The role of endothelial factors in the pathogenesis of hemorrhagic fever with renal syndrome

Hemoragična mrzlica z renalnim sindromom (HMRS) je sistemska bolezen, ki prizadene različne organe in organske sisteme. Okužba s hantavirusi lahko poteka brez kliničnih znakov, kot blaga bolezen ali kot huda bolezen z večorgansko prizadetostjo in smrtnim izidom. Patogeneza HMRS je slabo poznana. Značilnost bolezni je disfunkcija endotelija s prehodno povečano prepustnostjo kapilar, s posledičnim edemom tkiv in organsko prizadetostjo. Izsledki raziskav kažejo, da imajo pri patogenezi HMRS pomembno vlogo različni citokini in kemokini, ki vplivajo na potek in izid bolezni. V slovstvu so zaradi zapletene funkcije citokinov in njihovega delovanja preko različnih mehanizmov, pogosto prišli do nasprotujočih zaključkov glede vpliva na izid bolezni. V doktorski nalogi smo proučevali dnevno spreminjanje kliničnih in laboratorijskih kazalcev, ki so značilni za HMRS ter pomen dejavnikov endotelijske prepustnosti v patogenezi HMRS. S prospektivnim, dnevnim spremljanjem izbranih kliničnih in laboratorijskih kazalcev smo želeli pridobiti natančen vpogled v potek HMRS ter ugotoviti razlike med bolniki, okuženimi z virusom Puumala (PUU) oziroma virusom Dobrava (DOB). Eden od pomembnejših citokinov, ki uravnava žilno prepustnost je VEGF, zato smo v doktorski nalogi proučevali dinamiko VEGF in receptorja sVEGFR2 v plazmi in seču bolnikov v času bolnišničnega zdravljenja. Zanimalo nas je, ali lahko povežemo potek bolezni, virusno breme ter izbrane klinične in laboratorijske kazalce s koncentracijami VEGF oziroma sVEGFR2. Klinični znaki, ki so bili prisotni prvi dan bolezni, so bili povišana telesna temperatura, glavobol in bolečine v mišicah. Prisotni so bili pri 86 %, 65 % in 40 % bolnikov in so v povprečju trajali 4, 4 oziroma 5,5 dni. Sledili so akutna kratkovidnost, ki se je pojavila 5. dan bolezni, nespečnost (6. dan), oligurija/anurija (6. dan), poliurija (9. dan) in sinusna bradikardija (9,5. dan). Prisotni so bili pri 35 %, 30 %, 28 %, 91 % in 35 % bolnikov z mediano trajanja 2, 2, 2, 7 oziroma 1 dan. V laboratorijskih izvidih smo ob sprejemu v bolnišnico ugotovili trombocitopenijo, povečane vrednosti alanin aminotransferaze, C-reaktivnega proteina, prokalcitonina, kreatinina, zmanjšane vrednosti glomerulne filtracije in levkocitozo. Dokazali smo jih pri 95 %, 87 %, 99 %, 91 %, 94 %, 87 % oziroma 55 % bolnikov. V povprečju so trajali 4, 3, 7, 3, 9, 8 oziroma 2 dni. Primerjava kliničnih in laboratorijskih ugotovitev pri bolnikih, okuženih z virusom DOB in virusom PUU, je pokazala razlike v njihovi pojavnosti, jakosti in trajanju. Bolniki, okuženi z virusom DOB so v večjem deležu izpolnjevali kriterije za hujši potek bolezni (prisotnost oligurije oziroma anurije, potreba po zdravljenju s hemodializo, pogostejši dokaz ascitesa, plevralnega izliva, večjih krvavitev) ter daljšo hospitalizacijo. Klasičnega poteka bolezni v petih fazah (vročinska, hipotenzivna, oligurična, poliurična in faza konvalescence) pogosto nismo dokazali. Dnevno spreminjanje koncentracij VEGF in sVEGFR2 v plazmi bolnikov s HMRS ni znatno odstopala od vrednosti, izmerjenih pri kontrolni skupini. Pri dinamiki VEGF smo ugotovili razlike v izločanju VEGF glede vrsto hantavirusa. Pri bolnikih, okuženih z virusom PUU, smo izmerili največje koncentracije VEGF v plazmi med 10. in 15. dnevom bolezni, v času, ko bolniki običajno vstopijo v fazo konvalescence in so odpuščeni iz bolnišnice. V skupini bolnikov z DOB virusno okužbo takšnega vrha izločanja VEGF nismo ugotovili. Pri bolnikih z DOB virusno okužbo smo ugotovili povečane koncentracije VEGF v seču med celotno hospitalizacijo. Ko smo primerjali koncentracije VEGF v plazmi in seču bolnikov s HMRS, so bile le-te večje v seču. Porast plazemske koncentracije VEGF je sovpadal s porastom koncentracije trombocitov v krvi, ki je bil eden izmed prvih pokazateljev kliničnega izboljšanja. V nasprotju s koncentracijami v plazmi, so bile koncentracije VEGF v seču negativno povezane s koncentracijo trombocitov in z diurezo. Ti podatki kažejo na dvojno vlogo VEGF pri patofiziologiji ledvic: v začetnem obdobju bolezni nakazujejo na lokalno izločanje VEGF s posledično okvaro ledvic, kasneje v poteku bolezni pa ima VEGF aktivno vlogo pri popravilu in izboljšanju delovanja ledvic. Pri analizi sočasno odvzetih .vzorcev plazme in seča smo pri bolnikih s PUU virusno okužbo ugotovili povečane koncentracije VEGF v seču v prvih 5. dneh bolezni. V naši raziskavi so bile plazemske koncentracije sVEGFR2 v negativni povezavi z virusnim bremenom. Ugotovili smo tudi pozitivno linearno povezavo med plazemskimi koncentracijami sVEGFR2 ter koncentracijami trombocitov v krvi in diurezo, z večjimi koncentracijami v poliurični fazi bolezni. Pri bolnikih, okuženih z virusom PUU, so se koncentracije sVEGFR2 v seču hitro zmanjšale v nekaj dneh, medtem ko so bile pri bolnikih, okuženih z virusom DOB, povečane do 17. dne bolezni. Vrednosti sVEGFR2 v seču so bile negativno povezane s koncentracijami trombocitov in diureze pri obeh vrstah hantavirusov (PUU in DOB). Povečanje virusnega bremena je značilno sovpadalo z zmanjšanjem plazemskih koncentracij sVEGFR2. Pri bolnikih s krvavitvami so bile močno povečane koncentracije VEGF v plazmi in seču ter koncentracije sVEGFR2 v seču. Določitev lokalnega izločanja VEGF in sVEGFR2 v seču bi lahko bil potencialno uporaben biološki kazalec za prepoznavo bolnikov s HMRS, pri katerih bo bolezen potekala v hujši obliki. V nalogi smo potrdili le hipotezo, da je težji potek HMRS povezan s povečanimi koncentracijami VEGF v seču in da so v plazmi in seču spremenjene koncentracije sVEGFR2.Hemorrhagic fever with renal syndrome (HFRS) is a systemic disease targeting different organs and organ systems. The clinical spectrum ranges from asymptomatic infection to a severe course with fatal outcome. The pathogenesis of HFRS is only partially understood. Capillary leakage is the characteristic of hantavirus disease, resulting in tissue and organ failure. Cytokines may play more than one role by exerting various functions in local and time-dependent mannersuch multifactorial function might explain why different studies have shown their diverse impacts on the disease outcome. One such cytokine, assesed in several studies is vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR2). The aim of our study was to gain detailed insight into HFRS dynamics in patients infected with Puumala (PUU) and Dobrava (DOB) virus by analyzing sequential values of selected clinical and laboratory parameters. We also evaluated the importance of factors causing endothelial dysfunction in the pathogenesis os HFRS. To study kinetics of secretion of VEGF and sVEGFR2 serial blood and serial urine samples were analyzed. Levels of VEGF and sVEGFR2 in plasma and urine samples were compared with selected clinical and laboratory parameters to evaluate the usefulness of VEGF and sVEGFR2 as biomarkers for disease severity. The initial signs/symptoms, appearing on median day 1 of illness were fever, headache, and myalgia. These were present in 86 %, 65 %, and 40 % of patients and had a median duration of 4, 4, and 5.5 days, respectively. The signs/symptoms were followed by myopia (appearance on day 5), insomnia (day 6), oliguria/anuria (day 6), polyuria (day 9), and sinus bradycardia (day 9.5). These were present in 35 %, 30 %, 28 %, 91 %, and 35 % of patientstheir median duration was 2, 2, 2, 7, and 1 day, respectively. Laboratory abnormalities, including thrombocytopenia, elevated alanine aminotransferase, CRP, procalcitonin, creatinine, diminished glomerular filtration rate, and leukocytosis, were ascertained on admission to hospital or on the following day (day 5 or 6 of illness) and were established in 95 %, 87 %, 99 %, 91 %, 94 %, 87 %, and 55 % of patients, and had a median duration of 4, 3, 7, 3, 9, 8, and 2 days, respectively. Comparison of patients infected with DOB and PUU viruses found several differences in the frequency, magnitude, and duration of abnormalities, indicating that Dobrava virus causes the more severe HFRS. In the majority of patients, the classic clinical distinction into febrile, hypotonic, oliguric, polyuric, and convalescent phases of illness is unclear. In assessment of VEGF and sVEGFR2 kinetics in serial plasma samples from HFRS patients, most plasma VEGF levels were not significatly higher than in the control group. However, differences were observed in VEGF secretion in relation to virus species. In PUU-infected group, VEGF levels peaked between day 10 and day 15 of illness, at the time when patients usually enter the recovery stage of disease and are discharged from hospitalhowever, in DOB-infected patients no peak was seen. In DOB-infected patients, VEGF levels in urine were considerably elevated throughout the disease course. Comparison of VEGF dynamics in plasma and urine showed its pronounced secretion in the urine. Increase in plasma VEGF was associated with an increased platelet count, which is one of the first markers of clinical improvementin contrast, the urine VEGF level was negatively associated with platelet count and diuresis. These results suggest a dual role of VEGF: first, local secretion of VEGF in kidneys following by renal impairmentand second, involvement in the repair, as implied by higher levels of plasma VEGF and improvement of clinical parameters. In concomitant samples of plasma and urine, in PUU-infected patients, higher VEGF levels were detected in urine samples, with peak in the first days of illness. The finding of normal plasma VEGF levels and lower plasma sVEGFR2 levels suggests a substantial difference in the circulating active VEGF levels. A decrease of plasma sVEGFR2 could be a potential mechanism for VEGF-directed systemic permeability, where VEGF cotributes to capillary leak by not being inactivated by receptor. In our study, the plasma level of sVEGFR2 was negatively associated with viral load, but positive linear association between plasma sVEGFR2 levels and both, platelet count and diuresis, was recognized, with higher levels in the polyuric stage of the disease. The urine levels of sVEGFR2 in PUU-infected patients decreased rapidly in a few days, but in DOB-infected patients they remained high up to day 17 of illness. In urine, sVEGFR2 levels negatively associated with platelet count and diuresis in both virus species groups (PUU and DOB). Significant association was confirmed between sVEGFR2 and viral load, where an increase of viral load correlated with decresed plasma sVEGFR2. Patients with hemorrhagic manifestations had very high plasma and urine VEGF levels and also high levels of urine sVEGFR2. Measuring a local secretion of sVEGFR2 in urine might be a useful biomarker for identifying those HFRS patients who will progress to severe disease. In our work, we only confirmed the thesis, that the severe course of HFRS is associated with elevated VEGF concentrations in urine and that sVEGFR2 concentrations are altered in urine in plasma