphorylation status of Smad2/3 and Smad1/5/8 was assessed. To establish a causal relationship, the effect of SIS3 (an inhibitor for Samd2/3) and BMP-7 (an agonist for Smad1/5/8) was evaluated on Germ2-induced podocyte apoptosis. RESULTS: BTBRob/ob mice showed elevated urinary protein levels. Renal tissues of BTBRob/ob mice showed an increased expression of Grem2; both glomerular and tubular cells displayed enhanced Grem2 expression. In vitro studies, high glucose increased Grem2 expression in cultured human podocytes, whereas, Grem2 silencing partially protected podocyte from high glucose-induced apoptosis. Overexpression of Grem2 in podocytes not only increased Bax/Bcl2 expression ratio but also promoted podocyte apoptosis; moreover, an overexpression of Grem2 increased the phosphorylation of Smad2/3 and decreased the phosphorylation of Smad1/5/8; furthermore, SIS3 and BMP-7 attenuated Grem2-induced podocyte apoptosis. CONCLUSIONS: High glucose increases Grem2 expression in kidney cells. Grem2 mediates podocyte apoptosis through Smads.

Nicotine enhances mesangial cell proliferation and fibronectin production in high glucose milieu via activation of Wnt/ß-catenin pathway.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Clinic reports indicate cigarette smoking is an independent risk factor for chronic kidney disease including DN; however, the underlying molecular mechanisms are not clear. Recent studies have demonstrated that nicotine, one of the active compounds in cigarette smoke, contributes to the pathogenesis of the cigarette smoking-accelerated chronic kidney disease. One of the characteristics of DN is the expansion of mesangium, a precursor of glomerular sclerosis. In the present study, we examined the involvement of Wnt/ß-catenin pathway in nicotine-mediated mesangial cell growth in high glucose milieu. Primary human renal mesangial cells were treated with nicotine in the presence of normal (5 mM) or high glucose (30 mM) followed by evaluation for cell growth. In the presence of normal glucose, nicotine increased both the total cell numbers and Ki-67 positive cell ratio, indicating that nicotine stimulated mesangial cell proliferation. Although high glucose itself also stimulated mesangial cell proliferation, nicotine further enhanced the mitogenic effect of high glucose. Similarly, nicotine increased the expression of Wnts, ß-catenin, and fibronectin in normal glucose medium, but further increased mesangial cell expression of these proteins in high glucose milieu. Pharmacological inhibition or genetic knockdown of ß-catenin activity or expression with specific inhibitor FH535 or siRNA significantly impaired the nicotine/glucose-stimulated cell proliferation and fibronectin production. We conclude that nicotine may enhance renal mesangial cell proliferation and fibronectin production under high glucose milieus partly through activating Wnt/ß-catenin pathway. Our study provides insight into molecular mechanisms involved in DN.

Imiquimod-induced psoriasis-like inflammation in differentiated Human keratinocytes: Its evaluation using curcumin.

Psoriasis is considered to be a systemic disease of immune dysfunction. It is still unclear what triggers the inflammatory cascade associated with psoriasis but recent evidences suggest the vital role of IL-23/IL-17A cytokine axis in etiology of psoriasis. Several studies have been conducted in psoriatic-like animal models but ethical issues and complexity surrounding it halts the screening of new anti-psoriatic drug candidates. Hence, in this study, we developed a new in-vitro model for psoriasis using imiquimod (IMQ) induced differentiated HaCaT cells which could be used for screening of new anti-psoriatic drug candidates. The differentiated HaCaT cells were treated with IMQ (100µM) to induce psoriatic like inflammation and its effect was investigated using a natural anti-psoriatic compound, curcumin. The proliferation of psoriatic-like cells was inhibited by curcumin at 25 and 50µM concentrations. The psoriatic-like cells decreased in number with increase in apoptotic and dead cells upon curcumin treatment. Curcumin inhibited the proliferation of IMQ-induced differentiated HaCaT cells (Psoriatic-like cells) by down-regulation of pro-inflammatory cytokines, interleukin-17, tumor necrosis factor-α, interferon-γ, and interleukin-6. Apart from this, curcumin significantly enhanced the skin-barrier function by up-regulation of involucrin (iNV) and filaggrin (FLG), the regulators of epidermal skin barrier. The IMQ-induced differentiated HaCaT in vitro model recapitulated some aspects of the psoriasis pathogenesis similar to murine model. Henceforth, we conclude that this model may be used for rapid screening of anti-psoriatic drug candidates and warrant further mechanistic studies.

Effect of APOL1 disease risk variants on APOL1 gene product.

Gene sequence mutations may alter mRNA transcription, transcript stability, protein translation, protein stability and protein folding. Apolipoprotein L1 (APOL1) has two sets of sequence variants that are risk factors for kidney disease development, APOL1G1 (substitution mutation) and APOL1G2 (deletion mutation). Our present study focuses on the impact of these variants on APOL1 mRNA transcription and translation. APOL1 plasmids (EV, G0, G1 and G2) were transfected into human embryonic kidney (HEK) 293T cells. APOL1 variant expression was observed to be significantly lower than that of APOL1G0. Podocyte cell lines stably expressing APOL1 transgenes also showed lower levels of APOL1 expression of APOL1 variants (G1 and G2) compared with APOL1G0 by Western blotting and FACS analysis. The enhanced expression of GRP78 by podocytes expressing APOL1 variants would indicate endoplasmic reticulum (ER) stress. Bioinformatics evaluation using two different programs (MUPro and I-Mutant 2.0) predicted that APOL1 variants were less stable than APOL1G0. Concomitant with protein levels, APOL1 mRNA levels were also depressed following induction of APOL1 variant compared with APOL1G0 in both proliferating and differentiated podocytes. APOL1 mRNA transcript stability was tested after actinomycin D pulsing; APOL1G1 and APOL1G2 mRNAs transcript decayed 10-15% and 15-20% (within a period of 0.5-3 h) respectively. Our data suggest that down-regulated APOL1 protein expression in APOL1 variants is due to compromised transcription and decay of the APOL1 variant transcripts.

Silk fibroin-Thelebolan matrix: A promising chemopreventive scaffold for soft tissue cancer.

Research of improved functional bio-mimetic matrix for regenerative medicine is currently one of the rapidly growing fields in tissue engineering and medical sciences. This study reports a novel bio-polymeric matrix, which is fabricated using silk protein fibroin from Bombyx mori silkworm and fungal exopolysaccharide Thelebolan from Antarctic fungus Thelebolus sp. IITKGP-BT12 by solvent evaporation and freeze drying method. Natural cross linker genipin is used to imprison the Thelebolan within the fibroin network. Different cross-linked and non-cross-linked fibroin/Thelebolan matrices are fabricated and biophysically characterized. Cross-linked thin films show robustness, good mechanical strength and high temperature stability in comparison to non-cross-linked and pure matrices. The 3D sponge matrices demonstrate good cytocompatibility. Interestingly, sustained release of the Thelebolan from the cross-linked matrices induce apoptosis in colon cancer cell line (HT-29) in time dependent manner while it is nontoxic to the normal fibroblast cells (L929).The findings indicate that the cross-linked fibroin/Thelebolan matrices can be used as potential topical chemopreventive scaffold for preclusion of soft tissue carcinoma.

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes.

Human skin is body's vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 µg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 µg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations.

Identification and structural characterization of a new pro-apoptotic cyclic octapeptide cyclosaplin from somatic seedlings of Santalum album L.

Small cyclic peptides exhibiting potent biological activity have great potential for anticancer therapy. An antiproliferative cyclic octapeptide, cyclosaplin was purified from somatic seedlings of Santalum album L. (sandalwood) using gel filtration and RP-HPLC separation process. The molecular mass of purified peptide was found to be 858 Da and the sequence was determined by MALDI-ToF-PSD-MS as 'RLGDGCTR' (cyclic). The cytotoxic activity of the peptide was tested against human breast cancer (MDA-MB-231) cell line in a dose and time-dependent manner. The purified peptide exhibited significant antiproliferative activity with an IC50 2.06 µg/mL. In a mechanistic approach, apoptosis was observed in differential microscopic studies for peptide treated MDA-MB-231 cells, which was further confirmed by mitochondrial membrane potential, DNA fragmentation assay, cell cycle analysis and caspase 3 activities. The modeling and docking experiments revealed strong affinity (kcal/mol) of peptide toward EGFR and procaspase 3. The co-localization studies revealed that the peptide sensitizes MDA-MB-231 cells by possibly binding to EGFR and induces apoptosis. This unique cyclic octapeptide revealed to be a favorable candidate for development of anticancer agents.

Isolation and characterization of extracellular polysaccharide Thelebolan produced by a newly isolated psychrophilic Antarctic fungus Thelebolus.

The present investigation is on a newly isolated psychrophilic Antarctic filamentous Ascomycetous fungus that has been identified as Thelebolus sp. and given the designation of Thelebolus sp. IITKGP-BT12. The culture was primarily identified through morphological studies, and was further confirmed by 18S rRNA sequencing (GenBank Accession No. KC191572), which revealed its close relatedness with Thelebolus microsporus. The exopolysaccharide (EPS) produced (1.94 g L(-1)) by the fungus was isolated, purified and characterized as glucan having an average molecular mass of 5×10(5)Da. The structure of EPS was determined by gas chromatography with tandem mass spectrometry (GC-MS/MS), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) studies ((1)H, (13)C and HSQC). NMR analysis indicated the presence of (1→3)-linked ß-d-glucan backbone with (1→6)-linked branches of ß-d-glucopyranosyl units. Antiproliferative activity of EPS was demonstrated in B16-F0 cells, with IC50 of 275.42 µg m L(-1). Flow cytometry analysis and DNA fragmentation studies revealed that the cytotoxic action of the EPS mediated apoptosis in cancer cells. This is the first ever report on bioactive EPS thelebolan from Thelebolus sp.