PART Ⅰ：The Inflammatory Process Modulates The Expression And Localization Of WT1 in Podocytes Leading To Kidney Damage
Mar 23, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
MARIELA ARELLANO-RODRÍGUEZ, PABLO ZAPATA-BENAVIDES & ET AL.
Introduction
Wilm's tumor-1 gene(WTI) is one of the main genes involved in the survival of podocytes and inefficient renal filtration. Inflammatory processes lead to kidney failure and this might possibly occur through modulation of WT1(Wilm's tumor-1 gene). WT1(Wilm's tumor-1 gene) encodes a transcription factor, a zinc finger protein that has four major isoforms by alternative splicing in exon 5(17AA±)and exon 9(KTS±)(1). The KTS-isoform has a higher affinity for DNA, KTS+ co-localizes with spliceosome proteins in the cytoplasm (2, 3).WT1(Wilm's tumor-1 gene) plays a crucial role in embryogenesis, primarily in gonadal and kidney development; in adults, its expression is limited to specialized visceral cells in the kidney (podocytes)and is essential for maintenance and modulating several genes, such as podocalyxin, nephrin(NPHS1), and paired-box transcription factor (4-6). Mutations or reduction of WTl expression may lead to reduced expression of nephrin and podocalyxin and is associated with nephropathies such as glomerulosclerosis(5, 7-10).
Nephrotic syndrome is the most frequent renal syndrome in childhood, its histological classification is Minimal nephrotic change, focal segmental glomerulosclerosis, and diffuse mesangial sclerosis, and according to its extent of the response to steroids is classified as a sensitive and resistant nephrotic syndrome (11).
effects of cistanche: anti-inflammation
Mutation of WT1(Wilm's tumor-1 gene) is commonly reported in children with steroid-resistant nephrotic syndrome, a rare disease that affects 10-15% of patients with nephrotic syndrome(12,13). About 80% of all children with sporadic nephrotic syndrome respond to steroid treatment, however, no mutations were found in WT1(Wilm's tumor-1 gene) in a large cohort study(14).Reduction in expression of steroid-resistant nephrotic syndrome and to a lesser extent in patients with steroid-sensitive nephrotic syndrome(15).In a murine model of systemic inflammation induced by lipopolysaccharide(LPS),24 h after LPS administration, WT1(Wilm's tumor-1 gene) was translocated to the cytoplasm and was associated with a decrease in the expression of nephrin, a key component for maintaining the slit diaphragm in podocytes and essential for correct glomerular filtration, thereby showing WT1(Wilm's tumor-1 gene) acts as a transcriptional factor for up-regulating nephrin gene(16).
In A459 lung cancer cells treated with tumor necrosis factor-alpha (TNFa), translocation of WT1(Wilm's tumor-1 gene) protein to cytoplasm has been observed, suggesting that an inflammatory stimulus can alter its intracellular localization and transcriptional function (17). Phosphorylation may play a role in modulating the transcriptional regulatory activity of WT1(Wilm's tumor-1 gene) through interference with nuclear translocation, as well as by inhibition of WT1(Wilm's tumor-1 gene) DNA-binding(18)by cAMP-dependent protein kinase-mediated phosphorylation of Ser-365 and Ser-393 in the zinc finger domain (19).
Sepsis is a result of the systemic inflammatory response and is characterized by the release of pro-and anti-inflammatory cytokines induced by infection. Acute renal injury is common in critically ill patients in the presence of acute inflammation and has a strong link to the progression of chronic renal damage (20,21). There is evidence that inflammation plays an important role in damage to various organs, including kidneys(22).In this work, we evaluated the effect of pro-inflammatory cytokines on the modulation of WT1(Wilm's tumor-1 gene) expression and its translocation to the cytoplasm.
cistanche tubulosa benefit: antit-cncer and anti-tumor
Materials and Methods
Animal model. Female BALB/c mice(8-10 weeks old) were obtained from Harland Mexico(Mexico City, Mexico). The mice were caged under controlled room temperature, humidity, and light(12/12h light-dark cycle) with water and food ad libitum.LPS of Escherichia coli O111:B4(Sigma Aldrich, Toluca, Mexico City, Mexico)and a single injection of LPS was administered at a dose of 8 mg/kg intraperitoneally(16); the saline solution was used for control, untreated mice. The mice were sacrificed with an intraperitoneal injection of 200 mg/kg of sodium pentobarbital solution at 12,24,36,48 and 72 h, with n=5 at each time point, and then kidney tissue and blood were collected. The urine was collected before sacrificing the animals.
Two mice for kidney explants were injected with 1 mg/kg of LPS every 2 days to induce chronic kidney injury as a positive control (21). All experiments were carried out in accordance with prior approval from the Institutional Animal Care and Use Committee of the Faculty of Biological Sciences (CEIBA-2016-034).
Kidney explants. From the kidney of BALB/c mice, tissue slices 250-300 um thick were prepared with a Krumdieck tissue slicer(Alabama Research and Development, Munford, AL, USA)at 4℃ with the constant flow of buffer of Krebs Henseleit bicarbonate (KB) that was aerated with 5% CO2. The slices were harvested in KB buffer at 4°℃. Kidney explants were plated in 12-well plates with Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum and 5% antibiotic-antifungal agent, and incubated at 37℃C, with 5% CO, and stirring at 25 rpm. The explants were treated with 10 ng/ml of TNFa(R& D Systems, Minneapolis, MN, USA),20 ng/ml of interleukin 1β (IL1β; R &D Systems) and 100 ng/ml of LPS and analyzed at 6, 12 and 24 h later.
Urinary protein. Urine samples were collected at each timepoint above. The protein concentration was measured using the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA)and albuminuria was evaluated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and based on the band size of bovine serum albumin (16).
Pro-inflammatory cytokines. Levels of IL6, IL1β, and TNFa were measured using sandwich enzyme-linked immunosorbent assay kits (PeproTech, Mexico City, Mexico).
Quantitative polymerase chain reaction(qPCR). Total RNA was isolated from renal tissues using Trizolw Reagent (Invitrogen, Carlsbad, CA, USA)according to the manufacturer's instructions. Single-stranded cDNA was synthesized by reverse transcription using High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, CA, USA)according to the manufacturer's instructions. Real-time PCR was performed using the 7500 Real-Time PCR System (Applied Biosystems) with TaqMan gene expression assays. Comparative real-time PCR assays were performed for each sample in triplicate. The primers for WT1(Wilm's tumor-1 gene) were forward:5-TCTGCGG AGCCCAATACAG-3'; reverse:5-CACATCCTGAATGCCTCT GAAGA-3';and probe FAM:5-CACCGTGCGTGTGTATT-3'NFQ;the protocol was performed for 95°C for 10 min and 40 cycles at 94℃for 30 s and 64℃C for the 30s. The relative expression of the WT1(Wilm's tumor-1 gene) gene was calculated using the equation 2-AACT(23) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene(Applied Biosystems)being used for normalization.
treat kidney disease cistanche extract
Expression of nephrin mRNA was determined with 3 ug of cDNA and the following primers: Forward: 5'-CCCCAACATCG ACTTCACTT-3'and reverse:5'-GGCAGGACATCCATGTAGAG-3'at 94℃ for 30 s,60°℃ for 30 s and 72℃℃ for 30s for 30 cycles(372 bp)(16).For the expression of GAPDH, primers used were: Forward:5'-ACCACAGTCCATGCCATCAC-3',reverse:5'-TCCACCACCC TGTTGCTGTA-3', with the following reaction conditions:94°C for 40s,60℃ for 30s and 72℃ for40s for 35 cycles(452 bp).Expression of WT1(Wilm's tumor-1 gene):Forward 5'-CACATGAGAGAAACGCCCCTTCATGTG-3', reverse 5'-TTTGAGCTGGTCTGAACGAGAAA-3'at 94°℃ for 40 s, 64℃ for 30s and 72°Cfor 30s for 35 cycles(160 bp).The expression of WT1(Wilm's tumor-1 gene) isoforms KTS±and 17 AA±(F2-R2 primers to detect 17 AA+/-and F3-R3 to detect KTS+/-splicing isoforms)was performed by conventional PCR according to Oji et.al.(24). The determination of the expression of β-actin was performed according to Lauxet al. (25). The products were observed using 10% polyacrylamide gel electrophoresis for the KTS±isoform and a 2% agarose gel for the 17AA±isoform.
Immunofluorescence. The kidneys from LPS-treated mice and controls were fixed in 10% formalin and processed for hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence.
To determine the location of WT1(Wilm's tumor-1 gene) in mice treated with LPS,4-um-thick sections of renal tissue mounted on microscope slides were used. The specimens were dewaxed, permeabilized with Triton X-100 [0.1% Triton with 1% sodium citrate in phosphate-buffered saline (PBS)], and washed three times with PBS. The tissues were blocked with 20% fetal bovine serum in PBS for 30 min and incubated at 4°C overnight with monoclonal antibody to WT1(Wilm's tumor-1 gene) sc-7585(F-6)(Santa Cruz Biotechnology, Santa Cruz, CA, USA)diluted 1:100 in 10%fetal bovine serum in PBS. After washes with PBS, the presence of bound antibody was identified by staining with goat anti-mouse IgG Texas Red sc-2979(Santa Cruz Biotechnology). The slides were counterstained with 4',6-diamidino-2-phenylindole. Finally, the slides were mounted and qualitatively examined using an Olympus BX61W1 confocal microscope with a 40×water immersion objective and Fluoview 4.0a software(Olympus, Tokyo, Japan).
Immunohistochemistry. To determine phosphorylated (p)-WT1(Wilm's tumor-1 gene) in podocytes from LPS-treated mice, polyclonal antibodies to p-WT1(Wilm's tumor-1 gene) serine 393,sc-12933 and 363,sc-12934-R(Santa Cruz Biotechnology, Dallas， TX， USA） were used. Slides containing 4-μm sections of renal tissue were dewaxed and permeabilized. Immunohistochemistry was performed using Universal Quick Kit (Vector Laboratories, Burlingame, CA, USA)according to the manufacturer's protocol. The slides were examined using a light microscope with a 100× oil immersion objective(Primo Star, Carl Zeiss, GmbH, Germany).
Western blot. Total protein was isolated with 200 μl of lysis buffer (1%Triton,150 mM NaCl, 25 mM Tris-HCl pH 7.6), and the concentration was measured using a DC Protein Assay kit(Bio-Rad). Protein(50 ug) was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by western blotting with WT1(Wilm's tumor-1 gene) [F6] antibody sc-7585(Santa Cruz Biotechnology). Samples were normalized using anti-GAPDH(AB2302; Sigma, San Louis, MO, USA). Proteins were visualized using a Lumi-Light Western Blotting system sc-2048(Santa Cruz Biotechnology).
benefit of cistanche: protect kidney
Results
Induction of kidney damage in BALB/c mice by LPS.It is well established that podocyte injury is associated with increased proteinuria. Albumin and total protein in urine were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand DC Protein Assay kit(Bio-Rad). A significant increase (p=0.001)of urinary albumin was observed 12 h after LPS treatment, with a maximum increase at 24 h(215.87±1.41 ug/ml）compared to the control（15.53±5.0 μg/ml）（Figure 1A）. The concentration of total urinary protein was observed to peak at 36 h(26.02±2.43 mg/ml) and was significantly higher compared to the control(p=0.01)as shown in Figure 1. The renal damage induced by LPS was analyzed by histology. At 24 h after treatment, focal glomerulonephritis was observed in kidney tissue, with the greatest damage being found at 36 h, increasing the size of the glomerulus, with mesangial cell proliferation. At 48 h after, a gradual recovery of the glomerulus it was observed until 72 h, as shown in Figure 1C.
Figure 1. Effect of systemic inflammation on the kidney. Concentrations of urinary albumin(A) and urinary protein (B)as markers of kidney damage in mice treated with lipopolysaccharide. C: A representative image of renal histology of mouse(hematoxylin and eosin staining) at different times (12,24,36,48, and 72 h)after lipopolysaccharide injection, with the comparison of a model of chronic inflammation, magnification 100×.Significantly different at: *n<0,05.**p<0,0] and***p<0.00 ].
Production of the pro-inflammatory cytokines TNFa and IL1βin serum from LPS-treated mice. To confirm kidney damage was induced by LPS, pro-inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay. An increase of TNFa and I1β was observed after 12 h(3.6±0.9 ng/ml, 11.9±5ng/ml) and up to 36h(4.44±0.1 ng/ml,18±0.9 ng/ml respectively)of treatment with LPS, beginning to decline thereafter(Figure 2A and B). IL6 expression was increased at 12 h(49.5 ng/ml) and 36 h(23.5 ng/ml)post-treatment,being significantly higher than the control (Figure 2C).
Figure 2. Time course of levels of pro-inflammatory cytokines tumor necrosis factor-alpha(TNFa). (A), interleukin 1β (LL1β)(B), and IL6(C) in serum from mice following intraperitoneal administration of lipopolysaccharide. Significantly different at: *p≤0.05,**p≤0.01 and ***p≤0.001.
WTI gene expression in kidney tissue fromLPS-treated mice. In order to answer our central question of whether WT1(Wilm's tumor-1 gene) was modulated by the inflammatory process, and this had renal effects, the total expression of the WT1(Wilm's tumor-1 gene) gene was determined in renal samples from mice with systemic inflammation at different times in the course of inflammation. WT1(Wilm's tumor-1 gene) mRNA was lower at all times analyzed with respect to the control, with the lowest relative expression at 36 h and the highest at 12 h after induction of systemic inflammation as shown in Figure 3A.In addition, it was determined whether the treatment with LPs induced modification of the expression of the isoform's exon5(17 AA±)and KTS±of WTI by PCR. The data showed that there was no deregulation of the expression of these isoforms as the same behavior was observed as the control group without treatment throughout the systemic inflammatory process induced by LPS (Figure 3B). WT1 protein was shown by western blotting to decrease throughout the inflammatory process, with a slight recovery at 36 h after treatment, however, the amount of WT1 was less than that of the untreated control (Figure 3C and D).
Figure 3. Effect of lipopolysaccharide(LPS)on Wilms'tumor I (WT1)gene expression in kidney tissue from mice with systemic inflammation. A: Relative expression of WTI mRNA.B: Relative expression of isoforms KTS±and 17IAA±of WT1.C: Levels of WTI protein by immunoblotting after LPS treatment and D: Density of immunoblotting for WT1.Significantly different at:**p≤0.01 and ***p≤0.001,
Phosphorylation and localization of WTI in kidney biopsies of LPS-treated mice. An important event in the post-translational modulation of WTI protein is the phosphorylation of amino acid 393, which induces the displacement of the protein to the cytoplasm. Phosphorylation and localization of WT1 in kidney samples from mice treated with LPS were determined. Phosphorylation of WT1 was analyzed by histochemistry, indicating the presence of phosphorylated WT1 in the cytoplasm of the cells in sections of renal tissue starting 12 h after the induction of systemic inflammation and being maintained until 72 h.WT1 was phosphorylated at amino acids 393 and 363 as shown in Figure 4A. The location of the WT1 protein was analyzed by immunofluorescence. The location of WT1 in podocytes in kidney samples from LPS-treated mice was mainly cytoplasmic, whereas in the podocytes from the control samples, the location was unclear(Figure 4B).
Figure 4. Phosphorylation and localization of Wilms'tumor I (WTI) in kidney samples from lipopolysaccharide-treated mice. A: Immunohistochemical staining for phosphorylated (p)-WTI showing an increase in phosphorylated at serine 393 and 363 starting 12 h after LPS administration (magnification 100× on the light microscope). B: Localization of WTI by immunofluorescence in the kidney; examples showing minor nuclear localization of WT1(squares)36 h after of the administration of lipopolysaccharide (magnification 40× on the confocal microscope).DAP1:4,6-Diamidino-2-phenylindole.
Modulation of the expression of WTI and nephrin in kidneys from LPS-treated mice. WT1 location in the cell influences its biological activity. It has been reported that cytoplasmic WT1 does not exert transcriptional activity; for this reason, the expression of WT1 and nephrin mRNA was analyzed by PCR in kidney samples from mice treated with LPS (Figure 5A).PCR indicated a decrease in nephrin mRNA from 24 to 48 h, with an increase at 72 h (Figure 5B), while the expression of WTI mRNA increased at 12 h and thereafter declined during the critical hours of inflammation and kidney damage (24 and 36 h) and (Figure 5B).
Figure 5. Analysis of nephrin (NPHS1)and Wilms'tumor 1(WT1)mRNA expression. A: In the analysis of mRNA expression, NPHSI and WTI decreased at 24,36 and 48 h after the induction of systemic inflammation using lipopolysaccharide. B: Quantification of the bands shown in A by densitometry and normalized by expression to glyceraldehyde 3-phosphate dehydrogenase.
Kidney explants treated with TNFa and IL1β modulate the expression of WTI and nephrin. To determine if the cytokines modulate the expression of WT1, renal explants were cultured and treated with the cytokines TNFa and IL1βfor 6,12, and 24 h to analyze the expression of WT1 and nephrin by PCR. Nephrin mRNA expression decreased and WTI mRNA expression increased at 24 h in renal explants treated with TNFa and ILlb (Figure 6).
Figure 6.Analysis of the expression of nephrin (NPHS1)and Wilms'tumor 1 (WT1)mRNA in renal explants. A: mRNA expression levels of NPHSi and WTI at different times in renal explants exposed to 10 ng/ml of tumor necrosis factor-alpha (TNFα), and 20 nglml of interleukin 16 (IL16)B: Relative expression obtained using densitometric analysis of the signal product. The bands were quantified by normalization to glyceraldehyde 3-phosphate dehydrogenase.
Localization of WTI in renal explants by immunofluorescence. To determine whether TNFa and ILβ were responsible for the change in localization of the WT1 protein, mouse kidney explants were treated with TNFa and L1β and LPS for 24 h. In explants treated with ILlb and LPS, a nucleus/cytoplasmic localization of WTl protein was apparent, and in the explants treated with TNFa, cytoplasmic localization of WT1 protein was observed; meanwhile, in untreated controls, mainly nuclear localization of WT1 was observed(Figure 7).
Figure 7. Localization of the Wilms'tumor 1(WTI) protein by immunofluorescence using Red TX (WT1) and 4',6-diamidino-2-phenylindole (DAPI)(nucleus) in podocytes from kidney explants treated with l0 ng/ml of tumor necrosis factor-alpha (TNFa),20 ng/ml of interleukin 16(L1B), and lipopolysaccharide (LPS).Reduced nuclear localization of WTas shown in the explants treated with TNFa compared to the nuclear/cytoplasmic localization of WTI under IL1β and LPS treatments. Magnification, 40×.
