Introduction: Wilms’ tumor 1 antigen (WT1) expression in podocytes has the important role of maintaining their integrity and glomerular function. Vimentin also plays a role in preserving podocyte function and in morphological changes observed after injury. Corticotropin-releasing factor (CRF) is important in stress and in maintaining homeostasis. According to our previous studies, tyrosine (Tyr) isoforms (meta- and ortho-Tyr) may play a role in the development of many diseases. Methods: Our aim was to investigate the expression of WT1, vimentin, and CRF in the human kidney and in HEK 293 cell cultures. Histological and clinical features of 42 focal segmental glomerulosclerosis (FSGS) patients were evaluated and compared to those of patients with thin basement membrane as a control group. Cells were cultured in medium containing para-, meta-, and ortho-Tyr, and their expression of WT1, vimentin, and CRF were determined by immunocytochemistry. Podocyte foot process effacement was investigated by electron microscope. Results: The intensity of WT1 staining in glomeruli was the same in FSGS and control groups, but it was lower in the tubulointerstitium of FSGS patients. Vimentin was lower in glomeruli of FSGS patients (p = 0.009), and it was higher in the tubulointerstitium compared to the control group (p = 0.003). CRF intensity was lower in the glomeruli (p = 0.002). Podocyte foot process effacement determined by electron microscope showed correlation with vimentin and CRF in glomeruli. WT1 staining intensity was lower in meta- and ortho-Tyr group (p = 0.001; p = 0.009). Vimentin was lower in the meta-Tyr group (p = 0.001). Discussion: Our observations on kidney biopsy samples support that the reduction of WT1 and vimentin could be characteristic for FSGS. Our results on HEK cells suggest that meta- and ortho-Tyr may play a role in the development of FSGS.
Focal segmental glomerulosclerosis (FSGS) is an important renal syndrome . Podocyte injury with foot process effacement (FPE) and podocyte detachment are typical morphological events in FSGS ; however, tubulointerstitial changes are less frequently investigated.
WT1 gene has an important role in the development of the kidneys. Decreased expression of WT1 genes plays an important role in the development of some types of glomerular diseases . WT1 may act as a transcription factor, cofactor, or post-transcriptional regulator . During nephrogenesis, increasing gene expression can be detected, but at the end of differentiation, it is downregulated except for podocytes , suggesting that WT1 plays an important role in maintaining their function.
In animal models, disruption of WT1 will lead to an early GBM thickening and finally glomerular sclerosis. In humans, WT1 reduction results in a reduced filtration area and permeability , with proteinuria and renal scarring .
Intermediate filaments are tubular structures with a diameter of 7–11 nm. They can be found in most nucleated cells and may serve as differentiation markers . Vimentin characterizes cells of mesenchymal origin and vascular smooth muscles. Podocytes in human glomeruli show a strong reaction to anti-vimentin. Vimentin plays an important role in maintaining podocyte integrity and in morphological changes in response to injury [7, 8]. To a lesser extent, mesangial and endothelial cells also react to anti-vimentin.
Vimentin may act as a sensitive marker for tubular damage. Tubular epithelial-to-mesenchymal transition (EMT) has been demonstrated by vimentin neo-expression . The immunofluorescent staining pattern of intermedier filaments may change under pathological conditions in the human glomerulus .
Corticotropin-releasing factor (CRF) has an important role in response to stress and maintaining homeostasis. CRF is thought to have both direct and indirect effects on the immune system: indirectly, CRF may downregulate inflammation through the release of glucocorticoids, whereas a direct pro-inflammatory action of CRF may occur in peripheral tissues . Relatively few data are available about the expression and biological role of CRF in kidneys.
Reactive oxygen species (ROS) are produced by several endogenous and exogenous processes. Oxidative stress occurs from the imbalance between ROS production and antioxidant defenses.
Oxidative stress plays an important role in the pathogenesis of numerous diseases including FSGS . It is present at the early stages of the diseases and contributes to their progression . NADPH oxidases and mitochondrial respiratory chain are the sources of the majority of ROS. Antioxidant enzymes act as defense mechanisms against reactive oxidants, causing modification of proteins, DNA, and lipids .
When hydroxyl free radicals are present, L-phenylalanine is converted into para-tyrosine (Tyr), nonphysiological meta-Tyr, and ortho-Tyr besides the physiological isomer para-Tyr . According to our previous studies, pathological amino acids are not merely markers of oxidative stress but also play a role in the development of many diseases [14‒18].
The aim of our study was to investigate the expression of WT1, vimentin, and CRF in glomeruli and tubulointerstitium of patients with FSGS and in a control group. Furthermore, the effect of meta- and ortho-Tyr on WT1, vimentin, and CRF expression in HEK 293 cells was also studied.
In our retrospective study, histological, clinical, and laboratory features of 42 patients with FSGS were evaluated. The study was approved by the Regional Committee for Research Ethics, University of Pécs (No: 9111/22). All of the renal biopsies were performed at the 2nd Department of Medicine and Nephrology-Diabetes Center, University of Pécs Medical School, Hungary. All patients were at least 18 years old with a mean age of 39.9 ± 18.6 years (23 males and 19 females).
As controls, kidney biopsy specimens from 7 patients with thin basement membrane disease were used. Their mean age was 45.1 ± 15.6 years (3 males and 4 females). Body mass index, blood pressure, laboratory data, and medical drugs taken at the time of biopsy were recorded. Baseline characteristics of FSGS and control groups are summarized in Table 1.
Cell Culture and Treatments
HEK 293 cell line (ATCC® CRL-11268TM) was cultured maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich CAT, number: D6046) with 10% fetal bovine serum (Gibco, CAT number: 16170-078), 100U/mL penicillin, 0.1 mg/mL streptomycin (Gibco, CAT number: 15070-063), 2 μg/mL fluconazole (Fresenius Kabi, Hungary). Dulbecco’s modified Eagle’s medium contains 36 mg/L para-Tyr. As a control, 72 mg/L para-Tyr (physiological Tyr) was added to the medium of the control group and 72 mg/L meta- or ortho-Tyr (unphysiological Tyr) to the medium of the other two groups. Cells were grown in a humidified incubator at 37°C and 5% CO2.
After 5 days, the cells were washed twice with PBS to remove any trace of the medium. Cells were fixed with 4% buffered paraformaldehyde (Sigma-Aldrich, CAT number: P-6148) in PBS for 20 min, followed by three washes with PBS with a 5-min incubation for each wash. Permeabilized with 0.3% Triton-X 100 (Sigma-Aldrich, CAT number: T-9284) in PBS for 10 min, it was followed by three washes with PBS with a 5-min incubation for each wash and blocked with 2.5% bovine serum albumin (Sigma-Aldrich, CAT number: A7906-100G) for 45 min. In the primary antibody staining, cells were incubated with the primary antibodies (WT1 monoclonal antibody, Abnova, CAT number: MAB14650; vimentin polyclonal antibody, Abcam, CAT number: ab24525; and CRF polyclonal antibody, Bioss, CAT number: bs-0246R) in PBS for 60 min followed by three washes with PBS with a 5-min incubation for each wash. In the secondary antibody staining, cells were incubated with secondary antibodies (goat anti-mouse IgG [H + L] cross-adsorbed secondary antibody, Alexa Fluor 488, Invitrogen, CAT number: A-11001; goat anti-rabbit IgG [H + L] highly cross-adsorbed secondary antibody, Alexa Fluor 350, Invitrogen, CAT number: A-21245; and goat anti-chicken IgY [H + L] secondary antibody, Alexa Fluor 647, Invitrogen, CAT number: A21449) in PBS for 60 min, followed by three washes with PBS with a 5-min incubation for each wash. Finally, the cells were mounted with VECTASHIELD Antifade Mounting Medium (H 1400-10).
Renal Biopsy Samples
Pathological, clinical, and laboratory features of 47 patients with FSGS or thin basement membrane disease were evaluated. Biopsy samples were frozen in liquid nitrogen and sliced in cryostat. The percentage of FPE was evaluated using an electron microscope as part of the routine histological examination. During the quantification of FPE, at least 10 glomerular capillary loops were analyzed separately, and the average percent of processes with effacement was recorded (Fig. 1).
The slices were permeabilized and blocked with 0.6% Triton-X 100 (Sigma-Aldrich, CAT number: T-9284) and 1% bovine serum albumin in 1X PBS (Sigma-Aldrich, CAT number: A7906-100G) for 30 min. In the primary antibody staining, slices were incubated with the primary antibodies (WT1 monoclonal antibody, Abnova, CAT number: MAB14650; vimentin polyclonal antibody, Abcam, CAT number: ab24525; and CRF polyclonal antibody, Bioss, CAT number: bs-0246R) for 90 min followed by three washes with 1× PBS with a 5-min incubation for each wash. In the secondary antibody staining, cells were incubated with secondary antibodies (goat anti-mouse IgG [H + L] cross-adsorbed secondary antibody, Alexa Fluor 488, Invitrogen, CAT number: A-11001; goat anti-rabbit IgG [H + L] highly cross-adsorbed secondary antibody, Alexa Fluor 350, Invitrogen, CAT number: A-21245; and goat anti-chicken IgY [H + L] secondary antibody, Alexa Fluor 647, Invitrogen, CAT number: A21449) in 1× PBS for 60 min, followed by three washes with 1× PBS with a 5-min incubation for each wash. Finally, the slices were mounted with VECTASHIELD Antifade Mounting Medium (H 1000-10).
Intensity Measurements and Statistical Analyses
Stained cells and renal samples were analyzed with a Nikon Confocal Laser Microscope system (Nikon, Tokyo, Japan). The intensity of staining was quantified with Nikon NIS element software. The regions of interest (ROIs) were selected manually on the images (Nikon Electronic Format 12-bit). All ROIs were duplicated (the area of duplicate ROI was equal with the original ROI), and the duplicate ROIs were placed in the background. All pixels within the ROI area were measured, and the intensity of the background ROI was subtracted from the original ROI. Finally, the intensity value of each ROI was corrected with the corresponding area.
Continuous variables with normal distribution were compared using independent t-sample test and variables with non-normal distributions compared using Mann-Whitney U test. Correlations between WT1, vimentin, CRF, and FPE were analyzed using Spearman’s rho correlation test. Categorical variables were compared with χ2 test. A p value of less than 0.05 was considered statistically significant. The unbalanced statistical test of the control group (7 patients) and the FSGS group (42 patients) comprises one of the limitations of our study.
Renal Biopsy Samples
WT1 staining intensity in glomeruli (expressed as arbitrary units) was the same in the control and FSGS groups (median [IQR] 951 [866–1,048] a.u. vs. 846 [517–1,235] a.u., p = NS), but it was significantly lower in the tubulointerstitium of FSGS patients (median [IQR] 450 [439–469] a.u. vs. 248 [156–319] a.u., p < 0.001, Fig 2a–c). The staining intensity of glomeruli was significantly higher than that of tubulointerstitium in both control and FSGS groups (p < 0.001, Fig. 2a–c). In Figure 3, the localization of WT1 in the glomerulus (Fig. 3a) and tubulointerstitium (Fig. 3b) is shown at a higher magnification.
In contrast, the staining intensity of vimentin in glomeruli of the control group was significantly higher compared to the FSGS group (median [IQR] 959 [740–1,114] a.u. vs. 545 [375–813] a.u., p = 0.009). The staining intensity of vimentin in the control group was lower in the tubulointerstitium compared to the FSGS group (median [IQR] 75 [63–106] a.u. vs. 206 [113–280] a.u., p = 0.003, Fig 2a, b, d). In the FSGS group, the staining intensity of vimentin in glomeruli was higher than the intensity of the tubulointerstitium (p < 0.001). The same result was found in the control group (p = 0.001, Fig. 2a, b, d). In Figure 3, the localization of vimentin in the glomerulus (Fig. 3a) and tubulointerstitium (Fig. 3b) is shown in a higher magnification.
The CRF staining intensity was almost twofold higher in the glomeruli of the control group compared to the FSGS group (median [IQR] 536 [525–632] a.u. vs. 332 [256–409] a.u., p = 0.002, Fig 2a, b, e). The staining intensity was also higher in the tubulointerstitium of the control group; however, the difference was not significant (median [IQR] 469 [364–714] a.u. vs. 329 [238–486] a.u., p = NS, Fig 2a, b, e). The difference in staining intensity in glomeruli and tubulointerstitium was not significant in the control group. The same result was found in patients with FSGS (Fig. 2a, b, e). In Figure 3, the localization of CRF in the glomerulus (Fig. 3a) and tubulointerstitium (Fig. 3b) is shown at a higher magnification.
PFE showed significant negative correlation with WT1 staining intensity in the tubulointerstitium (WT1TI) (Table 2, Fig. 4a), vimentin in glomeruli (vimentinglom) (Table 2; Fig. 4b), and CRF in glomeruli (CRFglom) (Table 2; Fig. 4c). WT1 in glomeruli (WT1glom) showed a strong positive correlation with WT1TI and a significant positive correlation with vimentinglom, CRFglom.
WT1TI showed a significant positive correlation with vimentinglom, CRFglom, and CRF in the tubulointerstitium (CRFTI). There was a significant positive correlation between vimentinglom, CRFglom and between CRFglom and CRFTI (Table 2).
HEK 293 Cells
In HEK cells, the WT1 staining intensity was significantly lower in meta-Tyr (median [IQR] 1,325 [942–1,879] a.u. vs. 1,196 [738–1,568] a.u., p = 0.001) and ortho-Tyr (median [IQR] 1,325 [942–1,879] a.u. vs. 1,017 [666–1,930], p = 0.009) group compared to para-Tyr group (Fig. 5a, b). The vimentin staining intensity was significantly lower in the meta-Tyr group compared to para-Tyr (median [IQR] 1,481 [935–2,071] a.u. vs. 1,304 [790–1,569] a.u., p = 0.001) and ortho-Tyr (median [IQR] 1,533 [916–2,752] a.u. vs. 1,304 [790–1,569] a.u., p = 0.001) group (Fig. 5a, c). In the case of CRF staining, we could not find any significant difference between para-Tyr, meta-Tyr, and ortho-Tyr group (Fig. 5a, d).
Our main findings are as follows:
(i) Glomerular WT1 intensity did not differ in FSGS and control groups. (ii) The WT1 intensity in the tubulointerstitium of FSGS patients was significantly lower compared to the controls. (iii) WT1 staining was significantly lower in the tubulointerstitium of both FSGS patients and the control group in contrast to the glomeruli of these groups. (iv) Abnormal Tyr isomers decrease WT1 protein expression in HEK cell culture.
(i) Glomerular vimentin intensity was significantly lower in FSGS compared to controls. (ii) The vimentin intensity in the tubulointerstitium of FSGS patients was significantly higher compared to controls. (iii) Vimentin staining was significantly lower in the tubulointerstitium of both the FSGS and control group compared to glomeruli. (iv) Abnormal Tyr isomers decrease vimentin expression in HEK cell culture.
(i) Decreased CRF expression was observed in the glomeruli of FSGS patients compared to controls. No difference was found in the tubulointerstitium between the two groups. (ii) Abnormal Tyr isomers had no influence on CRF expression in HEK cell culture.
Oxidative stress and WT1
According to animal models, one of the initial events in FSGS is podocyte injury . Podocytes have no regenerative capabilities. Although they are epithelial cells, they have some mesenchymal characteristics including motility and vimentin expression . Damage of podocytes can be caused by numerous events .
In the adult kidney, WT1 is expressed mainly in podocytes. WT1 is needed for maintaining the function of podocytes and in preserving glomerular function.
FSGS is characterized by extensive podocyte FPE, vacuolar degeneration, detachment, and downregulation of podocyte markers such as WT1, synaptopodin, nephrin, and podocalyxin . Decreased amount of epithelial markers is also observed in podocytes cultured with TGF-β and begin to express mesenchymal markers . In patients with diabetic nephropathy and FSGS, the same changes can be observed [21, 22].
Oxidative stress has a key role in the progression of chronic kidney diseases, including FSGS. Excess production of ROS and accumulation of protein products produced during oxidative stress (so-called advanced oxidation protein products) result in podocyte damage.
Increased oxidative stress may be in the background of tubulointerstitial fibrosis . TGF-beta activation in the podocytes is the other key event in the progression of FSGS [23, 24]. It may lead to increased oxidative stress and damage of mitochondrial function in endothelial cells, even before the ultrastructural damage of podocytes [19, 25]. In turn, oxidative stress induces and activates TGF-beta, causing a vicious circle .
Hydroxyl free radicals can hydroxylate the benzyl ring of the amino acid phenylalanine, which then produces para-Tyr and the abnormal Tyr isomers meta-Tyr and ortho-Tyr . Aberrant incorporation of these isomers into proteins is at least one of the mechanisms contributing to the toxic effects of m- and o-Tyr. Furthermore, other undiscovered cellular effects produced by the Tyr isomers may lead to the observed alterations in cellular function, including enhanced protein degradation, stimulation of apoptosis, and inhibition of mitogenic signaling pathways . In our study, glomerular WT1 intensity in patients with FSGS did not differ compared to the control group. Podocytes are not able to regenerate; however, in response to injury PECs, they are able to migrate and obtain podocyte characteristics [28, 29]. These cells can proliferate in response to podocyte loss and have the capability to replace injured podocytes . This kind of regenerative process might be responsible for the unchanged WT1 levels, at least at the early stages of FSGS.
In contrast to intense WT1 staining of glomeruli, tubules have only weak cytoplasmic staining . This observation is parallel with our study. WT1 staining was significantly lower in the tubulointerstitium of both the FSGS patient and control groups compared to the glomeruli of these groups. In FSGS, the chronic tubulointerstitial changes, interstitial fibrosis may cause decrease in WT1 staining, as it was observed in our study. A strong negative correlation between podocyte FPE and WT1TI also supports this observation. The WT1 intensity in the tubulointerstitium of FSGS patients was significantly lower compared to controls.
Inhibition of WT1 protein expression in a mouse model rapidly induced FSGS . In our study, WT1 staining intensity of HEK cells was decreased in culture containing meta-Tyr and ortho-Tyr compared to the control. It can be hypothesized that abnormal Tyr isomers caused by oxidative stress negatively affect the WT1 protein expression. This is in accordance with our observations in FSGS patients.
Oxidative Stress and Vimentin
Vimentin plays an important role in keeping the normal structure of podocytes and the formation of slit pores . The significantly lower level of mesenchymal marker vimentin in glomeruli of FSGS patients compared to the control group might be explained by the increased number of damaged podocytes, even without marked histological alterations.
This is in parallel with the strong negative correlation between podocyte FPE and vimentinglom and between WT1glom and vimentinglom. This may be an indication of podocyte damage in patients with a high percentage of podocyte FPE.
Vimentin may act as a sensitive marker of tubulointerstitial injury . In our observations, vimentin expression was significantly higher in the tubulointerstitium of patients with FSGS compared to the control group. The elevated vimentin level observed in the tubulointerstitium of FSGS patients may show the EMT in response to chronic injury.
According to Jercan et al. , vimentin can be a good parameter for the chronic histological changes in chronic kidney diseases, even in earlier stages. Some studies showed a good correlation between EMT markers and renal function in chronic tubulointerstitial lesions. Parallel with the decreased renal function, increased expression of EMT markers was observed [32‒34]. The elevated staining intensity with vimentin may reflect the initiation of EMT, which is in concordance with other investigations [32‒34].
High vimentin expression in glomeruli may be a sign of preserved structures; in the tubulointerstitium, it is rather a marker of tissue damage. Our result shows that the meta-Tyr in culture medium significantly reduces the labeling intensity of vimentin compared to para- and ortho-Tyr in HEK cells. This is in accordance with our results on kidney biopsy samples.
Oxidative Stress and CRF
CRF has a key role in response to stress. Furthermore, CRF has two effects on the immune system: an indirect anti-inflammatoric and a direct pro-inflammatoric effect . To our knowledge, CRF expression in the kidneys of patients with FSGS was not studied, and there is no evidence about CRF expression in healthy and diseased kidneys.
In our study, CRF expression was observed by immunofluorescence in both patients with FSGS and control groups. CRF staining intensity was significantly lower in the glomeruli of the FSGS group. CRF staining intensity was not different in the tubulointerstitium of the two groups.
In our study, HEK cells showed CRF positivity; however, culturing with meta-Tyr or ortho-Tyr had no effect on CRF staining. In summary, CRF presumably does not play a significant role in FSGS.
Nonphysiological Tyr isomers and byproducts of oxidative stress may cause damage to both HEK cells and podocytes in patients with FSGS and may contribute to EMT in damaged kidney tissue as demonstrated by the modified WT1 and vimentin protein expression in HEK cell cultures and kidney biopsy specimens in our investigations. The presence of nonphysiological Tyr isomers is likely to play a role in the development of kidney diseases, including FSGS. Because of the relatively low number of our cases, further studies are needed.
The authors would like to thank Professor Dr. József Andor for revision of language and style and Enikő Bodor for technical assistance.
Statement of Ethics
Ethical approval was obtained from the Local Ethics Committee of University of Pécs, Medical School, Hungary (No: 9111/22). Our study was retrospective, so we did not require written informed consent from patients. Written informed consent from participants was not required in accordance with local and national guidelines.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
No funding was obtained in connection with our presented manuscript.
The authors confirm contribution to the paper as follows. Study conception and design: Tibor Vas, István Wittmann, and Gábor Sütő; data collection: Tibor Vas and Roland Csurgyók; analysis and interpretation of results: Roland Csurgyók, Gábor Sütő, István Wittmann, and Tibor Vas; draft manuscript preparation: Roland Csurgyók, Gábor Sütő, István Wittmann, and Tibor Vas. All authors reviewed the results and approved the final version of the manuscript.
Data Availability Statement
All data generated or analyzed during the study are included in this article. Further inquiries can be directed to the corresponding author.