The Role Of Klotho In Cyst Formation Of Autosomal Dominant Polycystic Kidney Disease

Backgroundandobjective:Autosomaldominantpolycystickidney disease?ADPKD?is the most frequent inherited kidney disease,and happens 1 in 400 to 1in 1000 people.For most of the patients,the disease happens in their late thirties,and the main pathological changes are renal tubule progressive cyst growth,oppression of the normal kidney structure,the damage of kidney function,eventually leads to end-stage renal disease?ESRD?.Around 50%of ADPKD patients will progress into ESRD by the 6th decade of life,and need to refer to dialysis and kidney transplantation to prolong their lives.ADPKD is genetically heterogeneous with two loci identified,PKD1?16p13.3?,which encodes polycystin-1?PC1?,and PKD2?4q22?,which encodes PC2.Mutation in PKD1 is found in 85%of ADPKD cases,which have typically a more severe phenotype than those with a mutation in PKD2,accounting for 15%of cases.The main pathogenesis of ADPKD has been clear,however,due to Pkd1 gene encoding sequence is too long and mutations can be various,makes its clinical manifestations changing a lot between different families;However,with the study goes deeper,we find that the disease progression still changes a lot within the same family,even between twin brothers or sisters.This finding reminds us that not only the gene mutation itself influence the disease but also the post-transcriptional regulation plays an important role.And the gene methylation is a very important transcriptional regulatory mechanism.Based on these,our team process the DNA methylation sequencing in ADPKD kidney samples.The results show that the promoter region of Klotho has pretty high DNA methylation level,which means that the Klotho level might be down regulated in ADPKD patients.Klotho protein?also known as?-Klotho protein?is a kind of type?membraneprotein encoded by Kl gene,which is mainly expressed in distal renal tubules and brain.This protein is composed of KL1 and KL2,and can be cutted into different length fragments from membrane and released into blood,urine and cerebrospinal fluid by ADAM?A desintegrin and metalloproteinase?10 and ADAM17.The full length Klotho fragment is the most important functional Klotho protein.It can bind with different receptor and play a role as endocrine,autocrine and paracrine factor.In 1997,Kuro-o et al found that the mice with an injection mutation in 5'Klotho gene show several aging related symptoms and a shorter overall lifespan.Studies show that Klotho is not only related to aging but also various kinds of diseases such as cancer,type 2 diabetes mellitus,myocardial hypertrophy and chronic renal disease.One of the clinical research published in CJASN has shown us that serum Klotho values correlated inversely with cyst volume and kidney growth in ADPKD patients.Besides,Klotho also plays an anti-inflammation role which can inhibit the Akt,Wnt,TNF-?and TGF-?related pathways,playing an important role in the ADPKD progression.Thus,it is of significant importance to make clear the role Klotho protein plays in cyst growth.Our study plans to see the Klotho level changing in PKD cell line and kidney tissues,investigate the mechanism of Pkd1 mutation influencing the Klotho level and the role Klotho plays during the cyst development,in order to figure out new target for ADPKD treatment.Methods:?1?Collect samples of ADPKD patients kidney tissue and normal kidney tissue,Pkd1 conditional knockout mice kidney tissue samples and the samples of wild type mice kidney,Pkd1^+/-and Pkd1^-/-renal epithelial cell line,detecting Klotho protein expression with protein immunoblot,immunohistochemistry and RT-PCR technique.?2?By intraperitoneal injection of recombinant mouse Klotho protein,preliminary observe the role Klotho plays in Pkd1^fl/fl:Pkhd-cre mice?an early-onset ADPKD model?during cyst growth.?3?Build up late-onset PKD1^fl/fl:tamoxifen-cre mouse model,further observing the regulation of recombinant mouse Klotho protein in cyst formation.?4?Carry out the DNA methylation sequencing in kidney tissues of ADPKD patients and normal kidney tissues,investigating how Klotho protein is regulated in ADPKD;Carry out the chromatin immunoprecipitation to make sure of the binding between DNMT1 and Klotho promoter region as well as between MBD and Klotho promoter region;Examine the regulation between DNMT1 and Klotho with Western Blot.?5?Examine c-Myc,Cyclin D1,p-STAT3 protein levels with Western Blot to see the regulating function of Klotho on proliferation in ADPKD models.?6?Detect Smyd2,p-P65,TNF-?and Ccl-2 levels with Western Blot and RT-PCR to evaluate the regulating function of Klotho on inflammation process in ADPKD models.?7?To confirm the influence of Klotho in ferroptosis,we designed several experiments.First of all,we did the MTT to see if Klotho can influence the cell viability;Secondly,we treat the Pkd1^-/-renal epithelial cells with Klotho,Erastin,Fer-1,followed by C11:BODIPY 581/591 staining and run flow cytometry to see the percentage of positive cells;We also run the Western Blot to see the GPX-4 level in Pkd1^-/-cell line as well as kidneys of ADPKD mouse model.?8?Do Masson,?-SMA and Collagen-?staining in mouse kidney tissues and run TGF-?,Fibronectin,?-SMA,p-smad2/3 with Western Blot in mouse kidney tissues to see if Klotho treatment can influence the fibrosis progression in ADPKD;Test the inhibition function of TGF-?on Klotho expression in rat fibroblast cell line.Results:?1?The expression level of Klotho protein is down regulated in the kidney tissues of ADPKD patients,mouse Pkd1^-/-renal epithelial cells and kidney tissues of PKD1^fl/fl:Pkhd1-cre mice.?2?Klotho treatment in PKD1^fl/fl:Pkhd1-cre mice can delay the cyst growth.?3?In the late-onset PKD mouse model,Klotho treatment can also delay ADPKD progression,improving renal function and slow down the kidney volume growth.?4?DNA methylation sequencing shows that the promoter region as well as the gene body of Klotho is highly methylated,which leads to the gene silence.?5?The chromatin immunoprecipitation results show that both DNMT1 and MBD2 bind to the promoter region of Klotho gene.?6?Hydralazin?inhibitor of DNMT1?treatment can upregulate the Klotho expression in mouse Pkd1^-/-renal epithelial cells;Besides,mouse recombinant Klotho treatment in Pkd1^-/-renal epithelial cells can also downregulated the DNMT1expression level.?7?Mouse recombinant Klotho treatment can downregulate several protein markers in promotion related signaling pathways and regulate cell promotion.?8?Mouse recombinant Klotho treatment downregulates Smyd2 and p-P65 level to inhibite TNF-?and Ccl-2 expression,blocking the inflammation process in mouse Pkd1^-/-renal epithelial cells as well as mouse ADPKD kidney tissues.?9?Mouse recombinant Klotho treatment can lightly promote the cell viability,combining with the proliferation inhibiting function,showing that Klotho may influence some kind of cell death progression.?10?Mouse recombinant Klotho treatment can upregulate GPX-4 expression level in mouse Pkd1^-/-renal epithelial cells and kidney tissues of mouse ADPKD models.?11?The flow cytometry shows that Klotho treatment may decrease the percentage of C11:BODIPY581/591 positive cells,indicating Klotho may alleviate the lipid peroxidation.?12?Masson,?-SMA and Collagen-?staining shows that Klotho may relieve the fibrosis progression in ADPKD;At the same time,Klotho treatment can also downregulated the fibrosis related protein expression in ADPKD mouse model,only to confirm its fibrosis inhibiting function.?13?TGF-?treatment can inhibit Klotho expression in rat fibroblast cells.Conclusion:?1?Klotho protein plays an important role in cyst growth in ADPKD;?2?DNMT1 can methylate the promoter region of Klotho gene,which might be the main reason causing Klotho level drop in ADPKD;?3?Klotho can influence the ADPKD progression through cell proliferation,cell death,inflammation and fibrosis progression.