autosomal dominant polycystic kidney disease ( adpkd ) is a common inherited disease that affects approximately 1 in 1,000 individuals . the disease 's main clinical manifestations are bilateral renal cyst formation and hypertension , and the half of these patients reach end stage renal failure by the age of 60 yr ( 1 ) . adpkd is genetically heterogeneous and most of the afflicted families have mutations of either pkd1 on chromosome 16 ( -85% ) or pkd2 on chromosome 4 ( -15% ) ( 2 - 3 ) . pkd1 encodes polycystin-1 ( pc-1 ) , which is an integral membrane protein of 4,302 amino acids with an expected molecular mass of 462 kda ( 4,5 ) . pc-1 is widely expressed in the renal tubular epithelium and it is thought to be a cell - cell / matrix receptor molecule at the cell surface . pc-1 and pc-2 may heterodimerize to form a pc complex and this could function as the same signaling pathway ( 6,7 ) . however , there are controversies about the pc-1 expression and function when using different antibodies to study them . the large size and the low expression of pc-1 have made the study of pc-1 very difficult . there are also some doubts as to the specificity of the pc-1 antibodies ( 8,9 ) . as an initial approach towards studying adpkd and to obtain more insight into pc-1 expression , we have carried out immunoblot and immunochemical analyses of the pc-1 expression in the tubular cells of fetal , adult and adpkd kidneys . we used human embryonic kidney ( hek ) 293 cells and renal proximal tubular epithelial cellsr ( rptecs ) for performing immunoblottting . the cells were grown to 70 - 90% confluence and they were then lysed in phosphate buffered saline that contained 0.5% nonidet p-40 . a large amount of protein ( -400 g each ) was separated on 4% sds - page gel with or without boiling . the membranes were blocked with 5% nonfat dry milk for 1 hr and they were next incubated with primary antibodies diluted at 1:500 . we used two pc-1 antibodies ; c-20 ( santa cruz , sc-10372 , santa cruz , ca , u.s.a . ) was used as the c - terminal antibody and p-15 ( santa cruz , sc-10307 , santa cruz , ca , u.s.a . ) was used as the n - terminal antibody . after washing , the membranes were incubated for 1 hr at room temperature with the peroxidase - labeled secondary antibody ( anti - goat igg - hrp , santa cruz ) at 1:500 dilution . the membrane - bound antibodies were detected by using the enhanced chemiluminescence detection system ( amersham biosciences , bucks , u.k . ) . normal fetal kidney was obtained from autopsy ( gestation age : 20 weeks , weight : 305 grams ) after spontaneous abortion . adult kidneys were obtained from renal tumor nephrectomy patients ( a male 58 yr old and a male 63 yr old ) . the autosomal dominant polycystic kidneys were obtained from pkd1 patients ( a male 53 yr old and a male 46 yr old ) who had the typical clinical manifestations during transplant nephrectomy . all the tissues were embedded in optimal cutting temperature compound ( sakura tissue tek , torrance , ca , u.s.a . ) and stored at -70 until further use . cryosections 5 m thick were cut , and the tissues were fixed in acetone at -20 for 10 min and then washed with phosphate - buffered saline . after blocking the endogenous peroxidase activity , the primary antibodies ( c-20 and p-15 ) were diluted to 1:100 and the sections were incubated for 1 hr with them at room temperature . after washing , the sections were incubated for 45 min with the secondary antibody ( bionylated anti - goat ig , dako , glostrup , denmark ) . the reaction was developed with 3-amino-9-ethylcarbazol ( aec , red stain ) or with 3,3'diaminobenzidine ( dab , brown stain ) . we used human embryonic kidney ( hek ) 293 cells and renal proximal tubular epithelial cellsr ( rptecs ) for performing immunoblottting . the cells were grown to 70 - 90% confluence and they were then lysed in phosphate buffered saline that contained 0.5% nonidet p-40 . a large amount of protein ( -400 g each ) was separated on 4% sds - page gel with or without boiling . the membranes were blocked with 5% nonfat dry milk for 1 hr and they were next incubated with primary antibodies diluted at 1:500 . we used two pc-1 antibodies ; c-20 ( santa cruz , sc-10372 , santa cruz , ca , u.s.a . ) was used as the c - terminal antibody and p-15 ( santa cruz , sc-10307 , santa cruz , ca , u.s.a . ) was used as the n - terminal antibody . after washing , the membranes were incubated for 1 hr at room temperature with the peroxidase - labeled secondary antibody ( anti - goat igg - hrp , santa cruz ) at 1:500 dilution . the membrane - bound antibodies were detected by using the enhanced chemiluminescence detection system ( amersham biosciences , bucks , u.k . ) . normal fetal kidney was obtained from autopsy ( gestation age : 20 weeks , weight : 305 grams ) after spontaneous abortion . adult kidneys were obtained from renal tumor nephrectomy patients ( a male 58 yr old and a male 63 yr old ) . the autosomal dominant polycystic kidneys were obtained from pkd1 patients ( a male 53 yr old and a male 46 yr old ) who had the typical clinical manifestations during transplant nephrectomy . all the tissues were embedded in optimal cutting temperature compound ( sakura tissue tek , torrance , ca , u.s.a . ) and stored at -70 until further use . cryosections 5 m thick were cut , and the tissues were fixed in acetone at -20 for 10 min and then washed with phosphate - buffered saline . after blocking the endogenous peroxidase activity , the primary antibodies ( c-20 and p-15 ) were diluted to 1:100 and the sections were incubated for 1 hr with them at room temperature . after washing , the sections were incubated for 45 min with the secondary antibody ( bionylated anti - goat ig , dako , glostrup , denmark ) . the reaction was developed with 3-amino-9-ethylcarbazol ( aec , red stain ) or with 3,3'diaminobenzidine ( dab , brown stain ) . we confirmed that the c-20 antibody was sensitive for detecting pc-1 by performing immunoprecipitation ( 10 ) , and we confirmed the pc-1 expression in the hek 293 cells and in the rptec lysates ( fig . 1 ) . however , this was technically difficult because of the large size of the pc-1 molecule and its faint expression level . in the fetal and adult kidneys , the patterns of expression were diffuse and cytoplasmic at the proximal tubules , distal tubules and collecting ducts . the expression in the fetal kidney was more prominent in the proximal tubules ( fig . the pc-1 proteins were weakly expressed in the tubular or cystlining epithelial cells , as compared to that of the fetal and adult kidneys . we confirmed that the c-20 antibody was sensitive for detecting pc-1 by performing immunoprecipitation ( 10 ) , and we confirmed the pc-1 expression in the hek 293 cells and in the rptec lysates ( fig . however , this was technically difficult because of the large size of the pc-1 molecule and its faint expression level . in the fetal and adult kidneys , the staining was restricted to tubular epithelial cells cytoplasm ( fig . the patterns of expression were diffuse and cytoplasmic at the proximal tubules , distal tubules and collecting ducts . the expression in the fetal kidney was more prominent in the proximal tubules ( fig . the pc-1 proteins were weakly expressed in the tubular or cystlining epithelial cells , as compared to that of the fetal and adult kidneys . the identification of pc-1 , which is the major gene mutated protein seen in adpkd , is a major step for discovering the pathogenesis in this common hereditary disease . however , there 's been some controversy about the pc-1 expression when using different antibodies to detect it . the difficulties for studying pc-1 are due to the antibody specificity and the low levels of pc-1 expression in cells and tissues . moreover , two thirds of the pkd1 gene is duplicated on chromosome 16 and there are at least three homologus genes . our study showed that pc-1 may be very weakly expressed in the renal tubular cells , as observed by immunoblotting , and it is widely expressed in the fetal and adult renal tubular epithelial cells cytoplasm , as observed by immunochemistry . before this study , we confirmed that c-20 antibody was sensitive to detect the pkd-1 c - terminal by performing immunoprecipitation ( 10 ) . to verify the specificity of the immunohistochemical staining , we performed immunoblot analysis of the kidney cells ( hek 293 cells and rptecs ) , the kidney tissues ( fetal and adults ) and the adpkd tissues . we were unable to detect the pc-1 band in the kidney tissues and the adpkd tissues . this is suggested that the pc-1 is either not expressed at detectable levels in normal and adpkd kidneys , or it is lost during sample preparation . however , we were able to detect low levels of pc-1 in the kidney cells . according to the previous reports ( 8,9 ) , immunoblotting of pc-1 was technically difficult because of the large size of the pc-1 molecule and the faint level of expression . a number of published papers have described the pc-1 expression in normal renal and cystic tissue . 11 ) have reported that in the early fetal kidney , pc-1 was localized to the plasma membranes of the ureteric buds and the s - shaped bodies . however , in the late fetal kidney and in the adpkd kidney , the majority of pc-1 staining was intracellular . they detected several bands in the fetal kidney and no bands were detected in the adult kidney by immunoblotting . they detected about a 400 kda pc-1 band in the fetal and adult mouse and human kidneys . pc-1 was noted to be localized at the plasma membrane and pc-1 expression was seen in most ( -90% ) of the adpkd cysts . ong et al . ( 14 ) reported that in adult pkd1 tissue , the majority of cysts ( -80% ) showed pc-1 expression , although pc-1 staining was absent in a variable , but significant minority of the cysts ( -20% ) . leeuwen et al . ( 15 ) found that the mutant pkd1 mice that had a reduced pkd1 gene expression showed polycystic kidney disease . they generated a novel mouse model with a hypomorphic pkd1 allele , and the pathologic features of this mouse were similar to the human adpkd phenotype . recently , roitbak et al . ( 16 ) have reported that pc-1 formed a complex with e - cadherin and -catenin at both the cell membrane and intracellularly . enhanced phosphorylation of pc-1 changed its subcellular localization and its ability to form protein complexes . second , the pc-1 is expressed in the tubular epithelium and in the adpkd cyst epithelium . third , the subcellular localization of pc-1 is mainly on the cell surface membrane and it may be changed in some conditions . our results showed similar patterns of pc-1 expression for the fetal , adult and adpkd kidneys , but our immunohistochmical study showed a cytoplasmic staining pattern of pc-1 . however , in the hek 293 cells grown to 80% confluency , we found that the pc-1 was localized on the cell surface membrane by performing immunofluorescent staining with using c-20 antibody ( 10 ) . . the epitope recognized by these antibodies may be not the full length , but rather , they are only a fraction of the pc-1 . pc-1 is the pkd 1 gene translated protein , and it is a 4,302 amino acids glycoprotein with an expected molecular mass of 460 kda . experimental studies have shown that pc-1 is a highly glycosylated 520 kda polypeptide that is present in the plasma membrane ( 6,7 ) . by performing amino acid sequence homology analysis , pc-1 was found to be a membrane receptor that is capable of binding and interacting with ligands . recent research have shown that pc-1 is also localized to the renal cilia and to the protein - mediated mechanosensation in the primary cilium of the kidney ( 17,18 ) in summary , we have found by performing immunoblotting that pc-1 is expressed at a very low level in the renal tubular cells . however , pc-1 is widely expressed in the fetal and adult renal tubular epithelial cells cytoplasm , as was noted upon performing the immunohistochemistry . in the adpkd kidney , the pc-1 proteins were heterogenously and weakly expressed in the tubular or cyst lining epithelial cells . a reduced pc-1 expression our data suggests that the development of kidney may regulate the expression of pc-1 , and the altered pc-1 expression may contribute to cyst formation seen in adpkd .
the mutation of the pkd1 gene causes autosomal dominant polycystic kidney disease ( adpkd ) , and the pkd1 gene encodes polycystin-1 ( pc-1 ) . 
 pc-1 is thought to be a cell - cell / matrix adhesion receptor molecule at the cell surface that is widely expressed in the kidney . however , there are controversies about the role of pc-1 protein and its expression when using different antibodies to detect it . 
 we used two pc-1 antibodies ; c-20 ( santa cruz , sc-10372 ) as the c - terminal antibody , and p-15 ( santa cruz , sc-10307 ) as the n - terminal antibody . 
 we evaluated the pc-1 expression by performing immunoblotting on the human embryonic kidney ( hek ) 293 cells and the renal proximal tubular epithelial cell ( rptec ) lysates . 
 we characterized the expression of pc-1 in the fetal , adult and polycystic kidneys tissues by performing immunohistochemistry . 
 we confirmed the pc-1 expression in the hek 293 cells and the rptec lysates , but the expression was very low . 
 the pc-1 proteins were diffusely expressed in the tubular epithelial cells cytoplasm in the fetal and adult kidneys , and the pc-1 expression was more prominent in the proximal tubules of the fetal kidney . in the adpkd kidney , the pc-1 proteins were heterogenously and weakly expressed in the tubular or cyst lining epithelial cells . 
 our data suggests that the development of the kidney may regulate the expression of pc-1 , and an altered pc-1 expression may contribute to cyst formation in adpkd .