preparing paraffin sections of kidney and peyer&#39;s patch isolated from an igan murine model for histological and immunohistochemical analyses

Treating Immunoglobulin A Nephropathy (IgAN) with Dioscin in a Murine Model

IgA Nephropathy (IgAN) is the most common type of primary glomerulonephritis, for which there is no specific treatment, and it remains a significant cause of end-stage renal disease1. Although the pathogenesis of IgAN is still not fully understood, the &#34;multi-hit hypothesis&#34; is generally accepted and supported by a large body of clinical and experimental research evidence2. The pathogenesis of IgAN involves activating B cells and producing Galactose-deficient IgA1 (Gd-IgA1)3. The increase in circulating Gd-IgA1 due to the excessive proliferation and activation of IgA-secreting cells during the mucosal immune response is a critical link in the pathogenesis of IgAN4,5,6. As the central place for the proliferation and activation of B lymphocyte phenotype conversion to IgA-secreting cells, Peyer&#39;s patch is the primary source of IgA secretion, closely related to the occurrence and development of IgAN7,8. In addition, the proliferation of IgA1-secreting cells, as well as expression of Core 1&#946;-1,3-galactosyltransferase(C1GalT1) and C1GalT1-specific molecular chaperone (Cosmc), were associated with abnormal glycosylation of IgA1, which causes GD-IgA1 production in IgAN patients6,9.
Clinical study on IgAN treatment with herbal medicine has progressed in recent years. Yiqi Qingjie Formula is an essential formula for treating IgAN by the Department of Nephrology of Guang&#39;anmen Hospital. The previous study of our group found that Gd-IgA1 decreased in the serum of IgAN patients after treatment with Yiqi Qingjie Formula. As one of the most used herbs in the Yiqi Qingjie Formula, Dioscoreae Nipponicae Rhizoma (DNR) is the dried rhizome of Dioscorea Nipponica Makino, which has various functions such as regulating immunity, suppressing inflammation, relieving cough and asthma10,11. Several scholars treated IgAN with DNR and achieved good results12,13,14. As the main active ingredient in DNR15, Dioscin (DIO) lowers uric acid, inhibits fibrosis, inhibits inflammatory response, and anti-oxidative stress16,17. Therefore, DIO may have a novel action mechanism to inhibit the cellular secretion of excessive Gd-IgA1 and exert specific kidney protection effects. Still, no study has been reported on DIO&#39;s action mechanism for treating IgAN.
To explore the potential therapeutic mechanism of DIO on IgAN and provide a new method for the treatment of IgAN, we carried out experiments for the therapeutic effects of DIO on IgAN in vivo and in vitro.

Author Spotlight: Investigating the Potential of Chinese Herbal Medicinal Active Dioscin in Treating IgA Nephropathy

Dioscin Mediated IgA Nephropathy Alleviation by Inhibiting B Cell Activation In Vivo and Decreasing Galactose-Deficient IgA1 Production In Vitro

Our main research area is how Chinese herbal medicine works to treat chronic kidney disease like IGA nephropathy. In this study, based on our clinical experience and treatment of IGA nephropathy, using Dioscorea Nipponica Rhizoma, abbreviation DNR, and our research result of the main active substance of DNR, we try to confirm the potential value of dioscin as one of the main active substance of DNR. And Discover is possible mechanism of action in the treatment of IGA nephropathy through in vivo and in vitro experiments.Abnormal IgA1 production, that is the increased lactose deficient IgA1, is important in the pathogenesis of IgA nephropathy, and there is strong evidence that glycan associated lymphoid tissue plays a significant role in the production of galactose-deficient IgA1. In vitro experiments, we found that dioscin has a very distinct inhibitory effect on the proliferation of IgA-secreting cells, dark cell line at relatively low concentration, as well as an inhibitory effect on the secretion of GD-IgA1 by dark cells. These findings indicate that dioscin may have a potential therapeutic value for IGA nephropathy as well as disease linked to excessive B cell activation, such as systemic lupus erythematosus.Due to the complexity of Chinese herbal ingredients, the study of the therapeutic mechanism of Chinese herbal medicine faces considerable difficulties, especially in cell experiments in which a mechanism discussion like ours, using the main active ingredient of a non-herbal medicine as the representative substance of the drug may be commonly adopted research method. And we hope these kinds of works may provide the foundation for the discovery of new drugs for natural medicine.

Preparing Paraffin Sections of Kidney and Peyer&#39;s Patch Isolated from an IgAN Murine Model for Histological and Immunohistochemical Analyses

To begin preparing the paraffin sections of the kidney and Peyer's patch isolated from properly-anesthetized IgA neuropathy model mouse, first fix one Peyer's patch or three-millimeter-thick kidney tissues with 4%paraformaldehyde for 24 hours. Then dehydrate them using a gradient of ethanol and xylene before washing them with water. Submerge the tissues in wax for two hours before sealing and freezing them.Next, cut four-micron-thick Peyer's patch sections or two-micron-thick kidney sections and place them in warm water to unfold. Using a clean glass slide, gently pick up the unfolded slices and incubate them in a constant temperature oven at 40 degrees Celsius for one hour before staining.

preparing-paraffin-sections-kidney-peyers-patch-isolated-from-an-igan

Research

JoVE Journal

Medicine

231 vistas. Para comenzar a preparar las secciones de parafina del riñón y el parche de Peyer aislados de un ratón modelo de neuropatía IgA debidamente anestesiado, primero fije un parche de Peyer o tejidos renales de tres milímetros de grosor con 4% de paraformaldehído durante 24 horas. A continuación, deshidratarlos con un gradiente de etanol y xileno antes de lavarlos con agua. Sumerge los pañuelos en cera durante dos horas antes de sellarlos y congelarlos.

Video: Preparación de secciones de parafina de riñón y parche de Peyer aisladas de un modelo murino de IgAN para análisis histológicos e inmunohistoquímicos

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal immune responses, which is a critical link in the pathogenesis of IgA nephropathy (IgAN). Peyer&#39;s patch, the prominent place where B lymphocytes are transformed into IgA-secreting plasma cells, is the primary source of IgA. In addition, the lower expression of core 1&#946;-1,3-galactosyltransferase (C1GalT1) and its molecular chaperone, C1GalT1-specific molecular chaperone (Cosmc), is related to abnormal glycosylation of IgA1 in IgAN patients. Our clinical experience shows that Dioscoreae Nipponicae Rhizoma&#39;s (DNR) herbal medicine can relieve proteinuria and hematuria and improve renal function in IgAN patients. Dioscin (DIO) is one of the main active ingredients of DNR, which has various pharmacological activities. This study explores DIO&#39;s possible mechanism in treating IgAN.The IgAN model mouse was established by mucosal immune induction. The mice were divided into the control, model, and DIO gavage groups. The glomerular IgA deposition in mice, renal pathological changes, and B cell markers CD20 and CXCR5 expression in Peyer&#39;s patch were detected by immunofluorescence and immunohistochemistry. After lipopolysaccharide (LPS) stimulation, DIO&#39;s effects on DAKIKI cells proliferation, IgA and Gd-IgA1 secretion, C1GalT1, and Cosmc expression were studied by cell counting kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA) test, quantitative real-time polymerase chain reaction (QRT-PCR), and western blotting (WB). In in vivo studies, IgA deposition accompanied by glomerular mesangial hyperplasia and increased expression of CD20 and CXCR5 in Peyer&#39;s patch in the IgAN model mouse was alleviated by DIO. In vitro studies showed 0.25 &#181;g/mL to 1.0 &#181;g/mL DIO inhibited LPS-induced DAKIKI cell proliferation, IgA and Gd-IgA1 secretion, and up-regulated the mRNA and protein expression of C1GalT1 and Cosmc. This study demonstrates that DIO may reduce Gd-IgA1 production by inhibiting excessive activation of IgA-secreting cells and up-regulating C1GALT1/Cosmc expression.

This study provides experimental data for treating immunoglobulin A nephropathy (IgAN) with Dioscin (DIO), the active ingredient of Dioscoreae Nipponicae Rhizoma (DNR), and a paradigm for studying herbal medicine's effects and underlying mechanisms in vivo and in vitro.

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal ...

Preparing IgAN Model Mice for the Dioscin-Mediated IgA Nephropathy Alleviation Experiment

Begin preparing the immunoglobulin A, or IgA nephropathy and control models after subjecting the mice to one week of adaptive rearing in a laboratory cage. Then for nine weeks, feed the model mice with 0.1%bovine gamma globulin, or BGG solution, in six millimolar aqueous hydrochloric acid. Next, inject 0.1 milliliters of 0.1%BGG solution in saline into the model animals'tail vein for three consecutive days while continuing the feeding as before.After successfully preparing the IgA nephropathy model, on week nine, begin the dioscin or DIO intervention. To half of the mice from the model group selected as the DIO gavage group, feed appropriate drinks to the groups as demonstrated for eight weeks before proceeding with the subsequent analysis.

Histological Analysis of Kidney Tissue and Peyer&#39;s Patch Isolated from an IgAN Murine Model

After preparing the paraffin sections of the kidney and Peyer's patch isolated from immunoglobulin A or IgA nephropathy model mouse, proceed to dewax and stain the desired paraffin section using periodic acid solution at room temperature for 10 minutes while avoiding exposure to light. Rinse the section with distilled water before blotting it dry. Then stain the dry section with Schiff's staining solution for 20 to 30 minutes while avoiding exposure to light.Rinse the section with distilled water until it appears red under the microscope. Immerse the rinsed section in hematoxylin staining solution for three minutes to stain the nuclei and then rinse it under running water until the section becomes colorless. After performing routine dehydration with xylene and ethanol gradients, seal the section with neutral gum to examine it under a microscope.Compared with the control group, the mucosal immune induced IgA nephropathy mice model has visible IgA deposition in the entire mesangial region. PAS staining of the renal tissue showed mesangial cell proliferation and stromal hyperplasia in the model group, which was reduced in the dioscin gavage group.

Immunohistochemical Analysis of Peyer&#39;s Patch Isolated from an IgAN Murine Model

To begin dewaxing previously prepared paraffin sections of the Peyer's patch isolated from immunoglobulin A or IgA neuropathy model mouse, submerge each section sequentially in xylene and ethanol gradients for five minutes. Then, rinse the section in distilled water. For antigen retrieval, heat sodium citrate solution for two minutes in an autoclave.Then, place the tissue slice in the solution and heat it at a high temperature for five minutes. Once cooled down, incubate the section in a 3%hydrogen peroxide solution for 15 minutes at room temperature away from light. Then, apply 10%goat serum evenly on the tissue section and incubate for 30 minutes at room temperature to block the section.After shaking off the blocking solution, apply an appropriate amount of the prepared primary antibody to each section. Incubate the sections laid flat in a wet box overnight at four degrees Celsius. On the next day, cover the tissue with a drop of the HRP-labeled secondary antibody and incubate it at room temperature for 50 minutes.Then, apply freshly prepared DAB chromogenic solution to each section and wait til color development. Restain the section with hematoxylin for approximately one minute. Subsequently rinse the sections for 10 minutes with tap water until they return to a blue color.For dehydration, place each section sequentially in gradients of ethanol and xylene for five minutes each. Once all the sections are dried slightly, seal each section with neutral gum. Immunohistochemical results showed that B-cell markers CD20 and CXCR5 expression were significantly higher in the IgA neuropathy model group, compared with the control group.However, dioscin could inhibit the expression of the molecular markers.

Assays for Screening Safe Concentrations and Effects of DIO on Normal DAKIKI Cells

To begin, seed DAKIKI cells in 96-well plates at a density of 0.4 million cells per well. Set up the different experimental groups, and after, the corresponding treatments based on the grouping method. Incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide.For the lactate dehydrogenase cytotoxicity assay, add five microliters of lysate to each well in the high control group. And incubate the plate at 5%carbon dioxide and 37 degrees Celsius for 15 minutes. Then, add 100 microliters of the reaction mixture to each well.To quench the reaction, add 50 microliters of the stop reaction solution per well. For the Cell Counting Kit-8, or CCK-8, assay, after the initial 24-hour incubation, add 20 microliters of CCK-8 reagent to each well. And return the plates to the incubator at 5%carbon dioxide and 37 degrees Celsius for two hours.The results of the lactate dehydrogenase cytotoxicity assay showed insignificant cytotoxicity induced by dioscin at concentrations of 0.25 to 1.0 micrograms per milliliter, which had lactate dehydrogenase release rates below 10%CCK-8 assay showed that, compared with the model group, dioscin inhibited LPS-induced DAKIKI cell proliferation in a concentration-dependent manner, with the concentrations of 0.5 and 1.0 micrograms per milliliter showing significant inhibition.

Detecting the Effect of DIO on the mRNA and Protein Levels of C1GALT1 and Cosmc in DAKIKI Cells

To begin, seed DAKIKI cells in a six-well plate at a density of six million cells per well. Set up the different experimental groups, and after the corresponding treatment, based on the grouping method, incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Extract total RNA from the DAKIKI cells, following the instructions in the total RNA extraction kit.Take one microliter of the extracted RNA from each group of samples to measure its concentration. Reverse transcribe one microgram of total RNA from each sample into complementary DNA, following the kit instructions. Perform reverse transcription polymerase chain reaction, or RT-PCR amplification to detect the expression of each gene.For western blotting after the initial 24-hour incubation, collect the cells from each group. Add an appropriate volume of cell lysis solution to the collected cells before incubating them on ice for 30 minutes. Centrifuge the samples at 13, 500g for 10 minutes at four degrees Celsius, and collect the supernatant for further analysis.Vortex the protein samples with 5X SDS-PAGE Loading Buffer at a ratio of four to one and heat the mixed samples at 100 degrees Celsius for five minutes to denature the protein before electrophoresis. Once the run is complete, transfer the gel onto the PVDF membranes. To block the membrane, incubate it in 5%nonfat milk for two hours at room temperature.Then, incubate the membrane with the corresponding primary antibodies for 24 hours while using beta-actin antibody as an internal control. Subsequently, incubate the membranes with the corresponding secondary goat anti-rabbit IgG antibody at room temperature for two hours. Finally, treat the membranes with an appropriate amount of enhanced chemoluminescence working solution for protein band detection.The quantitative RT-PCR results showed that the relative mRNA expression of C1GalT1 and Cosmc was downregulated in DAKIKI cells in the model group, compared with the control group. Dioscin upregulated the relative mRNA of both two different degrees compared with the model group. Western blotting results also showed that compared with the control group, the protein expression of C1GalT1 and Cosmc in DAKIKI cells in the model group decreased.The protein expressions were upregulated after dioscin intervention.