Single-Channel Analysis of TRPC Channels in the Podocytes of Freshly Isolated Glomeruli

Ilatovskaya, Daria V.; Staruschenko, Alexander

doi:10.1007/978-1-62703-351-0_28

Cited by 23 publications

(27 citation statements)

References 33 publications

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“…The glomeruli were separated from the renal biopsy tissues, which allowed us to more precisely identify the differentially expressed transcripts specifically to the glomerulus of IgAN. The glomeruli separating techniques were stable and commonly used in mice researches [21, 22]. Because human samples are difficult to obtain, there are no studies on the human glomerulus.…”

Section: Discussionmentioning

confidence: 99%

Functional networks of aging markers in the glomeruli of IgA nephropathy: a new therapeutic opportunity

et al. 2016

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IgA nephropathy(IgAN) is the most common primary glomerular disease in China. Primary infections always occur before IgAN. However, the pathology of IgAN is still unclear. Previously we found that LL37, a protein secreted by senescent cells, was specific for the progression of IgAN, and also played a role in the neutrophil function. So we hypothesized that the infiltration of neutrophils, inflammation factors, and aging markers, which were modulated by functional networks, induced the immune response and renal injury. RNA-Sequencing (RNA-seq) can be used to study the whole transcriptome and detect splicing variants that are expressed in a specific cell type or tissue. We separate glomerulus from the renal biopsy tissues. After RNA extraction, the sequences were analyzed with Illumina HiSeq 2000/2500. 381 genes with differential expression between the IgAN patients and the healthy controls were identified. Only PLAU, JUN, and FOS were related to DNA damage, telomere dysfunction-induced aging markers, neutrophil function and IgA nephropathy. The networks showed the possibility of these genes being connected. We conclude that DNA damage and telomere dysfunction could play important roles in IgA nephropathy. In addition, neutrophils are also important factors in this disease. The networks of these markers showed the mechanism pathways that are involved in the duration of the occurrence and progression of IgA nephropathy and might be a new therapeutic opportunity for disease treatment.

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Section: Discussionmentioning

confidence: 99%

Functional networks of aging markers in the glomeruli of IgA nephropathy: a new therapeutic opportunity

et al. 2016

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“…These microscope systems are less affordable but provide higher quality imaging and are more sensitive than widefield microscopy. Similar approach was also applied to study TRPC channels activity and calcium signaling in podocytes of freshly isolated glomeruli 17,46,47 . Other calcium imaging that could be applied include utilization of Fluo-8 as demonstrated on Figure 4B or ratiometric confocal measurement with Fluo4/FuraRed fluorescent dyes as described in Ilatovskaya et al 46 Technically, it is possible to perform both patch-clamp and calcium imaging simultaneously on the same cell if microscope is equipped with both setups.…”

Section: Discussionmentioning

confidence: 99%

“…2. Perform laparotomy and flush the kidneys with phosphate buffered saline (PBS) through the abdominal aorta 17 . 1.…”

Section: Isolation Of Renal Cysts and Connecting Tubules (Cnt)/collecmentioning

confidence: 99%

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Pavlov

Ilatovskaya

Palygin

et al. 2015

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Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca 2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca 2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays. Video LinkThe video component of this article can be found at

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“…For electrophysiological analysis, standard patch clamp approach in the cell attached configuration was used as previously described (9). Nicardipine (5 mM), niflumic acid (100 µM) and DIDS (100 µM) were added to the pipette solution directly before experiments to block N-type Ca 2+ and Ca 2+ -activated Cl − channels (10). …”

Section: Methodsmentioning

confidence: 99%

Two-photon imaging of endothelin-1-mediated intracellular Ca 2+ handling in smooth muscle cells of rat renal resistance arteries

et al. 2016

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Aims Endothelin-1 (ET-1) is a potent vasoconstrictor which regulates the physiology of cardiorenal system. The aim of this study was to evaluate ET-1-mediated elevation of intracellular Ca2+ in smooth muscle cells (SMC) of renal resistance arteries. Main methods In in vitro studies of primary SMC, which were isolated from rat renal microvessels, the levels of intracellular Ca2+ were calculated from the ratio of emissions at 340 and 380 nm after loading cells with Fura 2-AM dye. In ex vivo studies we used two-photon imaging of intracellular Ca2+ handling in renal resistance arteries excised from rat kidneys and loaded with fluorescent Ca2+ indicator Fluo-4 AM. Key findings The two-photon imaging demonstrates that treatment of isolated rat renal resistance arteries with ET-1 causes a rapid increase of intracellular Ca2+ concentration in smooth muscle vasculature of these vessels. These ex vivo observations are in accordance with in vitro findings indicating that ET-1 mediates activation of TRPC channels and increases the level of intracellular Ca2+ in cultured SMC to 510±83 nM. Significance ET-1-mediated elevation of intracellular Ca2+ is strongly linked to renal microvascular contraction and is crucial for ET-1-induced contraction of SMC. The two-photon imaging of intracellular Ca2+ in intact SMC of rat renal resistance arteries is a powerful technique which allows the detailed ex vivo analysis of intracellular Ca2+ handling by ET-1, an important player in hypertension-related kidney diseases.

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Single-Channel Analysis of TRPC Channels in the Podocytes of Freshly Isolated Glomeruli

Cited by 23 publications

References 33 publications

Functional networks of aging markers in the glomeruli of IgA nephropathy: a new therapeutic opportunity

Functional networks of aging markers in the glomeruli of IgA nephropathy: a new therapeutic opportunity

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Two-photon imaging of endothelin-1-mediated intracellular Ca 2+ handling in smooth muscle cells of rat renal resistance arteries

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