Patent Application: US-54019305-A

Abstract:
the present invention relates to a method of identification and quantification of proteins , isoforms of angiotensin i converting enzymes , 190 - kda , specially of 90kda in tissues , cells and biological fluids , specially in urine , genetic marker and prognostic agent of hypertension and primary or secondary renal lesion and kit for using in the diagnosis , risk stratification and therapeutical decision in arterial hypertension . one the aims of the present invention is to check the potential of the 90 kda isoform as a hypertension genetic marker isoform and as a prognostic for hypertension .

Description:
in order to carry out the method of identification and quantification of proteins , isoforms of the angiotensin i converting enzyme , specifically 190 - kda aces , specially 90 kda and 65 kda in tissues , cells and biological fluids , specially in urine , according to the present invention , it starts with collecting fluids , such as urine , tissues or cells from living organisms , submit them to a chromatographic separation ( aca44 and / or aca 34 resin ; reverse phase column c - 18 , in mass spectrometer ) and by western blotting ( using a specific antibody against somatic ace and n - domain ace [ 90 kda , genetic marker for hypertension and 65 kda ] of 190 kda , 90 kda and 65 kda isoforms . normal individuals have the 190 kda and 65 kda isoforms while the 90 kda isoform ( hypertension genetic marker ) will characterize those individual predisposed to develop hypertension and lesion in characteristic target organs ( heart , nervous system , vascular system and kidney ). the method of the present invention considers that an aliquot of fluid ( for example , fresh or concentrated urine ), cells and tissues are processed and analyzed by high performance liquid chromatography and detection by mass spectrometry ( hplc - ms ) or directly in the mass detector , where the sample is analyzed and compared with the previously established standards for 190 kda , 90 kda ( hypertension genetic marker ) and 65 kda ace isoforms . an aliquot of fluid ( for example , fresh or concentrated urine ), cells and tissue are processed and analyzed by western blotting or another immunoprecipitation method ) using specific antibodies against 190 kda and n - domain ace ( 90 kda hypertension genetic marker and 65 kda ), using as a control analysis the ace isoforms prepared as standards as well as the eca recombinant enzyme . in order to reach the results proposed by the present invention , the researches on ace , started in 1983 , when essential ( light and / or mild forms ) arterial hypertensive patients were analyzed after using captopril ( 50 to 150 mg ) orally administered in one - day dose . three days study , each day , each six hours collect duration ; samples of blood and urine were collected at the final basal period ( 1 hour ), after they reach the supine position , as well as at the end of the study ( after 6 hours ). the results show a 50 % inhibition on the enzyme activity , in a period between 1 and 2 hours after captopril administration , returning to the basal levels at the end of the studies . the enzymatic activity using hipuril - his - leu substrate was measured by friedland and silverstein method ( 1976 ). through ion exchange chromatography analysis , the collected urine ( collected after 6 hour ) two protein peaks were eluted with angiotensin i converting activity and inactivated for bradykinin , in conductivity of 0 . 7 ms ( 90 kda ) and 1 . 25 ms ( 65 kda ), differing from the profile found in 190 kda e 65 kda ace in normotensive individuals . based on this , studies have been developed with a higher number of light hypertensive patients , where it was found the presence of two protein peaks with angiotensin i converting activity , as cited above ( casarini et al , 1991 ). following the studies as referred above ( project support by fapesp , no 95 / 9168 - 1 ) the following study groups were organized : normal parents / normal children ; normal parents / hypertensive children ; hypertensive parents / hypertensive children and hypertensive parents / normal children . it was found that the group ( normal parents / hypertensive children ) hypertensive parents / hypertensive children , the children urine was presented two 90 e 65 kda forms ; in the normal parents / normal children group , the children urine presented the 190 and 65 kda forms ; and , finally , in the hypertensive parents / normal children , the children urine presented the 190 , 90 and 65 kda forms , being these two last forms , n - domain fragments . from these results , it was concluded that 90 kda would possibly be a hypertension marker . in order to prove if this findings was a genetic factor or another fact related or not to pressure increasing ( physical ), data were validated in the same project in the experimental model for rats . for this purpose , it was studied wistar brown norway , lyon , shr , 1r1c and doca - salt rats urine . as a result , the wistar , doca - salt , 1r1c and brown norway rats presented 170 and 65 kda forms ; only shr and shr - sp rats presented the 90 and 65 kda forms . these results confirm those obtained for humans , thus , being , the 90 kda ace isoform a hypertensive genetic marker ( fapesp 97 / 00198 - 0 , marques 1999 ). in 1997 , researchers of the present invention described that the mesangial cells in a culture that expresses the ace rnam ( casarini et al , 1997 ). this enzyme is detected in the intracellular ( 136 kda and 65 kda ) and secreted ( 136 kda e 65 kda ), indicating a potential effect of the local production of angiotensin ii in the function of these cells ( fapesp 95 / 9168 - 1 ; andrade et al , 1998 ). it was observed latter that the intracellular and the culture means of the shr mesangial cells presented the same profile of ( 90 and 65 kda ) ace isoforms , which were found in urine of such rats ( fapesp 99 / 01531 - 1 ); therefore , this confirms the results of the previous researches . from these results , it was observed by the authors of the present invention , that these isoforms are expressed in the lung , adrenal , heart , aorta and liver of wistar rats ( 136 kda and 69 kda ) and shr ( 96 kda and 69 kda ) and are not restricted to kidney ( ronchi , 2002 ); emphasizing that the 80 / 90 / 96 kda hypertension genetic marker is expressed in the various tissues and , therefore , bringing to conclude that these isoforms can contribute for a regulation of the specific organ ( it should be stressed that when de 80 or 96 or 90 kda enzymes are referred , it should be understood , the same enzymes with small alteration in the glycolization process ). the present invention starts with fluid collect as for example urine , tissue or living organisms cells , that are submitted to a chromatographic separation ( resin aca44 and / or aca 34 ; phase reverse column c - 18 , mass spectrometer ) and by western blotting ( using a specific antibody against ace somatic and against ace n - domain [ 90 kda , hypertension genetic marker and 65 kda ] of 190 kda , 90 kda and 65 kda isoforms . the 190 kda and 65 kda isoforms will be present normal individuals ( normal rats , cells and / or tissue from normal rats ); while the 90 kda ( hypertension genetic marker ) isoform will characterize those ( individuals or animals , etc ) predisposed to develop hypertension and lesions in characteristics target organs ( heart , nervous system , vascular system and kidney ). a aliquot of fluid ( for example , fresh or concentrated urine ), cells and tissue are processed and analyzed by high performance liquid chromatography method and detection by using mass spectrometry ( hplc - ms ) or directly in the mass detector , where the sample is compared with the standards established for ace of isoforms 190 kda , 90 kda ( hypertension genetic markers ) and 65 kda . an aliquot of fluid ( for example , fresh or concentrated urine ), cells and tissue are processed and analyzed by western blotting using specific antibodies against 190 kda ace and n - domain aces ( 90 kda ), hypertension genetic marker and 65 kda ), using as analysis control the ace isoforms prepared as standards and the recombinant ace enzyme . isoform of angiotensin i converting enzyme ( 90 kda , n - domain ) as a hypertension genetic marker produced by human urine : based on the previous studies , the researchers of the present invention found in normotensive individuals urine and using ion exchange chromatography , two peaks of angiotensin i converting activity with molecular mass of 196 kda and 65 kda . when hypertension patients urine is processed , it was obtained a profile where two peaks were eluted with angiostensin i converting activity in the 90 kda and of 65 kda molecular weight , not being detected the 190 kda form ( hypertension 26 : 1145 - 1148 , 1995 ). one of the objectives of the present invention consists in confirming the potential of the de 90 kda isoform as a hypertension genetic marker and as a hypertension prognostic . the collected urines were concentrated separately and dialyzed with tris - hcl 50 mm buffer , ph 8 . 0 and then submitted gel filtration in aca - 34 column equilibrated with tris - hcl 50 mm buffer , containing nacl 150 mm , ph 8 . 0 . the collected fraction ( 2 ml ) have been monitored by reading the absorbance in 280 nm and by the angiotensin i converting activity , using hipuril - l - his - l - leu - and z - phe - his - leu as substrates . the following results was obtained : normotensive individuals with normotensive parents presented two isoforms with ace activity ( 190 kda and 65 kda ) ( n = 21 ); normotensive individuals with hypertensive parents presented three ( 190 kda , 90 kda and 65 kda ) ( n = 13 ) isoforms and hypertensive individuals with hypertensive parents presented two isoforms ( 90 kda and 65 kda ) ( n = 13 ). as expected , it was not found anybody that would constitute the hypertensive group with normotensive parents . two individuals that presented 190 kda , 90 kda and 65 kda isoforms , normal pressure , and that were in contact with the research group , were monitored for 4 years . in the forth year after detection of isoforms in the urine , they became hypertensive ; this proves , therefore , that the 90 kda isoform is really a hypertensive genetic marker . considering that the urine of normotensive individuals with hypertensive parents presented the three ace isoforms with molecular weight of 190 kda , 90 kda and 65 kda , shows that the 90 kda isoform which early appears , is a prognostic that these individuals could be a hypertensive person , thus , being , a hypertensive genetic marker . quantification and identification of the ace isoform , hypertension genetic marker by mass espectrometry and western blotting urine was collected from a single time in the presence of a “ pool ” ( several inhibitors ) of proteases , then , it was concentrated and 100 ug was submitted to a 7 . 5 % polyacrylamide gel electrophoresis , followed by western blotting with pvdf membrane , then it was incubated with the polyclonal antibody y1 against human ace . line 1 : urine of normal individual , line 2 : ace wild recombinant , line 3 : ace recombinant secreted , line 4 : hypertensive individual urine . raw urine was centrifuged for 10 minutes , at 3000 rpm , 4 ° c ., followed by 4 × concentration in ultrafree ( millipore ) tube , then centrifuged for 5 minutes at 3000 rpm , 4 ° c ., dialyzed with centricon tris / hcl 1 mm buffer , ph 8 . 0 . then , it is centrifuged for 5 minutes , at 3000 rpm , 4 ° c . concentrated and dialyzed urine was , then , obtained and therefore , the prepared sample was analyzed in hplc - ms . the solvents used in the hplc system were : solvent a , which consists of 0 . 1 % trifluoroacetic acid ( tfa , merck , germany ). the urines were separated in a nova pak c 18 ( waters ) reverse phase column , for 15 minutes with a 1 . 5 ml / min flux . the conditions are still been standardized in order to improve the method resolution . isoform of angiotensin i converting enzyme ( 90 kda ) as a hypertenssive genetic marker secreted in rats urine . protocol design to prove the findings ( affirmation of genetic marker for the 90 kda protein ) with human urine . eca isoforms presented in isogenic normotensive rats ( wky and brown norway ) urine have been identified as well as in normotensive , isogenic hypertensive ( shr , shr - sp , lyon ), isogenic normotensive , experimentally hypertensive ( 1k1c and doca - salt ) and isogenic hypertensive rats , which were treated with antihypertensives drugs ( shr + enalapril ), aiming at to compare the obtained chromatography profiles , and with the objective to characterize the 90 kda form as an arterial hypertensive genetic marker . from wky rats urine , two peaks of ai converting activity have been separated by gel filtration in aca - 44 resin : the first , wk - 1 , corresponds to a high molecular enzyme ( 190 kda ) and the second one , wk - 2 , corresponds to a low molecular weight ( 65 kda ); these data were confirmed by western blotting . in the srh group , the chromatographic profile have presented different results from the previous group ( wk ), being identified an ace called s - 1 , with molecular weight 80 kda , and a second one , s - 2 , molecular weight of 65 kda , similar to those found in hypertensive patients urine that was not 90 kda and 65 kda treated ( casarini et al ., 1991 ). the molecular mass differences between the 80 kda enzyme from rat and the 90 kda enzyme of human urine occur due to the glycolization process ( data not shown ). in the third group ( 1k1c ), a renovascular induced hypertension model , the chromatography profile was similar to the one found in rats used as control ( wky ). in this group two peaks of ai converting activity were obtained : the first , c - 1 , corresponds to a 190 kda enzyme and the second one , c - 2 , to 65 kda . two peaks of ai converting activity were separated by gel filtration in aca - 44 resin , from shr - sp rats urine : the first one , called sp - 1 , corresponds to a 80 kda enzyme and a second one , called sp - 2 , which corresponds to the 65 kda enzyme , similar to that found in shr rats urine and also found in not - treated hypertensive patients urine ( casarini et al , 1991 , 1995 ). on the other hand , the shr rats , which were treated with enalapril , show that although having their pressure under control , they carry the 80 kda isoform ; this fact shows that the isoform profile is linked to genetic factors . in the group of rats used as control , the doca - salt model , in which it was not administered the hypertensive treatment , the chromatographic profile was similar to that found for normotensive wk rats . two peaks with ai converting activity were obtained : the first , cd - 1 , corresponds to a 190 kda enzyme , and the second , cd - 2 , corresponds to the 65 kda . the doca - salt model , with reduced hypertension induced by doca and saline administration , presented a chromatographic profile similar to that found in doca - salt and wk control rats . two peaks of ai converting activity were obtained : the first one , d - 1 , corresponds to 190 kda eca , and the second one , d - 2 , to 65 kda eca . this result shows that the 80 kda presence is linked to a genetic factor not being consequence of the increasing of pressure . the result of the gel filtration in brown norway normotensive rats urine was similar to the profiles found in normotensive rats urine . two peaks with ace activity have been obtained : bn - 1 , which corresponds to the 190 kda enzyme , and the second one , bn - 2 , which corresponds to the 65 kda enzyme , showing that different strain of normotensive presents the same chromatographic profile . comparing the chromatographic profiles of wk rats ( normal control ) urine , 1k1c ( experimental hypertensive — goldblatt ), doca - salt ( control ), doca - salt ( experimental hypertensive ) and normotensive brown norway with the urine of shr rats , lyon and shr - sp ( genetically hypertensive ), it can be affirmed that the basic difference is the presence de 80 kda isoforms in the genetically hypertensive rats urine . the fact that the 80 kda isoform do nor appear in the 1k1c and doca - salt rats , whose hypertension is induced ( physical factor ), reinforces the hypothesis that the same is linked to a genetic factor . the results found suggest that rats , genetically predisposed to hypertension , the 80 kda form would be detected instead of 190 kda ; this would be used to , as a consequence , as an early genetic marker for hypertension . segregation of the isoform of angiotensin i converting enzyme ( 90 kda , n - domain ), hypertension genetic marker in rat urine . protocol design to show the presence ( segregation of the hypertension genetic marker ( protein of 90 kda ) in rats urine . rats crossing expontaneously hypertenses ( shr ) and brown norway ( bn ). in a previous project it was characterized the different isoforms of low molecular weight in rats urine in different experimental models ( wistar - kyoto , shr , 1r1c , doca - salt control , doca - salt , shr - sp and brown norway ). it was observed that the 90 kda form only appears in shr and shr - sp rats , showing a genetic factor for the presence of such a form . in this project , it is studied the isoforms gene transmission of 190 kda , 80 kda and 65 kda of the angiotensin i converting enzyme , genotype and phenotype analysis in rats urine generated from the crossings and backcrossings among shr and brown norway races . crossings were carried out between brown norway and shr ( bn × shr ), rats generating a group of heterozygotes rats called f1sb01 to f1sb04 ; from this group two animals were chosen ( f1sb01 e f1sb03 ), males in order to carry out the backcrossing with the shr rat ( female ). for phenotyping , the urine of the animals was collected and concentrated , then , it was submitted to a aca - 34 gel filtration column chromatography , together with tris / hcl 0 . 05m buffer , ph 8 . 0 , containing nacl 0 . 15m . fractions of 2 . 0 ml have been eluted under a 20 ml / h flux , being monitored by absorbance measured in a280 nm and by the ace enzymatic activity using z - phe - his - leu ( zphehl ) as a substrate . parents : two peaks with ace activity were eluted from bn rats urine and submitted to aca - 44 , bn - 1 e bn - 2 column chromatography , with molecular weight estimated of 190 kda and 65 kda , respectively . on the other hand , in shr rats urine it was found two peaks with converting activity , however , with molecular weight estimated of 90 kda and 65 kda , respectively . in f1 , 39 animals were generated , from which 100 % were phenotyped as heterozygotes for the three , 190 , 90 and 65 kda enzyme forms . from the backcrossing animals were generated from which 85 % present the three enzyme isoforms ( nh group ) and 15 % presented the 90 and 65 kda forms ( h group ). through the obtained results , it is suggested that the 90 kda isoforms ( arterial hypertension genetic marker ) continue to be present in the generations originated by the crossings and backcrossings . expression of the hypertension genetic marker , 90 kda isoforms in tissues ( aorta , adrenal , heart , liver , lung , kidney , pancreas ) of rats expontaneouly hypertensives compared with to the wistar and isogenic wistar . it as been identified in previous studies 190 and 65 kda in wistar , whose profile , similar to those described for normotensive individuals . 80 and 65 kda isoforms have been identified in shr rats urine while n - terminal eca fragments repeats the profile , which was found for light hypertensive individuals . the homogenates were submitted to a gel filtration chromatography and two peaks have been detected , having activity on the hhl substrate in different w and w1 rats tissue , whose molecular weights of 137 and 69 kda are similar to those referred for w rats ( table i ). the shr rats tissues also presents two peaks of activity whose estimate molecular weights are 96 e 69 kda , and this profile corresponded to the one found for the enzymes contained in the urine of said rats . the protein expression of the 137 and 69 kda isoforms was observed in all tissues , which have been studied , obtained from wistar and isogenic wistar rats through the western blotting technique . by using the same technique , 96 and 69 kda isoforms expression of all tissues of the shr rats have been confirmed ( table i ). the obtained results show the expression of the 69 kda isoforms ( besides the 137 kda isoforms ) in the w and wi tissues , as the 96 and 69 kda isoforms in shr rats tissues , bringing to the conclusion that the expression of n - domain isoforms , more specifically the 96 kda isoform ( hypertensive genetic marker ) is not restricted only to urine and / or kidney , but are also present , locally , in the studied tissues . table i sumary of the study as to elution fractions and estimated molecular masses , showing the 80 kda eca as hypertension genetic marker . estimated elution molecular weight strains enzymes fraction ( n °) ( kda ) wky wk - 1 32 190 wk - 2 54 65 shr s - 1 50 80 s - 2 55 65 1k1c c - 1 32 140 c - 2 54 65 doca - salt d - 1 34 190 d - 2 52 65 doca - salt cd - 1 34 190 control cd - 2 52 65 shr - sp sp - 1 50 90 sp - 2 55 65 bn bn - 1 32 190 bn - 2 54 65 shr s - 1 50 80 enalapril s - 2 55 65 lyon l - 1 50 80 l - 2 55 65 expression of the hypertension genetic marker , 90 kda isoforms in the mesangial cells in rats culture , expontaneously hypertensesives compared to the wistar rats the glomerulus has been isolated from wistar or shr rats , according to greenspon and krakomer method ( 1950 ). the rats were put under sulfuric ether atmosphere and submitted to a bilateral nefrectomy . kidneys were decapsulated and cortical macrodissecation was carried out . the cortex was separated from the medula and then , fragments were passed through series of sieves which differ in size according to the meshes openings ( 60 , 100 and 200 mesh ). the glomerulus were collected from the surface of the third sieve and forced to pass through a ( 25 × 7 ) needle ( aiming at to decapsulate the glomerulus . the decapsulated glomerulus were counted by using newbauer chamber and divided ( density of ˜ 300 glomerulus / cm 2 ) in 25 cm 2 bottles , using rpmi 1640 supplemented with 20 % of bovine fetal serum , penicillin ( 50 u / ml ), hepes ( 2 . 6 g ) and glutamin 2 mm . the culture bottles were maintained into co 2 ( 5 %), at 37 ° c . each 36 h the medium was changed . after 3 weeks approximately the primary culture of the mesangial cells were submitted to trypsin . the subcultures grew in the same medium . this procedure was repeated up to the third subcultured , when cells were prepared for the experiments : mesangial cells ( mc ) ( 3 ° subcultured ) were incubated for 20 hours with rpmi , without bovine fetal serum ; further , the mc and the medium was collected separately . the collected mean in the 3 ° subcultured was concentrated in an amicon concentrator . the concentrated medium ( 2 . 0 ml ) was submitted to a gel filtration in aca - 44 ( 1 . 5 × 100 . 8 cm column ; volume 178 . 0 ml ), equilibrated with tris - hcl 50 mm buffer , ph 8 . 0 , containing nacl 150 mm . fractions of 2 . 0 ml under flux of 20 ml / h were collected . elution was carried out under a 20 ml flux by one hour . fractions of 2 ml were collected , and monitored by absorbance measurements in 280 nm and the enzymatic activity was quantified , by using hippuryl - his - leu ( hhl ). the cm collected were lysed with 4 ml tris - hcl 50 mm buffer , ph 8 . 0 , containing triton x - 114 1 % and pmfs 0 . 5 mm , through mechanical agitation , by one hour , at 4 ° c . after this period , the lysed cells were centrifuged and the supernatant was collected and concentrated an amicon concentrator , under nitrogen pressure , at 3 kgf / cm 2 . further , 2ml was submitted to a gel filtration chromatography . the results obtained for mc in wistar and shr rats culture presented the same chromatographic profile obtained for human urine of normal individuals and moderated hypertension patients as well as for urine of wistar and shr rats , thus , confirming that in kidneys , more specifically in the glomerulus , the different isoforms , already mentioned are produced ( table ii ). table ii summary of the study groups as to determinate molecular masses . tissues wistar wistar isogenic shr adrenal 137 137 96 69 69 69 aorta 137 137 96 69 69 69 heart 137 137 96 69 69 69 liver 137 137 96 69 69 69 lung 137 137 96 69 69 69 kidney 137 137 96 69 69 69 testicle 137 137 96 69 69 69 based on the fact that 90 kda ace only appears in hypertensive patients / mc of shr / urine of shr rats , it is suggested that it could have an important and specific role as a hypertension genetic marker . based on the studies obtained with the parents and children there was observed that 190 , 90 e 65 kda isoforms are present in normal individuals from hypertensive parents showing that a segregation of this isoform , thus , it could be characterized as a hypertensive predictor . these data were confirmed in crossing and backcrossing of brown norway and shr rats ( table iii ). lanzillo j j , fanburg b l . low molecular weight angiotensin i - 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