The changes of the tubular epithelium phenotype in the contralateral kidney nephrons while developing unilateral ureteral obstruction: an experimental study

Introduction

A high rate of kidney diseases caused by ureteral obstruction leads to a need for experimental and clinical studies on the mechanisms and consistencies of compensatory processes development in patients with renal disorders [1][2][3][4][5][6][7]. A kidney is a complicated organ of narrow specialization that is capable of restoring its functions after a certain degree of damage [8]. There is a concept of renal counterbalance that can be described as a state of increased functional load on the undamaged kidney that is proportional to a decrease in the function of the kidney, damaged because of a unilateral ureteral obstruction (UUO) [9, 10]. However, morphological and functional changes in the contralateral kidney are understudied. Meanwhile, available publications show that changes in the unaffected kidney reflect the adaptive capacity of the organ not only to the increased load. Besides, they target to maintain homeostasis during developing oxidative stress and mitochondrial dysfunction caused by UUO [11][12][13].

The mechanisms underlying the adaptive capacity of kidneys are still disputable. One of such mechanisms is an epithelial-mesenchymal transition (EMT). EMТ leads to changes in the shape, loss in polarity, and an increase in the mobility of epithelial cells associated with enhanced synthesis of collagen. Besides, highly invasive, migrating, and elongated mesenchymal cells [14]. Changes in the polarity and morphology of cells are associated with a decrease in the expression of markers of epithelial phenotype and the appearance of mesenchymal markers [15][16]. The loss of sigs of tissue cell differentiation affects the functioning of the organ in general.

Therefore, a search for key processes that lead to irreversible damage of the obstructed kidney and processes that provide compensatory functioning of the contralateral kidney is a relevant task in precise diagnostic, treatment, and prognosis of obstructive renal diseases. An experimental model shows a series of changes in the damaged and contralateral kidney in the dynamics of obstruction development [17][18].

The study aimed to evaluate the changes in the epithelial nephron cell phenotype of a collateral kidney that occur during modeled unilateral ureteral obstruction.

Materials and methods

UUO was modeled in adult male rabbits aged 3.5 months old with a bodyweight of 2.40–2.75 kg by the method proposed by Giamarellors-Bourbalis et al. [19]. The study was performed according to ethical norms of animal handling approved by the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, Federation of European Laboratory Animal Science Associations, and International Council for Laboratory Animal Science. The study was approved by the Ethical Committee of Rostov State Medical University (Protocol No.21/15 dated 2015-10-12). Depending on the duration of the experiment, the rabbits were separated into 4 groups (6 animals in each group): group I – control group, group II – 7 days of ureteral obstruction, group III – 14 days of ureteral obstruction, group IV – 21 days of mechanical obstruction of the left ureter.

The samples of tissues of contralateral kidneys for morphological studies were fixed in a 10% buffered neutral formalin with further dehydration, waxing, and microtomy. Serial sections of tissue samples were hematoxylin and eosin-stained by a standard protocol for a microscopic study and analysis of morphological changes. For the evaluation of morphometric parameters of the contralateral kidneys, the prepared samples were scanned with a histological slide scanner (Aperio CS2 Leica, Germany) at magnification x400. The obtained images were used for measuring the structures of a nephron with a scale in an Aperio ImageSccope (v. 12.4.0).

Immunophenotyping of tissue samples of the contralateral kidney was performed for markers of epithelial and mesenchymal phenotypes using murine monoclonal antibodies. Markers of the epithelial phenotype were Anti-cytokeratin 7 (СК7) (Abcam 9021, USA, dilution 1:200) and E-cadherin (Abcam 233766, USA, dilution 1:150). The signs of the mesenchymal phenotype were detected with vimentin antibody (Vim) (Biorbyt 317381, UK, dilution 1:200) and α-smooth muscle actin antibody (α-SMA) (Biorbyt 334169, UK, dilution 1:250). The visualization of the formed complexes of antigen-antibody was performed with a system of detection EnVision FLEX (Dako, Denmark).

The microscopic and photo imaging was made using an automated system with LED Leica DM4000 B LED (Leica, Germany). The results of immunohistochemical (IHC) reactions were evaluated by the intensity of staining of each marker.

Results

An insignificant decrease in the urinal space of a renal corpuscle was observed due to an increase in the area of vascular glomerulus by day 7 of the experiment. Two weeks after the development of UUO, dystrophic alterations of the calcium channel were registered in the compensatory kidney that included hyaline-drop dystrophy and hydropic degeneration. Renal glomeruli had an increased area and diameter of the renal corpuscle in comparison with the control group. An insignificant decrease in the lumen of the proximal tubules was registered due to an increase in the epithelial height. On day 21 of UUO, the authors revealed changes in the stroma of renal tissue manifested as net fibrosis localized between separate tubular structures in the medullary substance of a kidney. Morphometric studies showed that changes in the zona glomerulosa and proximal tubules were associated with an increase in the epithelial height in the distal tubules.

Immunophenotyping of the contralateral kidney showed that the expression of epithelial markers (E-cadherin and CK7) remained at the level comparable with a healthy kidney throughout the experiment. During the experiment, E-cadherin was detected in the tubular segments of the nephron of the contralateral kidney in the proximal and distal tubules (Fig. 1А). CK7-positive cells were visualized both in the epithelium of tubular segments of a nephron and epithelium of collecting ducts throughout the experiment (Fig. 1 В).

During the experiment of modeled UUO, the levels of α-smooth muscle actin (α-SMA) positive cells corresponded with the control group (Fig. 2А). However, by day 21, the authors registered a visualization of vimentin not only in the renal corpuscles, which is normal but also in the proximal channels of the contralateral kidney (Fig. 2В). Thus, by day 21 of UUO, adequate expression of epithelial markers was accompanied by the appearance of a mesenchymal phenotype marker, which indicated the reaction of nephron epithelium to an increased load. Thus, by day 21 of UUO, diffused visualization of the mesenchymal marker vimentin in the epithelium of the nephron tubular part of the contralateral kidney was synchronized with the registered morphometric and histological changes that manifested as hydropic degradation and hyaline-drop dystrophy in these structures.

Discussion

An increase in the functional load on the contralateral kidney caused by UUO is a stress factor for epithelial cells of renal tubules. The conducted IHC study revealed some molecular-biological changes in the nephron epithelium. The obtained results were interconnected with earlier obtained parameters of the morphological study on the functioning of the contralateral kidney during OOU in the dynamics [20]. In particular, there were no changes in the immune phenotype of the nephron epithelium of the contralateral kidney for two weeks of the experiment. In turn, morphologic changes had adaptive character and remained within physiological norm considering an increase in load on the compensatory organ. The IHC study results on the epithelial phenotype, in particular, CK7 visualization and significant expression of E-cadherin in the epithelium of nephron tubules, confirmed that the structural integrity of epithelial cells and intercellular adhesion of the epithelial layer of tubular structures remained intact. This also confirmed the efficiency of their functioning. This conclusion comes from an important role of membrane protein E-cadherin in the preservation of intercellular adhesion in all the parts of a nephron [21][22]. In turn, epithelial keratins serve as sensitive indicators of stress. They provide structural and mechanical integrity of epithelial cells. However, a decrease in their expression increases epithelial sensitivity to damage, and their loss is considered to be a typical sign of EMT [23][24]. The visualization of two highly specific epithelial markers in the compensatory kidney indicates the preservation of nephron epithelium integrity throughout the experiment and successful physiological adaptation of the organ to an increased load.

The appearance of the mesenchymal phenotype marker vimentin by day 21 of UUO can be the initial stage of the process of transformation of epithelial cells cytoskeleton of nephron tubules in response to an increase in the load. Some authors believe that even insignificant changes in the phenotype of epithelial cells indicate the formation of the processes of adaptation of nephron to super load, including via EMT. Seccia et al. reported that the expression of mesenchymal proteins in the nephron epithelium reflected stress-resistance of a kidney under increased load and its capacity of maintaining the general homeostasis of the organism [25]. The authors suggest that an increase in the duration of obstruction leads to a gradual decrease in the adaptive reserve of the contralateral kidney and increasing irreversible damage of the obstructed kidney can significantly increase the risk of negative events in the contralateral kidney [26].

Conclusion

The study of the nephron phenotype of the contralateral kidney provides a deeper understanding of molecular mechanisms of their adaptation to an increased load during the development of UUO. The results of the experiments revealed a need for further molecular-biological studies for the improvement of diagnostic and treatment of obstructive uropathies.