Uticaj placentalnog hormona rasta i insulinu sličnog faktora rasta 1 na glomerule bubrega miševa rođenih sa intrauterusnom restrikcijom rasta

Abstract

Neonatusi koji su in utero izloženi aberantnim fetalnim, maternalnim ili placentalnim faktorima koji koče pravilan rast i razvoj fetusa pripadaju visokorizičnoj grupi novorođenčadi sa intrauterusnom restrikcijom rasta (IUGR). Prema konceptu razvojnog programiranja hroničnih bolesti, dugoročne konsekvence IUGR se protežu do odraslog doba. Patofiziološki mehanizmi ovog procesa su višestruki i delimično razjašnjeni, ali se zna da smanjeni broj nefrona na rođenju kod IUGR potomaka izaziva kompenzatornu hipertrofiju glomerula i pokreće patološke mehanizme koji mogu usloviti razvoj hipertenzije i hroničnu bubrežnu insuficijenciju. Cilj studije je da se na eksperimentalnom modelu intrauterusne restrikcije rasta kod miševa ispita da li IUGR utiče na smanjenje broja glomerula miševa, da li davanje placentalnog hormona rasta (PGH) i insulinu sličnog faktora rasta 1 (IGF1) u različitim dozama utiče na povećanje broja i veličine glomerula i da li je bubrežna funkcija miševa sa IUGR, koji su nakon rođenja dobili PGH i IGF1, u adultnoj dobi kada su žrtvovani, bolja u odnosu na one koji nisu dobili PGH i IGF1. Metode: Korišćen je eksperimentalni model IUGR u kome su gravidne ženke dobijale deksametazon u dozi od 100μg/kg/dan supkutano od 15-21. dana gestacije, tj. do dana porođaja. Po metodi slučajnog izbora mladunci su razvrstani u jednu od šest grupa, po deset miševa u svakoj i ostavljeni na prirodnoj ishrani. Žrtvovani su nakon mesec dana. Iz srca se uzelo 0,5ml krvi za analizu koncentracije cistatina C i kreatinina, a potom je kod svake žrtvovane jedinke odstranjen po jedan bubreg. Histološkom obradom su dobijene pločice koje su analizirane uz pomoć digitalnog mikroskopa, skenirane i prevedene u digitalnu formu. Upotrebom softverskih sistema izračunata je numerička gustina i volumen glomerula svake grupe pojedinačno, u sve tri zone kore bubrega. Rezultati: Miševi kod kojih je eksperimentom izazvan IUGR su imali manji broj glomerula u odnosu na kontrolnu grupu (27.700 vs 36.111). U grupi miševa koji su dobili veću dozu IGF1 (10μg) je bio povećan i broj i volumen glomerula, a koncentracija kreatinina najniža u odnosu na osve ostale grupe, sa statistički značajnom razlikom (r<0,033) u odnosu na kontrolnu grupu bez intervencije. Davanje manje doze IGF1 (1μg), kao i davanje manje (2μg) i veće (10μg) doze PGH nije izazvalo povećanje broja i volumena glomerula. Davanje 10μg PGH je dovelo do smanjenog broja glomerula u površinskoj i intermedijarnoj zoni sa statistički značajnom razlikom (r<0,05) u odnosu na grupu koja je dobila 10μg IGF1 i kontrolnu grupu bez intervencije, kao i do smanjenja volumena glomerula sa statistički značajnom razlikom (r<0,05) u odnosu na grupu koja je dobila 10μg IGF1. Analiza cistatina C je slična u svim ispitivanim grupama. Zaključak: Veća doza IGF1 (10μg) povećava numeričku gustinu i volumensku frakciju glomerula, ali su potrebne dugoročne studije i na životinjskim modelima i na humanoj populaciji da utvrde da li će u starosti glomeruloskleroza i posledična hronična bubrežna insuficijencija pre nastupiti kod IUGR potomaka koji nakon rođenja nisu dobili IGF1 u odnosu na one koji su dobili IGF1, tj. da li aplikovanje IGF1 nakon rođenja IUGR potomci ima protektivno dejstvo i može duže da očuva funkciju njihovih bubrega. U dostupnoj literaturi nema podataka o istraživanjima dejstva PGH na glomerule IUGR potomstva, te su potrebna dalja ispitivanja da bi se dobili relevantni podaci o patofiziološkom dejstvu PGH.

Objectives:Neonates that were in utero subject to aberrant fetal, maternal or placental factors that retard normal growth and development of the fetus are considered a high-risk group of newborns with intrauterine growth restriction (IUGR). According to the concept of developmental programing of chronic illness, long term consequences of IUGR are present up until adulthood. Pathophysiological mechanisms of this process are manifold and only partially understood, but it is accepted that lowered nephron number at birth in IUGR offspring causes compensatory hypertrophy of glomeruli and initiates pathological mechanisms that can cause the development of hypertension and chronic kidney failure. The aim of the study is to investigate, using an experimental model of intrauterine growth restriction in mice, whether IUGR affects the decrease in the number of glomeruli in mice, whether the administration of placental growth hormone (PGH) and insulin-like growth factor 1 (IGF1) in different doses affects the increase in number and size glomeruli and whether the kidney function of mice with IUGR, which received PGH and IGF1 after birth, in adulthood when they were sacrificed, is better compared to those that did not receive PGH and IGF1. Methods: An experimental model of IUGR was used in which pregnant females received dexamethasone at a dose of 100μg/kg/day subcutaneously from 15-21. days of gestation, i.e. until the day of delivery. According to the method of random selection, the cubs were divided into one of six groups, ten mice in each, and left on natural food. They were sacrificed after a month. 0.5 ml of blood was taken from the heart for analysis of cystatin C and creatinine concentration, and then one kidney was removed from each sacrificed individual. Plates were obtained through histological processing, which were analyzed with the help of a digital microscope, scanned and translated into digital form. Using software systems, the numerical density and volume of the glomeruli of each group was calculated individually, in all three zones of the kidney cortex. Results: Mice with experimentally induced IUGR had a lower number of glomeruli compared to the control group (27,700 vs 36,111). In the group of mice that received a higher dose of IGF1 (10μg), the number and volume of glomeruli increased, and the creatinine concentration was the lowest compared to all other groups, with a statistically significant difference (p<0.033) compared to the control group without intervention. Administration of a lower dose of IGF1 (1 μg), as well as administration of a lower (2 μg) and higher (10 μg) dose of PGH did not cause an increase in the number and volume of glomeruli. The administration of 10 μg of PGH led to a decreased number of glomeruli in the superficial and intermediate zone with a statistically significant difference (p<0.05) compared to the group that received 10 μg of IGF1 and the control group without intervention, as well as to a decrease in the volume of glomeruli with a statistically significant difference (p<0.05) compared to the group that received 10 μg of IGF1. Cystatin C analysis didn’t reveal any differences between studied groups. Conclusion: A higher dose of IGF1 (10 μg) increases the numerical density and volume fraction of glomeruli, but long-term studies in both animal models and human populations are needed to determine whether glomerulosclerosis and subsequent chronic renal failure in old age will occur earlier in IUGR offspring that did not receive after birth IGF1, compared to those who received IGF1, ie. whether applying IGF1 after birth to IUGR offspring has a protective effect and can preserve the function of their kidneys longer. In the available literature, there is no data on research on the effect of PGH on the glomeruli of IUGR offspring, and further studies are needed to obtain relevant data on the pathophysiological effect of PGH.