Pyramidal cell subtype-dependent cortical oscillatory activity regulates motor learning

The cortex processes information through intricate circuitry and outputs to multiple brain areas by different sets of pyramidal cells (PCs). PCs form intra- and inter-laminar subnetworks, depending on PC projection subtypes. However, it remains unknown how individual PC subtypes are involved in cortical network activity and, thereby, in distinct brain functions. Here, we examined the effects of optogenetic manipulations of specific PC subtypes on network activity in the motor cortex. In layer V, the beta/gamma frequency band of oscillation was evoked by photostimulation, depending on PC subtypes. Our experimental and simulation results suggest that oscillatory activity is generated in reciprocal connections between pyramidal tract (PT) and fast-spiking cells. A similar frequency band was also observed in local field potentials during a pattern learning task. Manipulation of PT cell activity affected beta/gamma band power and learning. Our results suggest that PT cell-dependent oscillations play important roles in motor learning.

Plasma Treated Water Solutions in Cancer Treatments: The Contrasting Role of RNS

Plasma Treated Water Solutions (PTWS) recently emerged as a novel tool for the generation of Reactive Oxygen and Nitrogen Species (ROS and RNS) in liquids. The presence of ROS with a strong oxidative power, like hydrogen peroxide (H2O2), has been proposed as the main effector for the cancer-killing properties of PTWS. A protective role has been postulated for RNS, with nitric oxide (NO) being involved in the activation of antioxidant responses and cell survival. However, recent evidences proved that NO-derivatives in proper mixtures with ROS in PTWS could enhance rather than reduce the selectivity of PTWS-induced cancer cell death through the inhibition of specific antioxidant cancer defenses. In this paper we discuss the formation of RNS in different liquids with a Dielectric Barrier Discharge (DBD), to show that NO is absent in PTWS of complex composition like plasma treated (PT)-cell culture media used for in vitro experiments, as well as its supposed protective role. Nitrite anions (NO2-) instead, present in our PTWS, were found to improve the selective death of Saos2 cancer cells compared to EA.hy926 cells by decreasing the cytotoxic threshold of H2O2 to non-toxic values for the endothelial cell line.

Renal proximal tubule cell state and metabolism are coupled by nuclear receptors

Kidney disease development is poorly understood due to the complex cellular interactions of more than 25 different cell types, each with specialized functions. Here, we used single cell RNA-sequencing to resolve cell type-specific and cell fraction changes in kidney disease.Whole kidney RNA-sequencing results were strongly influenced by cell fraction changes, but minimally informative to detect cell type-specific gene expression changes. Cell type-specific differential expression analysis identified proximal tubule cells (PT cells) as the key vulnerable cell type in diseased kidneys. Through unbiased cell trajectory analyses, we show that PT cell differentiation state is altered in disease state. Lipid metabolism (fatty acid oxidation and oxidative phosphorylation) in PT cells showed the strongest and reproducible association with PT cells state. The coupling of cell state and metabolism is established by nuclear receptors such as PPARA and ESRRA that not only control cellular metabolism but also the expression of PT cell-specific genes in mice and patient samples.

Proximal Tubular Transcription Factors in Acute Kidney Injury: Recent Advances

The proximal tubule (PT) is a major target in acute kidney injury (AKI), leading to profound changes in PT cell biology. Amongst the genes with early and robust changes in expression are many transcription factors (TFs), which themselves account for other transcriptomic changes. Potentially important TFs are being revealed in large sequencing datasets; however, to understand whether these TFs account for adaptive or maladaptive changes requires further mechanistic studies, which may reveal novel therapeutic targets. This mini review will highlight the identification and biology of 3 novel TFs in AKI: <i>Sox9</i>, <i>Foxm1</i>, and <i>Foxo3</i>.

RESULTS OF THE ULTRAMICROSCOPIC RESEARCH OF ADENOID VEGETATIONS ACCOMPANIED BY THE PATHOLOGIES OF NASAL CAVITY AND PARANASAL SINUSES IN ADULTS

The aim: Grounding on the electronic microscopy of PT make conclusions about the tonsil activity in adults depending on the accompanied pathology of nose and PS. Materials and methods: Ultramicroscopic examination of 111 patients with PT hypertrophy aged 18–55 was done. Depending on the nose and PS pathology (inflammatory, non-inflammatory) patients’ PS biopsic materials were distributed into 2 groups: 58 cases on inflammatory and 53 on non-inflammatory background. The control group consisted of 24 patients aged 18 without nose and PS pathology. The images of ultrathin PT sections were received with the help of transmission electronic microscope PEM – 125 with digital camera (SELMI, Sumy). Results: Great variations in PT cell condition, depending on the pathology were distinguished. Features of the adenoiditis in inflammatory and non-inflammatory diseases of nose and PS were proved. Conclusions: 1. PT ultra-microscopy in control group shows great activity of lymphocytes and high energy exchange of cells, with prevailing B-lymphocyte population. 2. The complex of PT ultra structural changes while nose and PS inflammatory diseases shows the activation of immune reaction in competent cells with T-lymphocyte increase in patients older than 25, which witnesses chronic inflammation. 3. In group with nose and PT non-inflammatory diseases, activity of PT B-lymphocytes is unchanged accompanied by the T-lymphocytes growth, which is also characteristic for chronic inflammation

Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model

BackgroundLowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn’t clear.MethodsWe examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish.ResultsOCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment.ConclusionsOur data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

Unraveling reno-protective effects of SGLT2 inhibition in human proximal tubular cells

Large clinical trials demonstrated that SGLT2 inhibitors (SGLT2i) slow the progression of kidney function decline in type 2 diabetes. Because the underlying molecular mechanisms are largely unknown, we studied the effects of SGLT2i on gene expression in two human proximal tubular (PT) cell lines under normoglycemic conditions, utilizing two SGLT2i, namely empagliflocin and canagliflocin. Genome-wide expression analysis did not reveal substantial differences between these two SGLT2i. Microarray hybridization analysis identified 94 genes that were both upregulated by TGF-β1 and downregulated by either of the two SGLT2i in HK-2 and RPTEC/TERT1 (renal proximal tubular epithelial cells/telomerase reverse transcriptase 1) cells. Extracellular matrix organization showed the highest significance in pathway enrichment analysis. Differential gene expression of three annotated genes of interest within this pathway was verified on mRNA level in both cell lines. Whereas TGF-β1 induced mRNA expression of thrombospondin 1 (THBS1; 4.3-fold), tenascin C (TNC; 8-fold), and platelet-derived growth factor subunit B (PDGF-B; 4.2-fold), SGLT2i downregulated basal mRNA expression of THBS1 (0.2-fold), TNC (0.5 fold), and PDGF-B (0.6-fold). Administration of SGLT2i in the presence of TGF-β1 resulted in a significant inhibition of TGF-β1-induced THBS1 and TNC mRNA expression and TGF-β1-induced THBS1, TNC, and PDGF-BB protein expression. We conclude that SGLT2i block basal and TGF-β1-induced expression of key mediators of renal fibrosis and kidney disease progression in two independent human PT cell lines.

3329 Hemoglobin selectively inhibits renal proximal tubular uptake of proteins: Implications for vitamin D deficiency and kidney disease in sickle cell disease

OBJECTIVES/SPECIFIC AIMS: While Hb-induced toxicity has been assumed to cause PT dysfunction, the development of tubular proteinuria from this dysfunction in SCD patients is not well understood. We previously found that free Hb, at concentrations predicted to be present chronically and during hemolytic crisis in the tubular filtrate of SCD patients, impairs uptake of albumin by PT cells via direct competition for binding to megalin and cubilin receptors. The purpose of this study is to further evaluate the consequences of increased filtered Hb concentrations on vitamin D reabsorption and activation. METHODS/STUDY POPULATION: We have developed a PT cell culture model that closely mimics in vivo PT cell structure, morphology, and endocytic capacity. Using this model, we treated cells with physiologic levels of cell-free Hb estimated in SCD and measured protein endocytosis and toxicity/oxidative stress. Endocytosis of fluorescently-tagged DBP and RBP were evaluated and quantified by confocal imaging and spectrofluorometry. Cellular toxicity and oxidative stress were assessed by measuring aconitase activity and accumulation of mitochondrial reactive oxygen species. RESULTS/ANTICIPATED RESULTS: PT cell uptake of DBP was significantly inhibited by both concentrations of Hb estimated to be filtered into the tubule lumen under chronic conditions (0.6μM Hb; 39% inhibition) and hemolytic crisis (≤20μM Hb; up to 92% inhibition) in SCD patients. ****p<0.0001 by one-way ANOVA, Dunnett’s multiple comparisons test. PT cell uptake of RBP was minimally affected by the same concentrations of Hb that profoundly inhibited internalization of DBP. RBP uptake was not significantly inhibited by all concentrations of Hb tested except the estimated hemolytic crisis maximum concentration (20μM; 27% inhibition). RBP uptake inhibition at 20μM Hb treatment was dramatically less than DBP uptake inhibition under the same treatment condition (27% RBP inhibition vs 92% DBP inhibition). *p<0.05 by one-way ANOVA, Dunnett’s multiple comparisons test. Mitochondrial oxidative stress, measured as a decrease in aconitase activity, was significantly increased in cells exposed to Hb (~43% aconitase activity reduction after 72h 20μM Hb treatment, and ~11% aconitase activity reduction after 72h 1μM Hb treatment). *p<0.05, **p<0.01 by one-sample t-test of the differences between treatment and untreated control conditions. We are currently assessing changes in PT cell vitamin D hydroxylase expression levels and vitamin D metabolism after exposure to chronic and hemolytic concentrations of Hb. Because oxidative stress has been previously reported to affect hydroxylase expression and activity, we expect to find a decrease in vitamin D hydroxylase expression and/or activity, resulting in decreased vitamin D activation. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that competition for megalin/cubilin binding between Hb and normally-filtered proteins, including DBP, may be the primary cause of tubular proteinuria in SCD patients. This inhibition appears to be selective for proteins that are largely α-helical in structure, such as albumin and DBP. Understanding the structural basis for Hb competition with filtered proteins for PT uptake could identify biomarkers to detect tubular proteinuria in SCD patients prior to the onset of kidney disease. This may also help develop therapeutic compounds that would selectively inhibit Hb binding to megalin/cubilin receptors. We have developed a highly sensitive fluorescence-based assay to test for such compounds. Inhibition of DBP uptake and vitamin D metabolism in the PT could lead or contribute to vitamin D deficiency. To our knowledge, our study is the first to suggest a mechanism for vitamin D deficiency commonly observed in SCD patients. Ongoing studies focus on measuring vitamin D metabolism in both cell and mouse models of SCD.

Variations in the adenohypophysis of the expression of proliferating cellular nuclear antigen, oestrogen and androgen receptors in relation to gonadal steroids during pregnancy of viscachas (Lagostomus maximus maximus)

Viscachas are native rodents of South America that present a long pregnancy of ~154 days. In this work, we analysed variations in the expression of proliferating cellular nuclear antigen, oestrogen and androgen receptors (ERα and AR) in pituitary pars distalis (PD) and pars tuberalis (PT) in relation to oestradiol and testosterone serum levels in non-pregnant and pregnant viscachas. In PD, cell proliferation increased with pregnancy and lactotrophs proliferated during mid-pregnancy (MP). ERα nuclear-immunoreactive cells (ERαn-ir) were maximal in late pregnancy and AR expression did not vary during pregnancy. In PT, cell proliferation and AR expression increased during pregnancy, but ERα expression was very scarce. The immunostaining pattern of receptors was different in PD and PT. The peak of serum oestradiol and testosterone occurred during MP. Our results suggest that cell proliferation and gonadal receptors might be differentially regulated in the pituitary by oestradiol and testosterone during viscacha pregnancy.