Butyrylcholinesterase signal sequence self-aggregates and enhances amyloid fibril formation in vitro.

Alzheimer's disease (AD) pathogenesis has been attributed to extracellular aggregates of amyloid ß (Aß) plaques and neurofibrillary tangles in the human brain. It has been reported that butyrylcholinesterase (BChE) also accumulates in the brain Aß plaques in AD. We have previously found that the BChE substitution in 5'UTR caused an in-frame N-terminal extension of 41 amino acids of the BChE signal peptide. The resultant variant with a 69 amino acid signal peptide, designated N-BChE, could play a role in AD development. Here, we report that the signal sequence of the BChE, if produced in an extended 69 aa version, can self-aggregate and could form seeds that enhance amyloid fibril formation in vitro in a dose-dependent manner and create larger co-aggregates. Similar phenomena could have been observed in the human brain if such an extended form of the signal sequence had been, in some circumstances, translated.

Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (LDLR) gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in the discovery of more than 3000 variants of the LDLR gene, but not all of them are clinically relevant. Therefore, functional studies of selected variants are needed for their proper classification. Here, a single-cell, kinetic, fluorescent LDL uptake assay was applied for the functional analysis of LDLR variants in a model of an LDLR-deficient human cell line. An LDLR-defective HEK293T cell line was established via a CRISPR/Cas9-mediated luciferase-puromycin knock-in. The expressing vector with the LDLR gene under the control of the regulated promoter and with a reporter gene has been designed to overproduce LDLR variants in the host cell. Moreover, an LDLR promoter-luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the LDLR gene, which can serve as a simple tool for screening and testing new HMG CoA reductase-inhibiting drugs for atherosclerosis therapy. The data presented here demonstrate that the obtained LDLR-deficient human cell line HEK293T-ldlrG1 and the dedicated pTetRedLDLRwt expression vector are valuable tools for studying LDL internalization and functional analysis of LDLR and its genetic variants. Using appropriate equipment, LDL uptake to a single cell can be measured in real time. Moreover, the luciferase gene knock-in downstream of the LDLR promotor allows the study of promoter regulation in response to diverse conditions or drugs. An analysis of four known LDLR variants previously classified as pathogenic and benign was performed to validate the LDLR-expressing system described herein with the dedicated LDLR-deficient human cell line, HEK293T-ldlrG1.

Endothelial dysfunction due to eNOS uncoupling: molecular mechanisms as potential therapeutic targets.

Nitric oxide (NO) is one of the most important molecules released by endothelial cells, and its antiatherogenic properties support cardiovascular homeostasis. Diminished NO bioavailability is a common hallmark of endothelial dysfunction underlying the pathogenesis of the cardiovascular disease. Vascular NO is synthesized by endothelial nitric oxide synthase (eNOS) from the substrate L-arginine (L-Arg), with tetrahydrobiopterin (BH4) as an essential cofactor. Cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, aging, or smoking increase vascular oxidative stress that strongly affects eNOS activity and leads to eNOS uncoupling. Uncoupled eNOS produces superoxide anion (O2-) instead of NO, thus becoming a source of harmful free radicals exacerbating the oxidative stress further. eNOS uncoupling is thought to be one of the major underlying causes of endothelial dysfunction observed in the pathogenesis of vascular diseases. Here, we discuss the main mechanisms of eNOS uncoupling, including oxidative depletion of the critical eNOS cofactor BH4, deficiency of eNOS substrate L-Arg, or accumulation of its analog asymmetrical dimethylarginine (ADMA), and eNOS S-glutathionylation. Moreover, potential therapeutic approaches that prevent eNOS uncoupling by improving cofactor availability, restoration of L-Arg/ADMA ratio, or modulation of eNOS S-glutathionylation are briefly outlined.

Endothelial Dysfunction Driven by Hypoxia-The Influence of Oxygen Deficiency on NO Bioavailability.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The initial stage of CVDs is characterized by endothelial dysfunction, defined as the limited bioavailability of nitric oxide (NO). Thus, any factors that interfere with the synthesis or metabolism of NO in endothelial cells are involved in CVD pathogenesis. It is well established that hypoxia is both the triggering factor as well as the accompanying factor in cardiovascular disease, and diminished tissue oxygen levels have been reported to influence endothelial NO bioavailability. In endothelial cells, NO is produced by endothelial nitric oxide synthase (eNOS) from L-Arg, with tetrahydrobiopterin (BH4) as an essential cofactor. Here, we discuss the mechanisms by which hypoxia affects NO bioavailability, including regulation of eNOS expression and activity. What is particularly important is the fact that hypoxia contributes to the depletion of cofactor BH4 and deficiency of substrate L-Arg, and thus elicits eNOS uncoupling-a state in which the enzyme produces superoxide instead of NO. eNOS uncoupling and the resulting oxidative stress is the major driver of endothelial dysfunction and atherogenesis. Moreover, hypoxia induces impairment in mitochondrial respiration and endothelial cell activation; thus, oxidative stress and inflammation, along with the hypoxic response, contribute to the development of endothelial dysfunction.

Pathogenesis of psoriasis in the "omic" era. Part IV. Epidemiology, genetics, immunopathogenesis, clinical manifestation and treatment of psoriatic arthritis.

Psoriatic arthritis (PsA) is a chronic, progressive, inflammatory arthropathy associated with psoriasis as well as a complex pathogenesis. Genetic and environmental factors trigger the development of the immune-mediated auto-inflammatory response in different sites: skin, bone marrow, entheses and synovial tissues. Studies of the last two decades have changed the view of PsA from a mild, non-progressive arthritis to an inflammatory systemic disease with serious health consequences, not only associated with joint dysfunction, but also with an increased risk of cardiovascular disease and socioeconomic consequences with significantly reduced quality of life. The joint damage starts early in the course of the disease, thus early recognition and treatment with modern biological treatments, which may modify the natural history and slow down progression of this debilitating disease, is essential for the patient long-term outcome.

Pathogenesis of psoriasis in the "omic" era. Part III. Metabolic disorders, metabolomics, nutrigenomics in psoriasis.

Psoriasis is a systemic disease that is strictly connected with metabolic disorders (insulin resistance, atherogenic dyslipidemia, arterial hypertension, and cardiovascular diseases). It occurs more often in patients with a more severe course of the disease. Obesity is specially an independent risk factor and it is associated with a worse treatment outcome because of the high inflammatory activity of visceral fatty tissue and the production of inflammatory mediators involved in the development of both psoriasis and metabolic disorders. However, in psoriasis the activation of the Th17/IL-17 and the abnormalities in the Th17/Treg balance axis are observed, but this pathomechanism does not fully explain the frequent occurrence of metabolic disorders. Therefore, there is a need to look for better biomarkers in the diagnosis, prognosis and monitoring of concomitant disorders and therapeutic effects in psoriasis. In addition, the education on the use of a proper diet as a prophylaxis for the development of the above disorders is an important element of holistic care for a patient with psoriasis. Diet may affect gene expression due to epigenetic modification which encompasses interactions of environment, nutrition and diseases. Patients with psoriasis should be advised to adopt proper diet and dietician support.

Pathogenesis of psoriasis in the "omic" era. Part II. Genetic, genomic and epigenetic changes in psoriasis.

Psoriasis is a multifactorial disease in which genetic, environmental and epigenetic factors regulating gene expression play a key role. In the "genomic era", genome-wide association studies together with target genotyping platforms performed in different ethnic populations have found more than 50 genetic susceptible markers associated with the risk of psoriasis which have been identified so far. Up till now, the strongest association with the risk of the disease has been proved for HLA-C*06 gene. The majority of other psoriasis risk SNPs are situated near the genes encoding molecules involved in adaptive and innate immunity, and skin barrier function. Many contemporary studies indicate that the epigenetic changes: histone modification, promoter methylations, long non-coding and micro-RNA hyperexpression are considered as factors contributing to psoriasis pathogenesis as they regulate abnormal keratinocyte differentiation and proliferation, aberrant keratinocytes - inflammatory cells communication, neoangiogenesis and chronic inflammation. The circulating miRNAs detected in the blood may become specific markers in the diagnosis, prognosis and response to the treatment of the disease. The inhibition of expression in selected miRNAs may be a new promising therapy option for patients with psoriasis.

Genome-wide mRNA profiling identifies RCAN1 and GADD45A as regulators of the transitional switch from survival to apoptosis during ER stress.

Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol-requiring protein 1, protein kinase RNA-like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human-immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18-h time course and utilized real-time monitoring of cell survival, next-generation sequencing, and quantitative real-time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage-inducible alpha (GADD45A), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT-enhancer-binding protein homologous protein (a.k.a DDIT3), BCL2-binding component 3 (a.k.a. BBC3), and phorbol-12-myristate-13-acetate-induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor.

PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells.

Small noncoding microRNAs (miRNAs) post-transcriptionally regulate a large portion of the human transcriptome. miRNAs have been shown to play an important role in the unfolded protein response (UPR), a cellular adaptive mechanism that is important in alleviating endoplasmic reticulum (ER) stress and promoting cell recovery. Another class of small noncoding RNAs, the Piwi-interacting RNAs (piRNAs) together with PIWI proteins, was originally shown to play a role as repressors of germline transposable elements. More recent studies, however, indicate that P-element induced WImpy proteins (PIWI proteins) and piRNAs also regulate mRNA levels in somatic tissues. Using genome-wide small RNA next generation sequencing, cell viability assays, and caspase activity assays in human airway epithelial cells, we demonstrate that ER stress specifically up-regulates total piRNA expression profiles, and these changes correlate with UPR-induced apoptosis as shown by up-regulation of two pro-apoptotic factor mRNAs, CHOP and NOXA. Furthermore, siRNA knockdown of PIWIL2 and PIWIL4, two proteins involved in piRNA function, attenuates UPR-related cell death, inhibits piRNA expression, and inhibits the up-regulation of CHOP and NOXA mRNA expression. Hence, we provide evidence that PIWIL2 and PIWIL4 proteins, and potentially the up-regulated piRNAs, constitute a novel epigenetic mechanism that control cellular fate during the UPR.

eNOS expression and NO release during hypoxia is inhibited by miR-200b in human endothelial cells.

The nitric oxide (NO) secreted by vascular endothelium is required for the maintenance of cardiovascular homeostasis. Diminished release of NO generated by endothelial NO synthase contributes to endothelial dysfunction. Hypoxia and ischemia reduce endothelial eNOS expression via posttranscriptional mechanisms that result in NOS3 transcript destabilization. Here, we examine whether microRNAs contribute to this mechanism. We followed the kinetics of hypoxia-induced changes in NOS3 mRNA and eNOS protein levels in primary human umbilical vein endothelial cells (HUVECs). Utilizing in silico predictive protocols to identify potential miRNAs that regulate eNOS expression, we identified miR-200b as a candidate. We established the functional miR-200b target sequence within the NOS3 3'UTR, and demonstrated that manipulation of the miRNA levels during hypoxia using miR-200b mimics and antagomirs regulates eNOS levels, and established that miR-200b physiologically limits eNOS expression during hypoxia. Furthermore, we demonstrated that the specific ablation of the hypoxic induction of miR-200b in HUVECs restored eNOS-driven hypoxic NO release to the normoxic levels. To determine whether miR-200b might be the only miRNA that had this effect, we utilized Next Generation Sequencing (NGS) to follow hypoxia-induced changes in the miRNA levels in HUVECS and found 83 novel hypoxamiRs, with two candidate miRNAs besides miR-200b that could potentially influence eNOS levels. Taken together, the data establish miR-200b-eNOS regulation as a first hypoxamiR-based mechanism that limits NO bioavailability during hypoxia in endothelial cells, and show that hypoxamiRs could become useful therapeutic targets for cardiovascular diseases and other hypoxic-related diseases including various types of cancer.

miRNAs regulate the HIF switch during hypoxia: a novel therapeutic target.

The decline of oxygen tension in the tissues below the physiological demand leads to the hypoxic adaptive response. This physiological consequence enables cells to recover from this cellular insult. Understanding the cellular pathways that mediate recovery from hypoxia is therefore critical for developing novel therapeutic approaches for cardiovascular diseases and cancer. The master regulators of oxygen homeostasis that control angiogenesis during hypoxia are hypoxia-inducible factors (HIFs). HIF-1 and HIF-2 function as transcriptional regulators and have both unique and overlapping target genes, whereas the role of HIF-3 is less clear. HIF-1 governs the acute adaptation to hypoxia, whereas HIF-2 and HIF-3 expressions begin during chronic hypoxia in human endothelium. When HIF-1 levels decline, HIF-2 and HIF-3 increase. This switch from HIF-1 to HIF-2 and HIF-3 signaling is required in order to adapt the endothelium to prolonged hypoxia. During prolonged hypoxia, the HIF-1 levels and activity are reduced, despite the lack of oxygen-dependent protein degradation. Although numerous protein factors have been proposed to modulate the HIF pathways, their application for HIF-targeted therapy is rather limited. Recently, the miRNAs that endogenously regulate gene expression via the RNA interference (RNAi) pathway have been shown to play critical roles in the hypoxia response pathways. Furthermore, these classes of RNAs provide therapeutic possibilities to selectively target HIFs and thus modulate the HIF switch. Here, we review the significance of the microRNAs on the relationship between the HIFs under both physiological and pathophysiological conditions.

miR-200b downregulates Kruppel Like Factor 2 (KLF2) during acute hypoxia in human endothelial cells.

The role of microRNAs in controlling angiogenesis is recognized as a promising therapeutic target in both cancer and cardiovascular disorders. However, understanding a miRNA's pleiotropic effects on angiogenesis is a limiting factor for these types of therapeutic approaches. Using genome-wide next-generation sequencing, we examined the role of an antiangiogenic miRNA, miR-200b, in primary human endothelial cells. The results indicate that miR-200b has complex effects on hypoxia-induced angiogenesis in human endothelia and importantly, that many of the reported miR-200b effects using miRNA overexpression may not be representative of the physiological role of this miRNA. We also identified the antiangiogenic KLF2 gene as a novel target of miR-200b. Our studies indicate that the physiological changes in miR-200b levels during acute hypoxia may actually have a proangiogenic effect through Klf2 downregulation and subsequent stabilization of HIF-1 signaling. Moreover, we provide a viable approach for differentiating direct from indirect miRNA effects in order to untangle the complexity of individual miRNA networks.

Activity and polymorphisms of butyrylcholinesterase in a Polish population.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 3'UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (Fluoride-Number) values outside the reference range (DN < 78 and FN < lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 5'UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity ≥6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes.

miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF)1A and HIF3A expression in human endothelial cells.

Hypoxia-inducible factors (HIF) are heterodimeric transcription factors that allow cells to adapt and survive during hypoxia. Regulation of HIF1A and HIF2A mRNA is well characterized, whereas HIF3A mRNA regulation and function are less clear. Using RNA-Seq analysis of primary human umbilical vein endothelial cells, we found two isoforms of HIF3A were expressed, HIF3A2 and HIF3A3. Comparing HIF3A expression profiles to HIF1A mRNA during 48 hours of hypoxia revealed that HIF1A message peaked at 4 hours, whereas HIF3A expression increased while HIF1A was decreasing. Given that HIF1A mRNA is regulated by miR-429, we tested miR-429 effects on both HIF3A isoforms and found that they too were regulated by miR-429. Analysis of a HIF-3 target, DNA-damage-inducible transcript 4, a key survival gene, indicated that DDIT4 mRNA is induced by HIF-3 and negatively regulated by miR-429 through miR-429's actions on HIF3A message. This provides a compelling model for how hypoxia-induced miR-429 regulates the switch between HIF-1 adaptive responses to HIF-3 survival responses by rapidly decreasing HIF1A levels while simultaneously slowing the progression of HIF3A expression until the miR-429 levels drop below normoxic levels. Since HIF-1 drives HIF3A and miR-429 expression, this establishes a regulatory network in which miR-429 plays a pivotal role.

Posttranscriptional and transcriptional regulation of endothelial nitric-oxide synthase during hypoxia: the role of microRNAs.

Understanding the cellular pathways that regulate endothelial nitric oxide (eNOS, NOS3) expression and consequently nitric oxide (NO) bioavailability during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. eNOS expression and eNOS-dependent NO cellular signaling during hypoxia promote an equilibrium of transcriptional and posttranscriptional molecular mechanisms that belong to both proapoptotic and survival pathways. Furthermore, NO bioavailability results not only from eNOS levels, but also relies on the presence of eNOS substrate and cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROS) that can inactivate eNOS. Since both NOS3 levels and these signaling pathways can also be a subject of posttranscriptional modulation by microRNAs (miRNAs), this class of short noncoding RNAs contribute another level of regulation for NO bioavailability. As miRNA antagomirs or specific target protectors could be used in therapeutic approaches to regulate NO levels, either by changing NOS3 mRNA stability or through factors governing eNOS activity, it is critical to understand their role in governing eNOS activity during hypoxa. In contrast to a large number of miRNAs reported to the change eNOS expression during hypoxia, only a few miRNAs modulate eNOS activity. Furthermore, impaired miRNA biogenesis leads to NOS3 mRNA stabilization under hypoxia. Here we discuss the recent studies that define miRNAs' role in maintaining endothelial NO bioavailability emphasizing those miRNAs that directly modulate NOS3 expression or eNOS activity.

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Concurrent DNA Copy-Number Alterations and Mutations in Genes Related to Maintenance of Genome Stability in Uninvolved Mammary Glandular Tissue from Breast Cancer Patients.

Somatic mosaicism for DNA copy-number alterations (SMC-CNAs) is defined as gain or loss of chromosomal segments in somatic cells within a single organism. As cells harboring SMC-CNAs can undergo clonal expansion, it has been proposed that SMC-CNAs may contribute to the predisposition of these cells to genetic disease including cancer. Herein, the gross genomic alterations (>500 kbp) were characterized in uninvolved mammary glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array-based comparative genomic hybridization showed 10% (6/59) of patients harbored one to 359 large SMC-CNAs (mean: 1,328 kbp; median: 961 kbp) in a substantial portion of glandular tissue cells, distal from the primary tumor site. SMC-CNAs were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-CNAs indicating genomic destabilization. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations and rare variants in genes related to maintenance of genomic integrity: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro), and TP53 (p.Arg306*). Co-occurrence of gross SMC-CNAs along with point mutations or rare variants in genes responsible for safeguarding genomic integrity highlights the temporal and spatial neoplastic potential of uninvolved glandular tissue in breast cancer patients.

Expression of membrane-bound NPP-type ecto-phosphodiesterases in rat podocytes cultured at normal and high glucose concentrations.

The ecto-nucleotide pyrophosphatase/phosphodiesterase family (E-NPPs) contains two membrane-bound members: E-NPP1 and E-NPP3. These enzymes mediate hydrolysis of extracellular nucleotides and their abnormal expression may affect intracellular signal transduction pathways, leading to cellular dysfunction, e.g., insulin resistance. Podocytes are insulin-dependent glomerular epithelial cells that regulate the glomerular filtration rate. Pathology of podocytes is a hallmark of diabetic nephropathy. Here, we investigated the expressions of E-NPP1 and E-NPP3 and activity of E-NPP enzymes in rat podocytes cultured with 5mM (NG) or 30 mM glucose (HG). Insulin resistance was determined by measuring changes in [1,2-(3)H]-deoxy-D-glucose uptake in response to insulin. mRNAs of E-NPP1 and E-NPP3 were detected within podocytes. The E-NPP expressions were confirmed at the protein level using western blot and immunofluorescence techniques. At NG, insulin (300 nM, 3 min) increased glucose uptake 1.5-fold; however, this effect was abolished at HG. The protein expressions of E-NPP1 and E-NPP3 were not affected at HG. The E-NPP activities were 24.68±0.72 and 26.51±1.55 nmol/min/mg protein at NG and HG, respectively. In conclusion, ecto-nucleotide pyrophosphatase/phosphodiesterase 1 and 3 are expressed on podocytes, but changes in expression of these enzymes are most likely not involved in etiology of insulin resistance in podocytes.