Efekti hronične primene losartana i tempola u eksperimentalnom modelu fokalno segmentne glomeruloskleroze kod spontano hipertenzivnih pacova

Previous studies have shown that renin angiotensin system (RAS) plays an important role in the pathogenesis and progression of focal segmental glomerulosclerosis (FSGS). Oxidative stress is involved in the development and progression of chronic kidney disease. However, the association of oxidative stress and RAS in the progression of FSGS has not been completely elucidated. In this study we investigated the effects of chronic tempol (free radical scavenger, SOD mimetic), losartan (selective angiotensin II type 1 receptor (AT1R) blocker), and their combined treatment in slowing down the progression of FSGS, in spontaneously hypertensive (SH) rats with adriamycin (ADR) nephropathy. Animals were initially divided into two experimental groups: control (SHC) and group that received ADR 2 mg/kg i.v. twice in an interval of 21 days. After the second injection of ADR, the animals were given tap water (SHADR), losartan (SHADR+L), tempol (SHADR+T) or combined treatment (SHADR+T+L) by gavage. Hemodynamic measurements were performed, blood, urine, and kidney samples were taken for biochemical and histopathological analysis. Immunohistochemical method was used for protein expression and localization of the nestin and vimentin, proteins of cytoskeleton network, and change in their protein expression is a marker of cell injury. Matrix metalloproteinase-1 (MMP-1), which participates in the degradation of the extracellular matrix components and preserving the integrity of the glomerulus, was determined by the ELISA method. The parameters of oxidative stress: lipid peroxidation, protein carbonyl content (PCOs); antioxidant defense: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and antioxidant capacity were analysed. Immunohistochemical, Western blot and ELISA method were used for analysis of the Nox2 and Nox4 protein expression, isoforms of the catalytic subunit of NADPH oxidase, the main source of reactive oxygen species in the cell, involved in the inflammation process. Protein expression of three isoforms of azote monoxide synthase, inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) were determined by Western blot and immunohistochemical method in the kidney. Total metabolites of NO (nitrites, nitrates) were measured in the kidney and urine...Dosadašnja istraživanja pokazala su da renin angiotenzin sistem (RAS) ima važnu ulogu u patogenezi i progresiji fokalno segmentne glomeruloskleroze (FSGS). Oksidativni stres je prisutan u hroničnoj bubrežnoj slabosti i doprinosi progresiji bolesti. Povezanost oksidativnog stresa i RAS u procesima progresije FSGS još je nedovoljno razjašnjena. Stoga su istraživanja u okviru ove disertacije bila usmerena ka ispitivanju efekata hronične primene tempola (sakupljač slobodnih radikala, SOD mimetik), losartana (blokator receptora za angiotenzin II tipa 1, AT1R), kao i njihove kombinacije, na usporavanje progresije FSGS izazvane adrijamicinom (ADR) kod spontano hipertenzivnih (SH) pacova. Životinje su inicijalno deljene u dve eksperimentalne grupe, kontrolnu (SHC) i grupu koja je primila ADR 2 mg/kg i.v. dva puta u intervalu od 21 dan. Nakon druge injekcije ADR, životinje su dobijale vodu (SHADR), losartan (SHADR+L), tempol (SHADR+T) i kombinovani tretman (SHADR+T+L) gavažom. Na kraju šeste nedelje tretmana vršena su hemodinamska merenja i uzorkovanje krvi, urina i bubrega. Na osnovu biohemijskih parametara vršena je procena lipidnog statusa i bubrežne funkcije. Urađena je histopatološka analiza bubrega. Imunihistohemijskom metodom ispitivani su proteini citosketeta, nestin i vimentin, čije izmenjene ekspresije su pokazatelj oštećenja bubrežnog tkiva. Matriksna metaloproteinaza-1 (MMP-1), koja učestvuje u degradaciji komponenti vanćelijskog mariksa i očuvanju integriteta glomerula, određivana je ELISA metodom. Ispitivani su parametri oksidativnog stresa: lipidna peroksidacija i nivo karbonilovanih proteina (PCOs); aktivnosti enzima antioksidativnog sistema: superoksid dismutaze (SOD), katalaze (CAT), glutation peroksidaze (GPx) i glutation reduktaze (GR); kao i antioksidativni kapacitet. Primenom imunohistohemijske, Western blot i ELISA metode u bubrezima su određivane ekspresije Nox2 i Nox4 izoforme katalitičke subjedinice NADPH oksidaze, glavni izvor reaktivnih kiseoničnih vrsta u ćeliji, uključene u proces inflamacije. Ekspresija proteina tri izoforme azot-monoksid sintaze, inducibilna (iNOS), endotelna (eNOS) i neuronska (nNOS), određivane su Western blot i imunohistohemijskom metodom u tkivu bubrega. Određivani su ukupni metaboliti NO-a (nitriti, nitrati) u urinu i bubrezima..

Effects of chronic losartan and tempol treatments on experimental focal segmental glomerulosclerosis in spontaneously hypertensive rats

Dosadašnja istraživanja pokazala su da renin angiotenzin sistem (RAS) ima važnu ulogu u patogenezi i progresiji fokalno segmentne glomeruloskleroze (FSGS). Oksidativni stres je prisutan u hroničnoj bubrežnoj slabosti i doprinosi progresiji bolesti. Povezanost oksidativnog stresa i RAS u procesima progresije FSGS još je nedovoljno razjašnjena. Stoga su istraživanja u okviru ove disertacije bila usmerena ka ispitivanju efekata hronične primene tempola (sakupljač slobodnih radikala, SOD mimetik), losartana (blokator receptora za angiotenzin II tipa 1, AT1R), kao i njihove kombinacije, na usporavanje progresije FSGS izazvane adrijamicinom (ADR) kod spontano hipertenzivnih (SH) pacova. Životinje su inicijalno deljene u dve eksperimentalne grupe, kontrolnu (SHC) i grupu koja je primila ADR 2 mg/kg i.v. dva puta u intervalu od 21 dan. Nakon druge injekcije ADR, životinje su dobijale vodu (SHADR), losartan (SHADR+L), tempol (SHADR+T) i kombinovani tretman (SHADR+T+L) gavažom. Na kraju šeste nedelje tretmana vršena su hemodinamska merenja i uzorkovanje krvi, urina i bubrega. Na osnovu biohemijskih parametara vršena je procena lipidnog statusa i bubrežne funkcije. Urađena je histopatološka analiza bubrega. Imunihistohemijskom metodom ispitivani su proteini citosketeta, nestin i vimentin, čije izmenjene ekspresije su pokazatelj oštećenja bubrežnog tkiva. Matriksna metaloproteinaza-1 (MMP-1), koja učestvuje u degradaciji komponenti vanćelijskog mariksa i očuvanju integriteta glomerula, određivana je ELISA metodom. Ispitivani su parametri oksidativnog stresa: lipidna peroksidacija i nivo karbonilovanih proteina (PCOs); aktivnosti enzima antioksidativnog sistema: superoksid dismutaze (SOD), katalaze (CAT), glutation peroksidaze (GPx) i glutation reduktaze (GR); kao i antioksidativni kapacitet. Primenom imunohistohemijske, Western blot i ELISA metode u bubrezima su određivane ekspresije Nox2 i Nox4 izoforme katalitičke subjedinice NADPH oksidaze, glavni izvor reaktivnih kiseoničnih vrsta u ćeliji, uključene u proces inflamacije. Ekspresija proteina tri izoforme azot-monoksid sintaze, inducibilna (iNOS), endotelna (eNOS) i neuronska (nNOS), određivane su Western blot i imunohistohemijskom metodom u tkivu bubrega. Određivani su ukupni metaboliti NO-a (nitriti, nitrati) u urinu i bubrezima...Previous studies have shown that renin angiotensin system (RAS) plays an important role in the pathogenesis and progression of focal segmental glomerulosclerosis (FSGS). Oxidative stress is involved in the development and progression of chronic kidney disease. However, the association of oxidative stress and RAS in the progression of FSGS has not been completely elucidated. In this study we investigated the effects of chronic tempol (free radical scavenger, SOD mimetic), losartan (selective angiotensin II type 1 receptor (AT1R) blocker), and their combined treatment in slowing down the progression of FSGS, in spontaneously hypertensive (SH) rats with adriamycin (ADR) nephropathy. Animals were initially divided into two experimental groups: control (SHC) and group that received ADR 2 mg/kg i.v. twice in an interval of 21 days. After the second injection of ADR, the animals were given tap water (SHADR), losartan (SHADR+L), tempol (SHADR+T) or combined treatment (SHADR+T+L) by gavage. Hemodynamic measurements were performed, blood, urine, and kidney samples were taken for biochemical and histopathological analysis. Immunohistochemical method was used for protein expression and localization of the nestin and vimentin, proteins of cytoskeleton network, and change in their protein expression is a marker of cell injury. Matrix metalloproteinase-1 (MMP-1), which participates in the degradation of the extracellular matrix components and preserving the integrity of the glomerulus, was determined by the ELISA method. The parameters of oxidative stress: lipid peroxidation, protein carbonyl content (PCOs); antioxidant defense: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and antioxidant capacity were analysed. Immunohistochemical, Western blot and ELISA method were used for analysis of the Nox2 and Nox4 protein expression, isoforms of the catalytic subunit of NADPH oxidase, the main source of reactive oxygen species in the cell, involved in the inflammation process. Protein expression of three isoforms of azote monoxide synthase, inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) were determined by Western blot and immunohistochemical method in the kidney. Total metabolites of NO (nitrites, nitrates) were measured in the kidney and urine..

Uticaj hronične promene vrednosti hematokrita na krvni pritisak i glomerulsku filtraciju kod pacova sa urođenom hipertenzijom

Many studies in hypertensive humans and animals have shown that increased blood viscosity is in direct relation with essential hypertension. The aim of our studies was to investigate the effects of chronic hematocrit value changes on arterial blood pressure and kidney function in genetically induced hypertension. To this end, we studied the effects of several interventions, designed to increase/decrease hematocrit, on hemodynamic parameters, vascular reactivity, glomerular filtration and renal function curve in spontaneously hypertensive rats (SHR). Results of our study show that chronic hematocrit value elevation increases blood pressure and peripheral vascular resistance in SHR. On the other hand, chronic hematocrit lowering elucidates blood pressure and peripheral vascular resistance decrease followed by cardiac output rising. Both hematocrit value changes significantly reduce vasodilatory vascular response. Hematocrit lowering induces acute renal failure. Sodium excretion is shifted to higher blood pressure values in high hematocrit value animals and opposite - lower blood pressure values in low hematocrit value animals. Repeated transfusions develop salt sensitive malignant hypertension in SHR. Further studies are necessary to evaluate the degree of kidney damage after chronic hematocrit value changes in SHR.Brojna ispitivanja izvedena na životinjama i ljudima su ukazala da je povećanje viskoznosti krvi u direktnoj vezi sa hipertenzijom. Cilj naše studije je bio da se istraži uticaj promene vrednosti hematokrita (Hct) na krvni pritisak i funkciju bubrega u urođenoj hipertenziji. U tom cilju, istraživan je uticaj hroničnog povećanja ili smanjenja vrednosti Hct na hemodinamske parametre, vaskularnu reaktivnost, glomerulsku filtraciju i krivu bubrežne funkcije kod pacova sa urođenom hipertenzijom (SHR). Rezultati naše studije su ukazali da hronično povećanje vrednosti Hct dovodi do povećanja arterijskog krvnog pritiska (AKP) i perifernog vaskularnog otpora (PVO) kod SHR . Nasuprot tome, hronično smanjenje vrednosti Hct dovodi do smanjenja AKP i PVO, praćenih povećanjem minutnog volumena srca. Obe promene vrednosti Hct dovode do smanjenja vaskularne reaktivnosti. Hronično smanjenje vrednosti Hct izaziva nastanak akutne bubrežne insuficijencije. Ekskrecija natrijuma je pomerena ka većim vrednostima krvnog pritiska kod pacova sa povećanim vrednostima Hct, a ka nižim kod pacova sa sniženim vrednostima Hct. Hronično povećanje vrednosti Hct dovodi do razvoja maligne hipertenzije kod SHR. Potrebne su dalje studije da bi se utvrdio tačan stepen i priroda oštećenja bubrega nakon hronične promene vrednosti Hct

Uticaj blokade receptora tip 1 za angotenzin II udružene sa uklanjanjem superoksidnog anjona na postishemični bubreg kod pacova sa urođenom hipertenzijom

Ischemic acute kidney injury is characterized by renal vasoconstriction, filtration failure, tubular obstruction, tubular backleak and overproduction of angiotensin II and reactive oxygen species. Considering this complexity, the aim of our study was to investigate the effects of angiotensin II type-1 receptor blocker - Losartan and superoxide anion scavenger - Tempol, in a combined treatment on acute kidney injury in postischemic hypertensive rats. The experiment was performed in anesthetized, adult male spontaneously hypertensive rats. The right kidney was removed and the left renal artery was occluded for 40 minutes. Experimental groups received combined treatment (Losartan + Tempol) or saline in the femoral vein 5 minutes before, during and 175 minutes after clamp removal. Hemodynamics and biochemical parameters were measured and kidney specimens were collected 24h after reperfusion. Histological examination was performed by optical microscopy. Combined treatment improves renal haemodynamics parameters which were exacerbated due to acute kidney injury. Acute kidney injury significantly decreased creatinine and urea clearance and increased lipid peroxidation in the plasma. Treatment with Losartan and Tempol induced a significant increase of creatinine and urea clearance. Lipid peroxidation in the plasma decreased and glutathione peroxidase enzyme activity in the erythrocytes increased after Losartan + Tempol treatment. This combined treatment reduced cortico-medullary necrosis and tubular dilatation in the kidney. Our results indicate that synergism of Losartan and Tempol treatment could have beneficial effects on blood pressure and kidney function, during postischemic acute kidney injury development in experimental hypertension.Glavne karakteristike ishemične akutne bubrežne slabosti su: renalna vazokonstrikcija, pad glomerulske filtracije, tubularna opstrukcija, vraćanje glomerulskog filtrata u intersticijum tubula i prekomerna produkcija angiotenzina II i reaktivnih vrsta kiseonika. Uzimajući u obzir kompleksnost ovog poremećaja, cilj ove studije je bio da istraži efekte kombinovanog tretmana blokatora angiotenzin II receptora tip 1 - Losartana i sakupljača superoksidnog anjona - Tempola u modelu ishemične bubrežne slabosti kod hipertenzivnih pacova. Eksperiment je urađen na odraslim anesteziranim mužjacima hipertenzivnih pacova. Desni bubreg je uklonjen, dok je na levoj renalnoj arteriji urađena okluzija u trajanju od 40 minuta. Eksperimentalne grupe su primile kombinovani tretman (Losartan+Tempol) ili fiziološki rastvor u femoralnu venu, 5 minuta pre i 175 minuta nakon uklanjanja kleme sa renalne arterije. Hemodinamski i biohemijski parametri su izmereni, a uzorci bubrega uzimani su 24 časa nakon reperfuzije. Histološka ispitivanja su rađena pomoću svetlosnog mikroskopa. Kombinovani tretman poboljšava renalnu hemodinamiku, poremećenu usled ishemične akutne bubrežne slabosti. U ovom modelu bubrežne slabosti dolazi do pada klirensa kreatinina i uree, i povećanja lipidne peroksidacije u plazmi.Tretman Losartanom i Tempolom dovodi do značajnog povećanja klirensa kreatinina i uree. Lipidna peroksidacija je smanjena, a aktivnost glutation peroksidaze u eritrocitima je povećana nakon kombinovanog tretmana sa Losartanom i Tempolom. Takođe, ovakav kombinovan tretman smanjuje kortiko-medularnu nekrozu i tubularnu dilataciju u bubregu. Naši rezultati ukazuju na to da sinergizam Losartana i Tempola može imati povoljan efekat na krvni pritisak i bubrežnu funkciju tokom razvoja postishemične akutne bubrežne slabosti u eksperimentalnoj hipertenziji

Vibroacustic microvibrations enhance kidney blood supply, glomerular filtration and glutathione peroxidase activity in spontaneously hypertensive rats

Limited numbers of studies include research of microvibration therapy in experimental models. We examined effects of chronic vibroacustic-microvibration treatment on haemodynamics and anti-oxidative defense in experimental hypertension. Study was performed on chronically treated hypertensive and normotensive Wistar rats. Mean arterial pressure (MAP), cardiac output (CO), renal blood flow (RBF), glomerular filtration and activity of anti-oxidative enzymes were determined after three weeks treatment. Vibroacustic treatment had no influence on MAP and CO, but RBF was increased in both groups of treated rats. Additionally, vibroacustic treatment enhanced diuresis and increased glomerular filtration in hypertensive rats. Glutathione peroxidase (GSH-Px) activity was elevated in both treated rat strains, but activity of superoxide dismutase was unchanged. We conclude that microvibration treatment doesn't ameliorate hypertension but improves renal blood supply (trough diminished renal vascular resistance), glomerular filtration, diuresis, and enhances glutathione dependent anti-oxidant defense with more important beneficials in hypertensive animals

Highly potent antioxidant olea europaea l. Leaf extract affects carotid and renal haemodynamics in experimental hypertension: the role of oleuropein

Haemodynamic alterations in carotid and renal arteries are associated with the severity of target organ damage in patients with hypertension. Dietary habits, such as the Mediterranean diet, regulate blood pressure and oxidative stress, thus reduce the mortality rate due to cardiovascular diseases. In this study, our aim was to evaluate the reducing activity, antioxidant capacity and metal chelating ability of standardized Olea europaea L. leaf extract (OLE), and to test its (5, 25, 50 mg/kg) acute in vivo effects, as well as oleuropein's (OP, 10 mg/kg) on oxidative stress, carotid, renal and systemic haemodynamic parameters (blood pressure, heart rate, cardiac output, peripheral resistance) in spontaneously hypertensive rats (SHR). OLE has a higher antioxidative capacity than BHT, higher reducing ability than vitamin C, and 23 times lower capacity for metal ion chelation than EDTA. All three doses of OLE, and OP, improved oxidative stress in SHR. OLE5 improved carotid and renal haemodynamics, without significant effects on systemic haemodynamics. Two different mechanisms of antihypertensive responses to OLE were observed, OLE25 was most effective in reducing cardiovascular risks by improving systemic and regional (carotid and renal) haemodynamics, peripheral and regional vascular resistance. OLE50 causes the improvement of blood pressure and cardiac performances, but tends to retain elevated vascular resistance, therefore, reducing the inflow of blood into the brain and kidneys of the SHR. The OP did not alter systemic or regional haemodynamics, suggesting others constituents responsible for changes of cardiac function, as well as carotid and renal haemodynamics in response to OLE50

Resveratrol improved kidney function and structure in malignantly hypertensive rats by restoration of antioxidant capacity and nitric oxide bioavailability

Objective Background: The main cause of death among patients with malignant hypertension is a kidney failure. The promising field in essential and malignant hypertension therapy could be centered on the amelioration of oxidative stress using antioxidant molecules like resveratrol. Resveratrol is a potent antioxidative agent naturally occurred in many plants that possess health-promoting properties. Methods: In the present study, we investigated the therapeutic potential of resveratrol, a polyphenol with anti-oxidative activity, in NG-L-Arginine Methyl Ester (L-NAME) treated spontaneously hypertensive rats (SHR) - malignantly hypertensive rats (MHR). Results: Resveratrol significantly improves oxidative damages by modulation of antioxidant enzymes and suppression of prooxidant factors in the kidney tissue of MHR. Enhanced antioxidant defense in the kidney improves renal function and ameliorates the morphological changes in this target organ. Besides, protective properties of resveratrol are followed by the restoration of the nitrogen oxide (NO) pathway. 4) Conclusion: Antioxidant therapy with resveratrol could represent promising therapeutical approach in hypertension, especially malignant, against kidney damage

Upregulation of Heme Oxygenase-1 in Response to Wild Thyme Treatment Protects against Hypertension and Oxidative Stress

High blood pressure is the most powerful contributor to the cardiovascular morbidity and mortality, and inverse correlation between consumption of polyphenol-rich foods or beverages and incidence of cardiovascular diseases gains more importance. Reactive oxygen species plays an important role in the development of hypertension. We found that wild thyme (a spice plant, rich in polyphenolic compounds) induced a significant decrease of blood pressure and vascular resistance in hypertensive rats. The inverse correlation between vascular resistance and plasma heme oxygenase-1 suggests that endogenous vasodilator carbon monoxide generated by heme oxidation could account for this normalization of blood pressure. Next product of heme oxidation, bilirubin (a chain-breaking antioxidant that acts as a lipid peroxyl radical scavenger), becomes significantly increased after wild thyme treatment and induces the reduction of plasma lipid peroxidation in hypertensive, but not in normotensive rats. The obtained results promote wild thyme as useful supplement for cardiovascular interventions

Effects of Single and Combined Losartan and Tempol Treatments on Oxidative Stress, Kidney Structure and Function in Spontaneously Hypertensive Rats with Early Course of Proteinuric Nephropathy

Oxidative stress has been widely implicated in both hypertension and chronic kidney disease (CKD). Hypertension is a major risk factor for CKD progression. In the present study we have investigated the effects of chronic single tempol (membrane-permeable radical scavenger) or losartan (angiotensin II type 1 receptor blocker) treatment, and their combination on systemic oxidative status (plasma thiobarbituric acid-reactive substances (pTBARS) production, plasma antioxidant capacity (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, pABTS), erythrocyte antioxidant enzymes activities) and kidney oxidative stress (kTBARS, kABTS, kidney antioxidant enzymes activities), kidney function and structure in spontaneously hypertensive rats (SHR) with the early course of adriamycin-induced nephropathy. Adult SHR were divided into five groups. The control group received vehicle, while the other groups received adriamycin (2 mg/kg, i.v.) twice in a 21-day interval, followed by vehicle, losartan (L, 10 mg/kg/day), tempol (T, 100 mg/kg/day) or combined T+L treatment (by gavage) during a six-week period. Adriamycin significantly increased proteinuria, plasma lipid peroxidation, kidney protein oxidation, nitrite excretion, matrix metalloproteinase-1 (MMP-1) protein expression and nestin immunostaining in the kidney. Also, it decreased kidney antioxidant defense, kidney NADPH oxidase 4 (kNox4) protein expression and abolished anti-inflammatory response due to significant reduction of kidney NADPH oxidase 2 (kNox2) protein expression in SHR. All treatments reduced protein-to-creatinine ratio (marker of proteinuria), pTBARS production, kidney protein carbonylation, nitrite excretion, increased antioxidant capacity and restored kidney nestin expression similar to control. Both single treatments significantly improved systemic and kidney antioxidant defense, bioavailability of renal nitric oxide, reduced kMMP-1 protein expression and renal injury, thus retarded CKD progression. Losartan improved blood pressure, as well astubular injury and restored anti-inflammatory defense by reverting kNox2 expression to the control level. Interestingly, tempol was more successful in reducing systemic oxidative stress, proteinuria, kMMP-1 and glomerulosclerosis. However, combined treatment failed to overcome the beneficial effects of single treatments in slowing down the progression of ADR-induced nephropathy in SHR

Clinicopathological Relevance of PAX8 Expression Patterns in Acute Kidney Injury and Chronic Kidney Diseases

Transcription factor PAX8, expressed during embryonic kidney development, has been previously detected in various kidney tumors. In order to investigate expression of PAX8 transcription factor in acute kidney injury (AKI) and chronic kidney diseases (CKD), immunohistochemical analysis was performed. Presence, location and extent of PAX8 expression were analyzed among 31 human kidney samples of AKI (25 autopsy cases, 5 kidney biopsies with unknown etiology and 1 AKI with confirmed myoglobin cast nephropathy), as well as in animals with induced postischemic AKI. Additionally, expression pattern was analyzed in 20 kidney biopsy samples of CKD. Our study demonstrates that various kidney diseases with chronic disease course that results in the formation of tubular atrophy and interstitial fibrosis, lead to PAX8 expression in the nuclei of proximal tubules. Furthermore, patients with PAX8 detected within the damaged proximal tubuli would be carefully monitored, since deterioration in kidney function was observed during follow-up. We also showed that myoglobin provoked acute kidney injury followed with large extent of renal damage, was associated with strong nuclear expression of PAX8 in proximal tubular cells. These results were supported and followed by data obtained in experimental model of induced postischemic acute kidney injury. Considering these findings, we can assume that PAX8 protein might be involved in regeneration process and recovery after acute kidney injury. Thus, accordingly, all investigation concerning PAX8 immunolabeling should be performed on biopsy samples of the living individuals