Renal Effects Following Introduction of Cryopreserved Placental Extract on the Background of Experimental Renal Failure

ул. Переяславская, 23, г. Харьков, Украина 61015; тел.: (+38 057) 373-30-34, факс: (+38 057) 373-30-84, электронная почта: seldsan@rambler.ru *To whom correspondence should be addressed: 23, Pereyaslavskaya str., Kharkov, Ukraine 61015; tel.:+380 57 373 3034, fax: +380 57 373 3084, e-mail: seldsan@rambler.ru Department of Cryomorphology, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, Ukraine Отдел криоморфологии, Институт проблем криобиологии и криомедицины НАН Украины, г. Харьков

The purpose of this study was to explore the possibility of a new approach to the correction of functional and morphological changes in the kidney tissues by introducing cryopreserved allogeneic placenta extract (CPE) on the background of the experimental ARF.To do this, renal excretory function and morphological features of kidney tissue were studied at all the stages of development and course of modelled disease, as well as following introduction of CPE.
Исследования функций почек, выполненные через 3 недели после введения глицерола, показали, что состояние острого нарушения выделительной функции почек оставалось стабильным.Об этом свидетельствовали олигоурия, значительное, почти двукратное, снижение СКФ, гиперкреатининемия (рисунок).Через 8 недель после введения глицерола отмечалось незначительное улучшение показателей функциональ-formation, being the basis for CRF) [4].Before sacrificing the experimental animals were placed in a metabolism chamber, where during the diurnal diuresis was assessed, and the collected urine was used to measure the level of creatinine.The level of creatinine was also determined in blood serum.Glomerular filtration rate (GFR) was calculated according Rehberg-Tareyev.Histological studies were done in samples treated in a standard way.The sections were stained with hematoxylin and eosin.Transmitted electron microscopy studies (microscope Selmi, Ukraine) were done in samples conducted through series of alcohols and embedded in epone-araldyte, semithin sections were stained according Sato, ultrathin sections were counterstained by osmium tetroxide.Cryopreserved extract of placenta was procured as follows: rat placenta was homogenized, thrice frozen down to -196°C and thawed, mixed with physiological saline (1:1), centrifuged, supernatant was collected and transferred to plastic 1.8 ml tubes (Nunc, USA), frozen and stored in liquid nitrogen until needed.
Statistical processing was performed using the Statgraph 2.0 software and nonparametric Mann-Whitney criterion.
It is known that glycerol is an osmotic diuretic, which main mechanism of action is a significant reduction in sodium reabsorption in the distal nephron.Glycerol intoxication results in an abrupt rise of clinical symptoms of ARF (anuria, increased creatinine level), the pathogenesis of which is the feature of high doses of osmotically active substances to cause marked vasoconstriction of the renal arterioles and the development of medullar ischemia [3].Moreover, rapidly growing osmolarity in the tubular system is accompanied with an increase in the chloride ions concentration in the space between the macula densa and mesangial glomeruli.As a feedback this shift launches renal blood flow regulatory mechanisms and also causes vasoconstriction of the afferent glomerular arterioles, a drop in GFR and the development of ARF.
Studies of renal function performed 3 weeks following the glycerol administration showed that state of acute excretory disorders remained stable.This was evidenced by oliguria, a significant almost two-fold decreased GFR, hypercreatininemia (Figure).In 8 weeks following the glycerol administration only insignificant improvement in renal function in experimental animals was found: diurnal diuresis, GFR and blood creatinine level were increased, urine creatinine level was decreased.
Introduction of CPE caused improvement even to the 3 rd week of experiment: blood creatinine level decreased, urine creatinine and GFR increased.After 8 weeks, diurnal diuresis and GFR were higher if compared with previous observation term, but still lower than the intact control values (Figure ).Apparently, the functional disorders of the kidney have been associated with impaired renal blood flow, caused by the action of the renin-angiotensin system.
Morphological studies have shown that on the background of blood circulatory disorders in kidney tissue 3 weeks later glycerol administration, secondary damages of tubular apparatus occurred in terms of dystrophy and necrosis of proximal and distal convoluted tubules epithelium.Dead and desquamated epithelium appeared in discharging tubules and blocked them, that led to atrophy of epithelial tubules and significant increase of their diameter (thyroid-like appearance of the kidney).Violated ultrafiltrate outflow caused significant (if compared with the norm) increase in the size of the Shumlyansky-Bowman's capsule.In its turn, this negatively affected blood filling of glomeruli, which was evidenced by the reduction of the capillaries area.These events led to the prevalence of dystrophic and necrotic processes   despite the presence of regene-rative processes in the epithelium of the distal and pro-ximal tubules.In 8 weeks after the glycerol administra-tion the metric parameters of glomeruli, capsule and vascular loops were significantly lower than the norm.Degeneration and necrosis of tubular epithelium were observed together with significant increase in their lumen.Moreover the sites of inflammation were found, leukocyte infiltration was observed in the interstitium as well as interstitial edema.Further increase in interstitial pressure led to a greater compression of tubules and poor blood circulation in the kidney.Accrementition of connective tissue at inflammation sites additionally in-creased the vascular compression of the interstitium, as evidenced by hemorrhages and venous plethora.Thus, the basis of ARF are dystrophic and necrotic processes in the epithelium of the proximal and distal tubules, as well as poor blood circulation.The transition to the CRF in 8 weeks of experiment has another basis, an interstitial nephritis and nephrosclerosis.
The study of sections obtained from the kidneys of animals treated with CPE revealed that hyalinosis was found only in several epithelial cells, and no interstitial inflammation was present.Dimensions of glomeruli did not differ from the intact control, and there were no signs of lymph and blood circulation disorders.
The conducted investigation allows to conclude that introduction of cryopreserved extract of allogeneic placenta in early terms of renal failure development ('shock kidney' stage) improves the functional characteristics of the kidneys, as well as prevents the development of structure changes and, finally, do not allow the disease to enter a chronic phase.