Evaluation of immunohistochemical markers CD44 and PAX8 in diagnosis of Focal Segmental Glomerulosclerosis suspected cases and its differentiation from Minimal Change disease in patients with nephrotic syndrome

Evaluation of immunohistochemical markers CD44 and PAX8 in diagnosis of Focal Segmental Glomerulosclerosis suspected cases and its differentiation from Minimal Change disease in patients with nephrotic syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of immunohistochemical markers CD44 and PAX8 in diagnosis of Focal Segmental Glomerulosclerosis suspected cases and its differentiation from Minimal Change disease in patients with nephrotic syndrome Samaneh Salarvand, Fatemeh Nili, Farshid Dehkhoda, Alireza Abdollahi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3822572/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Expression evaluation of specific markers PAX8 and CD44 on kidney podocyte cells or their progenitor cells can be very helpful in diagnosing and differentiating between types of podocytopathies. In present study, the positivity of immunohistochemical markers CD44 and PAX8 in parietal epithelial cells (PECs) was evaluated to diagnose suspected cases of Focal Segmental Glomerulosclerosis (FSGS) and differentiate it from minimal change disease (MCD). Methods This cross-sectional study was conducted on patients who underwent kidney biopsy due to nephrotic syndrome. 42 FSGS samples and 36 MCD samples were selected and biopsies were evaluated for CD44 and PAX8 markers. Suitable blocks for immunohistochemical staining that had enough tissue were selected and evaluated. Results The expression frequency of PAX8 marker in group with FSGS was estimated as 97.6% and in group with MCD as 52.8%, which was significantly higher in FSGS group. Also, regarding the expression of CD44, in two groups with FSGS and MCD, respectively, 1 + cases equal to 26.2% and 94.4%, 2 + cases equal to 40.5% and 5.6%, and 3 + cases equal to 33.3% and 0.0%, which indicated the higher intensity of CD44 expression in FSGS group compared to MCD. Conclusions The expression of CD44 in FSGS was influenced by patients age and a direct and significant relationship was observed. The increase in amount and intensity of two markers PAX8 and CD44 expression in PEC cells in patients with FSGS shows high sensitivity of these markers in diagnosis of FSGS and the decrease in expression level in MCD can play an important role in differentiating between types of podocytopathy disorders. kidney: Nephrotic syndrome Podocytes parietal epithelial cells Focal Segmental Glomerulosclerosis minimal change disease Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Background Kidney diseases severely affect public health and millions of people die every year due to chronic kidney diseases[ 1 , 2 ]. Among these, one of the types of chronic kidney complications is the change in Podocytes cells structure in kidney [ 3 ]. Podocytopathy is the most common type of glomerular disease, which is characterized by damage and destruction of podocyte cells, dysfunction of these cells, and finally occurrence of various clinical syndromes. Among the most common types of podocytopathies, minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are the most common types [ 4 ]. FSGS is the most common primary glomerulopathy leading to end stage kidney disease. This disease is characterized by focal and segmental destruction and fading of glomerular capillaries structure along with an increase in matrix thickness. FSGS is considered to be one of the most common causes of nephrotic syndrome, which is accompanied by extensive destruction of podocytes, and if there is a significant increase in the number of these cells, the occurrence of glomerulosclerosis can be expected [ 5 , 6 ]. In MCD, the damage level is less. Apart from MCD, most cases of podocytopathies have an unfavorable prognosis, which may eventually lead to end-stage kidney disease[ 4 ]. Regarding the diagnostic symptoms of these two FSGS diseases, unlike MCD, glomerular sclerosis lesions are observed. MCD mainly occurs in children and younger people. In the context of MCD, basic pathophysiology of this disease includes disappearance of foot processes without structural changes in glomeruli. These non-structural changes are associated with increased glomerular permeability and eventually proteinuria. According to some evidence, MCD itself eventually leads to FSGS, but the relationship between these two diseases is still not clear [ 7 ]. However, tracking activated PECs seems to be very effective in distinguishing between these two diseases[ 8 ]. Evaluating the expression of specific expressed cell markers with the aim of evaluating disorder and severity of the said cell's dysfunction has an important role in diagnosis and final confirmation of various diseases and their differentiation from other diseases[ 9 ]. Tracking some specific markers on kidney podocyte cells or their precursor cells can be very helpful in the diagnosis and differentiation between types of podocytopathies. In this regard, evidence shows that the evaluation of two specific markers PAX8 and CD44 on these cells in various types of podocytopathies such as FSGS and minimal change disease as two common podocyte disorders can be very helpful and diagnostic. [ 10 – 12 ]. Also, in FSGS disease, the tracking of PECs with high expression of CD44 marker is quite evident, such manifestation is less observed in MCD disease and can be a distinguishing feature for these two disorders from each other[ 10 ]. According to mentioned cases, it is possible that differentiation of PEC cells into podocyte cells contains high expression of markers such as CD44 and PAX8, but findings have had conflicting results. Considering this issue, the present study was conducted with the aim of evaluating positivity of CD44 and PAX8 immunohistochemical markers in parietal epithelial cells to detect suspected cases of FSGS and differentiate it from minimal histopathological abnormality in patients with nephrotic syndrome. 2. Material and methods 2.1. Case selection This cross-sectional study was conducted on patients who underwent kidney biopsy due to nephrotic syndrome in Imam Khomeini Hospital in 2018 − 1400. After searching in the archive of pathology reports of patients in pathology department, 36 samples of patients whose pathology was diagnosed as MCD (MCD group) and 42 samples of FSGS (FSGS group) were examined. The extracted slides were reviewed by a pathologist and suitable blocks were selected for immunohistochemical staining that had enough tissue. Samples that did not have enough tissue to perform immunohistochemical tests or did not have enough glomerulus, or patient information including laboratory information or required clinical findings were not available, were excluded from study. Before starting the research, ethical aspects were examined by ethics committee of university. 2.2. Immunohistochemical evaluation and data collection After selecting blocks, immunohistochemical staining was performed for two immunohistochemical markers, CD44 and PAX8. First, a 4-micron section of each block was prepared by a microtome on a glass slide. Then they were placed in an autoclave at a temperature of 60°C for one night. In deparaffinization and hydration step, the slides were placed three times in Gesylol for 15 minutes each time, and twice in 100% alcohol for 10 minutes each time, and finally twice in 96% alcohol for 10 minutes each time. After that, samples were washed with distilled water and placed in an autoclave at 95°C for 20 minutes with 1.5 cc of EDTA buffer with pH = 8 for antigen retrieval. Again, the slices were washed 2–3 times with distilled water and kept at room temperature for 20 minutes. Then, internal peroxidases were blocked using peroxidase solution for 10 minutes. Then the primary antibody was added to slides and kept at 37°C for 60 minutes. Then it was washed with TBS buffer and secondary antibody was placed on slides for 15 minutes. Then the slides were placed in contact with diaminobenzidine (DAB) chromogen for 10 minutes and stained with hematoxylin. Finally, slides were dehydrated and mounted. To interpret the results, the stained slides were examined and recorded by two pathologists. For CD44, staining intensity was used (Fig. 1 ). 1+: positive membrane staining in a limited number of visceral cells in glomerulus, which is considered negative staining. 2+: positive membranous staining with moderate intensity in more cells, 3+: positive and strong membranous staining in visceral and parietal cells, especially in glomerular sclerosis, in interpretation of results according to similar studies and findings Here, + 1 cases were considered negative and + 2 and + 3 results were considered positive[ 13 ]. Polyclonal PAX8 antibody) Proteintech, dilution 1: 50, catalog 11392-3-AP) was used to evaluate activated PEC from paraffin sections. (Fig. 2 ) The experiments were carried out as previously described [ 14 ]. Clinical and laboratory information of patients such as age, sex and clinical symptoms of patients were also recorded from file information. Data were collected in a pre-prepared checklist. 2.3. Statistics SPSS version 26 software was used for statistical analysis of data. Comparison between quantitative variables was done by t-test and Mann-Whitney test. Comparison between qualitative variables was also done using Chi-square test. A significance level of less than 0.05 was considered. 3. Results 3.1. Patient characteristics A total of 78 samples were obtained; 42 samples from FSGS patients and 36 samples from MCD patients (Table 1 ). In terms of glomeruli number, the average number of glomeruli was 18.17 and 24.83, respectively, and there was a significant difference between two groups (P = 0.02). The frequency of cases with foamy cells was 11 cases (26.2%) and 2 cases (5.6%), respectively; which was significantly higher in first group (P = 0.01). Regarding interstitial fibrosis and tubular atrophy (IFTA) in FSGS and MCD patients, there were 22 cases (8.46%) and 2 cases (5.6%), respectively, which were significantly more seen in FSGS group (P = 0.001). In terms of gender, sampling area, mesangial matrix expansion and GBM changes, no significant difference was observed between two groups (p > 0.05). Table 1 Background characteristics of two groups with FSGS and MCD parameter FSGS (n = 42) MCD (n = 36) p- Value age (years) mean 33.50 ± 3.51 15.03 ± 2.51 0.001 gender male 24 (57.1%) 22 (61.1%) 0.72 female 18 (42.9%) 14 (38.9%) Number of glomeruli mean 18.17 ± 19.76 24.83 ± 15.47 0.02 Number of glomeruli Between 0 and 10 11 (26.2%) 6 (16.7%) 0.04 Between 11 and 25 21 (50.0%) 13 (36.1%) Above 25 10 (23. 8%) 17 (47.2%) foamy cell 11 (26.2%) 2 (5.6%) 0.01 Expansion of the mesangial matrix 36 (85.7%) 30 (83.3%) 0/77 Variations of GBM 26 (61.9%) 17 (47.2%) 0.14 subendothelial widening 2 (4.8%) 0 (0.0%) 0/18 IFTA 22 (46.8%) 3 (6.5%) 0.001 The frequency of PAX8 expression in the subgroup of patients with FSGS in men and women was 95.8% and 100%, respectively, and in MCD it was 27.3% and 42.9%. There was a significant difference in the expression of PAX8 in FSGS and MCD groups according to gender (P < 0.001). But according to the position involved, this difference was not significant (P = 0.07). In total, the frequency of PAX8 expression in the group with FSGS was estimated as 97.6% and in the MCD group as 52.8%, which was significantly higher in the FSGS group (P < 0.001).(Table 2 ) (Fig. 3 ) Table 2 Frequency of PAX8 expression in FSGS and MCD groups according to gender and site of involvement Parameter Property FSGS MCD p - Value Gender male 95.8% 27.3% P < 0.001 female 100% 42.9% Engaged position Cortex 95.0% 30.0% 0.07 Corticomedullary 100% 34.6% Total 97.6% 52.8% P < 0.001 In patients with FSGS and MCD, the expression of CD44 + 1 was not related to the gender and involved position, and no significant difference was observed in this regard in the two groups of patients (p > 0.05). In fact, this finding indicated the lack of influence of gender and involved position on the expression of this marker in two diseases. In total, the overall expression of CD44, in the two groups with FSGS and MCD, 1 + cases, which were considered negative, was equal to 26.2% and 94.4%, respectively, 2 + and 3 + cases, which were considered positive, respectively. In FSGS and MCD, it was equal to 40.5%, 5.6%, 33.3% and 0.0%, which indicated the higher intensity of CD44 expression in FSGS group compared to MCD (p > 0.05). (Table 3 ) ( Fig. 4 ) Table 3 The frequency of CD44 expression in FSGS and MCD according to and site of involvement Parameter FSGS MCD gender 1+ 2+ 3+ 1+ 2+ 3+ Gender male 29. 2% 29. 2% 41.7% 95.5% 4.5% 0.0% female 22.2% 55.6% 22.2% 92.9% 7.1% 0.0% p- Value 0.21 0.74 Engaged position Cortex 20.0% 45.0% 35.0% 100% 0.0% 0.0% Corticomedullary 31.8% 36.4% 31.8% 92.3% 7.7% 0.0% p- Value 0.67 0.36 Total 26.2% 40.5% 33.3% 94.4% 5.46% 0.0% Average age in FSGS patients was not related to PAX8 expression (p > 0.05). Also, the average age in FSGS patients was related to the expression of CD44 + 1, CD44 + 2 and CD44 + 3, respectively 14.09 years, 41.47 years and 37.57 years; which shows that the expression of marker increases with increasing age (P 0.05). Also, the average age in two groups with and without CD44 expression was 55 and 12.68 years, respectively, which indicates a significant relationship between the marker and increasing age (P = 0.04).( Table 4 ) Table 4 Frequency of expression of CD44 and PAX8 in FSGS and MCD according to average age Parameter marker FSGS MCD PAX8 positive 32.78 ± 22.54 15.92 ± 4.15 negative 63 14.58 ± 3.20 p-Value 0.19 0.8 CD44 negative 18.45 ± 9.08 12.68 ± 7.61 Positive 38.84 ± 21.36 55 ± 7.07 p - Value 0.009 0.04 In patients with MCD, out of 19 patients with positive expression of PAX8, 11 patients (57.9%) had basement membrane changes in electron microscopy, and 8 patients (42.1%) were without basement membrane changes, while in patients without PAX8 expression, basement membrane changes were observed in 41.2% of cases (p > 0.05). Also, the expression of CD44 in these two groups of patients was 5.9% and 5.3%, respectively, which again does not show a significant difference (p > 0.05).( Table 5 ) Table 5 Frequency of expression of CD44 and PAX8 in FSGS and MCD according to basement membrane changes Variations of GBM FSGS MCD PAX8 CD44 PAX8 CD44 positive Negative positive Negative positive Negative positive Negative Yes 25 (96.2%) 1 (3.8%) 21 (80.8%) 5 (19.2%) 11 (61.1%) 7 (38.9%) 1 (5.9%) 16 (94.1%) No 16 (100%) 0 10 (62.5%) 6 (37.5%) 8 (44.4%) 10 (55.6%) 1 (5.3%) 18 (94.7%) P value 0.61 0.17 0.31 0.72 In 19 patients with PAX8-positive MCD, 16 patients (55.2%) had suspicious clinical and histological symptoms. Suspicious clinical and histological symptoms were observed in 13 cases (44.8%) in patients with negative PAX8 (p > 0.05). In the case of CD44, the positive expression of this marker was observed in 14.3% and 37.9% of cases with definite and doubtful diagnosis, respectively (p > 0.05).( Table 6 ) Table 6 The frequency of expression of CD44 and PAX8 in the MCD group according to the presence of clinical symptoms and microscopic changes suspicious for FSGS Diagnosis PAX8 CD44 positive negative positive negative Absence of clinical and microscopic symptoms suspicious of FSGS 3 (42.9%) 4 (57.1%) 1 (14.3%) 6 (85.7%) Presence of clinical and microscopic signs suspicious for FSGS 16 (55.2%) 13 (44.8%) 11 (37.9%) 18 (62.1%) P value 0.55 0.23 Considering that FSGS was diagnosed with gold standard optical and electron microscopy, it can be said that the sensitivity and specificity of PAX8 marker for FSGS diagnosis are 97.6% and 66.7%, respectively, and the sensitivity and specificity of CD44 is 73.8% and 94.4% respectively. 4. Discussion In this study, we demonstrated that PAX8 and CD44 are effective markers in diagnosing suspected cases of FSGS and differentiating it from MCD. The result of this study was that, firstly, we encountered a significant expression of PAX8 in patients with FSGS (97.6%). While the expression of this marker in the MCD group was detected in only one third of cases (52.7%), this makes it necessary to follow up patients in the future in terms of the possibility of sclerotic involvement in subsequent biopsies. In other words, tracking the PAX8 marker can not only be very sensitive for the diagnosis of FSGS, but also very helpful in distinguishing between FSGS and MCD. On the other hand, regarding the expression of CD44, this marker expression was reported in both diseases but with different degrees, however, the expression of this marker in patients with FSGS was much higher than in patients with MCD. In a way, the expression of + 3 was detected in one third of the patients with FSGS group, while this level of expression was not reported at all in the patients of the opposite group. In a general view, tracking and determining the expression level of both markers was very helpful for distinguishing between the two types of podocytopathy. In this way, we saw a significant expression of these markers in FSGS compared to MCD. This finding has been reported in previous studies. Similar investigations have shown that the increase in the number of podocytes with simultaneous expression of PAX8 marker in animal FSGS model has been detected and confirmed. Also, the occurrence of CD44 markers has been an important marker to show the activity of PECs in FSGS disease [ 9 ]. A study by Okamoto et al. showed an increase in CD44 expression in glomeruli involved in FSGS, while the amount of CD44 + cells indicated the degree of podocyte damage in FSGS[ 15 ]. In a parallel study, it was shown that CD44 + was significantly increased in FSGS compared to MCD, and this method can be used to differentiate early FSGS from MCD [ 10 ]. FSGS is the most common primary glomerulopathy leading to end-stage kidney disease. This disease is characterized by focal and segmental destruction and fading of glomerular capillaries structure along with an increase in matrix thickness. This disease is also accompanied by extensive destruction of podocytes, and if there is a significant improvement in number of these cells, the occurrence of glomerulosclerosis can be fully expected [ 5 , 6 ]. In present study, the increase in CD44 expression in patients with FSGS was probably a sign of podocytes loss and the activation of PEC. In a study by Husain et al., it was reported that in FSGS, increased expression of CD44 was observed in all cases, implying loss of podocytes and simultaneous activation of PEC[ 16 ]. CD44 positivity in PEC in FSGS patients is associated with worse prognosis and more impaired renal function[ 17 ]. Roca and his colleagues reported that FSGS lesions were observed in 83% of CD44 positive patients. They stated that increased expression of CD44 had a significant relationship with resistance to steroid therapy and worse prognosis, which can be an early sign of FSGS [ 18 ]. In another study, it was reported that CD44 activated PEC can be an indicator of primary podocyte damage in the development of FSGS and is a good marker for the early diagnosis of CNI-induced nephrotoxicity[ 12 ]. Based on previous studies, it was proven that active PEC cells, in addition to being the precursors of podocyte cells, play an effective role in creating glomerular sclerotic lesions, which is due to the role of active PEC cells in extracellular matrix and production of fibrosis and finally sclerotic lesions [ 17 ]. Findings have shown that the PAX2 gene plays an effective role in kidney organogenesis and mutation in this gene is an important cause of kidney disorders of unknown cause[ 19 ]. Based on previous studies, PAX8 is a very sensitive marker and can identify microsclerosis and precursor lesions and is a very good substitute for PAS dye (8). According to our findings, the sensitivity of PAX8 marker to detect sclerotic lesions in FSGS is 100% in total of two biopsies, which shows the necessity of re-biopsy in patients suspected of FSGS based on other findings. In the study of Smeet et al., PEC markers including PAX8 and CD44 were present in 87% of patient's biopsies diagnosed with primary FSGS in capillary tufts, while no PEC activation markers were found in MCD patients (8). In another study, FSGS was diagnosed in 83.3% of PAX8 positive patients, while all PAX8 negative patients were diagnosed with MCD. A number of FSGS patients who were negative for PAX8 in the first biopsy were stained with PAX8 marker in the second biopsy, and finally all patients with FSGS were positive for PAX8 marker in at least one of the two biopsies performed[ 20 ]. In present study, the sensitivity and specificity of PAX8 marker for FSGS diagnosis were 97.6% and 66.7%, respectively. And the sensitivity and specificity of CD44 were 73.8% and 94.4%, respectively. Of course, the specificity of PAX8 marker is probably higher than mentioned number. Because according to clinical findings and electron microscopy, a significant number of patients who were diagnosed with MCD in first biopsy probably had sclerotic lesions in subsequent follow-ups. Also, the negativity of PAX8 marker in one of the samples diagnosed with FSGS is probably due to the fact that repeated cuts on tissue led to removal of glomerulus sclerosis in IHC staining. As in similar studies, the cause of non-positive PEC markers in a small number of FSGS cases has been attributed to the focal and segmental nature of the lesions[ 8 ]. Because in order to find these lesions, it is necessary to examine multiple sections, while we only stained one section, and this section was taken for light microscopy after preparing the previous sections, which raises the possibility of removing the involved area. As it was seen in a study that re-biopsy made the PAX8 marker positive in FSGS patients, which was negative in the first biopsy, and finally, the examination of both biopsies showed that 100% of FSGS patients have positive PAX8 marker at least once [ 20 ]. In total, the two main findings in this study include the first high sensitivity of PEC markers, especially PAX8, in the diagnosis of FSGS lesions, which suggests the role of these cells in the pathogenesis of the disease. The second finding is that these markers were positive in a number of samples that were diagnosed with MCD in the initial examination with light microscopy, which suggests the need to follow up these patients in terms of the possibility of diagnosing FSGS in subsequent samplings. One of the limitations of this study was the lack of complete follow-up of the patients, which was effective in the definitive diagnosis of the MCD group. In fact, a significant number of people in this group may have sclerotic lesions in subsequent biopsies and fall into the FSGS group. If the presence of electron microscope changes in some of these patients increases the possibility of undiagnosed sclerotic lesions. 5. Conclusion PAX8 and CD44 are effective in diagnosing suspected cases of FSGS and distinguishing it from MCD. The high level and intensity of PAX8 and CD44 expression in PEC cells in Bowman's capsule and capillary tuft in patients with FSGS show the high sensitivity of these markers in diagnosis of FSGS and the low expression in MCD can play an important role in distinguish between types of podocytopathies. It is suggested to use this marker to differentiate MCD from FSGS in cases of diagnostic doubt, especially in the early stages of FSGS. Abbreviations Focal Segmental GlomeruloSclerosis (FSGS) Minimal Change Disease (MCD) Interstitial Fibrosis and Tubular Atrophy (IFTA) Parietal Epithelial Cell (PEC) Glomerular Basement Membrane (GBM) diaminobenzene (DAB) Declarations Ethics approval and consent to participate: In this study, all provisions of the Helsinki regulations were considered. Informed consent was obtained from all the people when the file was filed by the medical records archive for the possible use of the file information for research purposes. The design of the study was approved by ethics committee of research of Tehran University of Medical Sciences code: IR.TUMS.IKHC.REC.1399.383 Consent for publication: Not applicable Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests Funding: The authors received no financial support for the research, authorship and publication of this study. Authors' contributions: Samaneh Salarvand: Conceived and designed the analysis, performed the histological examination of the kidney, reporting the slides, IHC and EM reporting Fatemeh Nili: Conceived and designed the analysis, performed the histological examination of the kidney, reporting the slides, IHC and EM reporting Farshid Dehkhoda: analyzed and interpreted the patient data, Performed the analysis, wrote the paper Alireza Abdollahi: Conceived and designed the analysis, collected data, contributed data Azin Alemzadeh: Collected the data, contributed data, Performed the analysis Maryam Abedi: collected data, contributed data and analysis tools, contributed in the histological examination of the kidney, Wrote the paper All authors read and approved the final manuscript Acknowledgements: Not applicable References Harris DC, Davies SJ, Finkelstein FO, Jha V, Donner J-A, Abraham G, et al. Increasing access to integrated ESKD care as part of universal health coverage. 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Nephron Experimental Nephrology . 2014;124(3-4):11-18. Husain S, Ginawi I, Bashir AI, Kfoury H, Al Johani TE, Hagar H, et al. Focal and segmental glomerulosclerosis in murine models: a histological and ultrastructural characterization with immunohistochemistry correlation of glomerular CD44 and WT1 expression. Ultrastructural Pathology . 2018;42(5):430-439. Froes BP, de Almeida Araújo S, Bambirra EA, Oliveira EA, Simoes e Silva AC, Pinheiro SVB. Is CD44 in glomerular parietal epithelial cells a pathological marker of renal function deterioration in primary focal segmental glomerulosclerosis? Pediatric Nephrology . 2017;32:2165-2169. Roca N, Jatem E, Abo A, Santacana M, Cruz A, Madrid Á, et al. CD44-negative parietal–epithelial cell staining in minimal change disease: association with clinical features, response to corticosteroids and kidney outcome. Clinical Kidney Journal . 2022;15(3):545-552. Muntean C, Chirtes C, Baczoni B, Banescu C. PAX2 Gene Mutation in Pediatric Renal Disorders—A Narrative Review. International Journal of Molecular Sciences . 2023;24(16):12737. Suzuki T, Kohatsu K, Han W, Watanabe S, Yahagi K, Nakata M, et al. Morphological features of minimal change disease and focal segmental glomerulosclerosis using repeat biopsy and parietal epithelial cell marker. Kidney Diseases . 2020;6(2):119-124. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3822572","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264702749,"identity":"9a186400-a210-46bb-aaf4-c97d00dfd5b5","order_by":0,"name":"Samaneh Salarvand","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Samaneh","middleName":"","lastName":"Salarvand","suffix":""},{"id":264702750,"identity":"175a3ea1-d0b4-4987-b597-124059612a8f","order_by":1,"name":"Fatemeh Nili","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Nili","suffix":""},{"id":264702751,"identity":"cfc94d7d-8168-46b2-92c8-3c9752649360","order_by":2,"name":"Farshid Dehkhoda","email":"","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Farshid","middleName":"","lastName":"Dehkhoda","suffix":""},{"id":264702752,"identity":"a94ca4b0-1356-40a5-a32c-869da8e6965a","order_by":3,"name":"Alireza Abdollahi","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Abdollahi","suffix":""},{"id":264702753,"identity":"82e0e5a8-1e20-4728-acc9-d0a788f90b76","order_by":4,"name":"Azin Alemzadeh","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Azin","middleName":"","lastName":"Alemzadeh","suffix":""},{"id":264702754,"identity":"940bd1a1-1540-495e-ba02-f38be6755fdf","order_by":5,"name":"Maryam Abedi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYPCCAwwMh5mBBIOEDCla2BJAWnhI0HKAxwDEIqxFt/3wA4YfNXfk+I7zfH51o8aCh4H98NEN+LSYnUkzYOw59sxY8jDvNuucY0CH8aSl3cCr5UCCAQMP2+HEDUAtxjlsQC0SPGb4tZx//oHxz7/D9RsO8zwzzvlHjJYbOQbMvG2HEwwO8zA/zm0jSsubgsOyfc8MZx5mM2PO7ZPgYSPol/PpGx+++XZHnu/84cefc77VyfGzHz6GVwsIHIDSbBJgkpByZMD8gRTVo2AUjIJRMHIAAO8vTXmhasqCAAAAAElFTkSuQmCC","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Abedi","suffix":""}],"badges":[],"createdAt":"2023-12-29 22:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3822572/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3822572/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49128071,"identity":"017d2aa5-b74c-4c03-ab21-b80d29dab0ee","added_by":"auto","created_at":"2024-01-03 15:04:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":263548,"visible":true,"origin":"","legend":"\u003cp\u003eIntensity of CD44 staining in the tissue. a) minimal CD44 staining in visceral location (1+), b) moderate CD44 staining (2+) c) strong CD44 staining in sclerotic area (3+)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3822572/v1/276f92c1c6aad784fa987717.png"},{"id":49128070,"identity":"fd3ead61-88a7-4168-8d67-5ec87aa8468a","added_by":"auto","created_at":"2024-01-03 15:04:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":339032,"visible":true,"origin":"","legend":"\u003cp\u003e5-A-C positive PAX8 staining in glomerular cells\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3822572/v1/6d250f790c85028686be499e.png"},{"id":49128069,"identity":"0a8e69d3-b30b-42ca-9f07-7c5cdb75dc58","added_by":"auto","created_at":"2024-01-03 15:04:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45071,"visible":true,"origin":"","legend":"\u003cp\u003ePAX8 expression level in two groups of FSGS and MCD: a (total PAX8 expression level b) PAX8 expression level according to gender c) PAX8 expression level according to the involved site\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3822572/v1/25b22ef5c43e5055b50d2127.png"},{"id":49128068,"identity":"2bf9249b-2252-4283-9656-d9e21dd48734","added_by":"auto","created_at":"2024-01-03 15:04:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42017,"visible":true,"origin":"","legend":"\u003cp\u003eTotal expression of CD44 in FSGS and MCD: a (total expression of CD44 b) total expression of CD44 according to gender c) total expression of CD44 according to the involved site\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3822572/v1/7a372ce8cc21e3b541377d7f.png"},{"id":61382193,"identity":"9dcbd6ef-5ffb-47e9-ac70-847a84d09e61","added_by":"auto","created_at":"2024-07-30 06:07:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1409307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3822572/v1/36028e80-2401-426b-9d11-e51ede57c178.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of immunohistochemical markers CD44 and PAX8 in diagnosis of Focal Segmental Glomerulosclerosis suspected cases and its differentiation from Minimal Change disease in patients with nephrotic syndrome","fulltext":[{"header":"1. Background","content":"\u003cp\u003eKidney diseases severely affect public health and millions of people die every year due to chronic kidney diseases[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among these, one of the types of chronic kidney complications is the change in Podocytes cells structure in kidney [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Podocytopathy is the most common type of glomerular disease, which is characterized by damage and destruction of podocyte cells, dysfunction of these cells, and finally occurrence of various clinical syndromes. Among the most common types of podocytopathies, minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are the most common types [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. FSGS is the most common primary glomerulopathy leading to end stage kidney disease. This disease is characterized by focal and segmental destruction and fading of glomerular capillaries structure along with an increase in matrix thickness. FSGS is considered to be one of the most common causes of nephrotic syndrome, which is accompanied by extensive destruction of podocytes, and if there is a significant increase in the number of these cells, the occurrence of glomerulosclerosis can be expected [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In MCD, the damage level is less. Apart from MCD, most cases of podocytopathies have an unfavorable prognosis, which may eventually lead to end-stage kidney disease[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the diagnostic symptoms of these two FSGS diseases, unlike MCD, glomerular sclerosis lesions are observed. MCD mainly occurs in children and younger people. In the context of MCD, basic pathophysiology of this disease includes disappearance of foot processes without structural changes in glomeruli. These non-structural changes are associated with increased glomerular permeability and eventually proteinuria. According to some evidence, MCD itself eventually leads to FSGS, but the relationship between these two diseases is still not clear [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, tracking activated PECs seems to be very effective in distinguishing between these two diseases[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvaluating the expression of specific expressed cell markers with the aim of evaluating disorder and severity of the said cell's dysfunction has an important role in diagnosis and final confirmation of various diseases and their differentiation from other diseases[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Tracking some specific markers on kidney podocyte cells or their precursor cells can be very helpful in the diagnosis and differentiation between types of podocytopathies. In this regard, evidence shows that the evaluation of two specific markers PAX8 and CD44 on these cells in various types of podocytopathies such as FSGS and minimal change disease as two common podocyte disorders can be very helpful and diagnostic. [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Also, in FSGS disease, the tracking of PECs with high expression of CD44 marker is quite evident, such manifestation is less observed in MCD disease and can be a distinguishing feature for these two disorders from each other[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to mentioned cases, it is possible that differentiation of PEC cells into podocyte cells contains high expression of markers such as CD44 and PAX8, but findings have had conflicting results. Considering this issue, the present study was conducted with the aim of evaluating positivity of CD44 and PAX8 immunohistochemical markers in parietal epithelial cells to detect suspected cases of FSGS and differentiate it from minimal histopathological abnormality in patients with nephrotic syndrome.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Case selection\u003c/h2\u003e \u003cp\u003eThis cross-sectional study was conducted on patients who underwent kidney biopsy due to nephrotic syndrome in Imam Khomeini Hospital in 2018\u0026thinsp;\u0026minus;\u0026thinsp;1400. After searching in the archive of pathology reports of patients in pathology department, 36 samples of patients whose pathology was diagnosed as MCD (MCD group) and 42 samples of FSGS (FSGS group) were examined. The extracted slides were reviewed by a pathologist and suitable blocks were selected for immunohistochemical staining that had enough tissue. Samples that did not have enough tissue to perform immunohistochemical tests or did not have enough glomerulus, or patient information including laboratory information or required clinical findings were not available, were excluded from study. Before starting the research, ethical aspects were examined by ethics committee of university.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Immunohistochemical evaluation and data collection\u003c/h2\u003e \u003cp\u003eAfter selecting blocks, immunohistochemical staining was performed for two immunohistochemical markers, CD44 and PAX8. First, a 4-micron section of each block was prepared by a microtome on a glass slide. Then they were placed in an autoclave at a temperature of 60\u0026deg;C for one night. In deparaffinization and hydration step, the slides were placed three times in Gesylol for 15 minutes each time, and twice in 100% alcohol for 10 minutes each time, and finally twice in 96% alcohol for 10 minutes each time. After that, samples were washed with distilled water and placed in an autoclave at 95\u0026deg;C for 20 minutes with 1.5 cc of EDTA buffer with pH\u0026thinsp;=\u0026thinsp;8 for antigen retrieval. Again, the slices were washed 2\u0026ndash;3 times with distilled water and kept at room temperature for 20 minutes. Then, internal peroxidases were blocked using peroxidase solution for 10 minutes. Then the primary antibody was added to slides and kept at 37\u0026deg;C for 60 minutes. Then it was washed with TBS buffer and secondary antibody was placed on slides for 15 minutes. Then the slides were placed in contact with diaminobenzidine (DAB) chromogen for 10 minutes and stained with hematoxylin. Finally, slides were dehydrated and mounted. To interpret the results, the stained slides were examined and recorded by two pathologists.\u003c/p\u003e \u003cp\u003eFor CD44, staining intensity was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 1+: positive membrane staining in a limited number of visceral cells in glomerulus, which is considered negative staining. 2+: positive membranous staining with moderate intensity in more cells, 3+: positive and strong membranous staining in visceral and parietal cells, especially in glomerular sclerosis, in interpretation of results according to similar studies and findings Here, +\u0026thinsp;1 cases were considered negative and +\u0026thinsp;2 and +\u0026thinsp;3 results were considered positive[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePolyclonal PAX8 antibody) Proteintech, dilution 1: 50, catalog 11392-3-AP) was used to evaluate activated PEC from paraffin sections. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) The experiments were carried out as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eClinical and laboratory information of patients such as age, sex and clinical symptoms of patients were also recorded from file information. Data were collected in a pre-prepared checklist.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Statistics\u003c/h2\u003e \u003cp\u003eSPSS version 26 software was used for statistical analysis of data. Comparison between quantitative variables was done by t-test and Mann-Whitney test. Comparison between qualitative variables was also done using Chi-square test. A significance level of less than 0.05 was considered.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Patient characteristics\u003c/h2\u003e \u003cp\u003eA total of 78 samples were obtained; 42 samples from FSGS patients and 36 samples from MCD patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In terms of glomeruli number, the average number of glomeruli was 18.17 and 24.83, respectively, and there was a significant difference between two groups (P\u0026thinsp;=\u0026thinsp;0.02). The frequency of cases with foamy cells was 11 cases (26.2%) and 2 cases (5.6%), respectively; which was significantly higher in first group (P\u0026thinsp;=\u0026thinsp;0.01). Regarding interstitial fibrosis and tubular atrophy (IFTA) in FSGS and MCD patients, there were 22 cases (8.46%) and 2 cases (5.6%), respectively, which were significantly more seen in FSGS group (P\u0026thinsp;=\u0026thinsp;0.001). In terms of gender, sampling area, mesangial matrix expansion and GBM changes, no significant difference was observed between two groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBackground characteristics of two groups with FSGS and MCD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eparameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFSGS (n\u0026nbsp;\u003cem\u003e=\u003c/em\u003e\u0026nbsp;42)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMCD (n\u0026nbsp;\u003cem\u003e=\u003c/em\u003e\u0026nbsp;36)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep-\u003c/em\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eage (years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003emean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003egender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003emale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (57.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22 (61.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003efemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (42.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14 (38.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNumber of glomeruli\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003emean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.17\u0026thinsp;\u0026plusmn;\u0026thinsp;19.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.83\u0026thinsp;\u0026plusmn;\u0026thinsp;15.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eNumber of glomeruli\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eBetween 0 and 10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (26.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (16.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eBetween 11 and 25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 (50.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13 (36.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAbove 25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (23. 8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17 (47.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003efoamy cell\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (26.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExpansion of the mesangial matrix\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (85.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 (83.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariations of GBM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (61.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17 (47.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003esubendothelial widening\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (4.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIFTA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (46.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (6.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe frequency of PAX8 expression in the subgroup of patients with FSGS in men and women was 95.8% and 100%, respectively, and in MCD it was 27.3% and 42.9%. There was a significant difference in the expression of PAX8 in FSGS and MCD groups according to gender (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). But according to the position involved, this difference was not significant (P\u0026thinsp;=\u0026thinsp;0.07).\u003c/p\u003e \u003cp\u003eIn total, the frequency of PAX8 expression in the group with FSGS was estimated as 97.6% and in the MCD group as 52.8%, which was significantly higher in the FSGS group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).(Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequency of PAX8 expression in FSGS and MCD groups according to gender and site of involvement\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProperty\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFSGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMCD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u003cem\u003e-\u003c/em\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003emale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003efemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEngaged position\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCortex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCorticomedullary\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e52.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn patients with FSGS and MCD, the expression of CD44\u0026thinsp;+\u0026thinsp;1 was not related to the gender and involved position, and no significant difference was observed in this regard in the two groups of patients (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In fact, this finding indicated the lack of influence of gender and involved position on the expression of this marker in two diseases. In total, the overall expression of CD44, in the two groups with FSGS and MCD, 1\u0026thinsp;+\u0026thinsp;cases, which were considered negative, was equal to 26.2% and 94.4%, respectively, 2\u0026thinsp;+\u0026thinsp;and 3\u0026thinsp;+\u0026thinsp;cases, which were considered positive, respectively. In FSGS and MCD, it was equal to 40.5%, 5.6%, 33.3% and 0.0%, which indicated the higher intensity of CD44 expression in FSGS group compared to MCD (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe frequency of CD44 expression in FSGS and MCD according to and site of involvement\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eFSGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eMCD gender\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2+\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3+\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003emale\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29. 2%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29. 2%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.7%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95.5%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.5%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003efemale\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.2%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.6%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.2%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.9%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.1%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep-\u003c/b\u003e\u003cb\u003eValue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEngaged position\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCortex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCorticomedullary\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep-\u003c/b\u003e\u003cb\u003eValue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.46%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAverage age in FSGS patients was not related to PAX8 expression (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Also, the average age in FSGS patients was related to the expression of CD44\u0026thinsp;+\u0026thinsp;1, CD44\u0026thinsp;+\u0026thinsp;2 and CD44\u0026thinsp;+\u0026thinsp;3, respectively 14.09 years, 41.47 years and 37.57 years; which shows that the expression of marker increases with increasing age (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In subgroup of patients with MCD, the average age in two groups with and without PAX8 expression was 15.92 and 14.58 years, respectively (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Also, the average age in two groups with and without CD44 expression was 55 and 12.68 years, respectively, which indicates a significant relationship between the marker and increasing age (P\u0026thinsp;=\u0026thinsp;0.04).( Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequency of expression of CD44 and PAX8 in FSGS and MCD according to average age\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003emarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFSGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMCD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePAX8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.78\u0026thinsp;\u0026plusmn;\u0026thinsp;22.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.92\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCD44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.45\u0026thinsp;\u0026plusmn;\u0026thinsp;9.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.68\u0026thinsp;\u0026plusmn;\u0026thinsp;7.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.84\u0026thinsp;\u0026plusmn;\u0026thinsp;21.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55\u0026thinsp;\u0026plusmn;\u0026thinsp;7.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u003cem\u003e-\u003c/em\u003eValue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn patients with MCD, out of 19 patients with positive expression of PAX8, 11 patients (57.9%) had basement membrane changes in electron microscopy, and 8 patients (42.1%) were without basement membrane changes, while in patients without PAX8 expression, basement membrane changes were observed in 41.2% of cases (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Also, the expression of CD44 in these two groups of patients was 5.9% and 5.3%, respectively, which again does not show a significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).( Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequency of expression of CD44 and PAX8 in FSGS and MCD according to basement membrane changes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eVariations of GBM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eFSGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eMCD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePAX8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCD44\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePAX8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eCD44\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 (96.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e(3.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003cp\u003e(80.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e(19.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11\u003c/p\u003e \u003cp\u003e(61.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003cp\u003e(38.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e(5.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(94.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e(62.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003cp\u003e(37.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e(44.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e(55.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e(5.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e(94.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn 19 patients with PAX8-positive MCD, 16 patients (55.2%) had suspicious clinical and histological symptoms. Suspicious clinical and histological symptoms were observed in 13 cases (44.8%) in patients with negative PAX8 (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the case of CD44, the positive expression of this marker was observed in 14.3% and 37.9% of cases with definite and doubtful diagnosis, respectively (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).( Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe frequency of expression of CD44 and PAX8 in the MCD group according to the presence of clinical symptoms and microscopic changes suspicious for FSGS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDiagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePAX8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCD44\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsence of clinical and microscopic symptoms suspicious of FSGS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (42.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (57.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (14.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (85.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresence of clinical and microscopic signs suspicious for FSGS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (55.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (44.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (37.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (62.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConsidering that FSGS was diagnosed with gold standard optical and electron microscopy, it can be said that the sensitivity and specificity of PAX8 marker for FSGS diagnosis are 97.6% and 66.7%, respectively, and the sensitivity and specificity of CD44 is 73.8% and 94.4% respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we demonstrated that PAX8 and CD44 are effective markers in diagnosing suspected cases of FSGS and differentiating it from MCD. The result of this study was that, firstly, we encountered a significant expression of PAX8 in patients with FSGS (97.6%). While the expression of this marker in the MCD group was detected in only one third of cases (52.7%), this makes it necessary to follow up patients in the future in terms of the possibility of sclerotic involvement in subsequent biopsies. In other words, tracking the PAX8 marker can not only be very sensitive for the diagnosis of FSGS, but also very helpful in distinguishing between FSGS and MCD. On the other hand, regarding the expression of CD44, this marker expression was reported in both diseases but with different degrees, however, the expression of this marker in patients with FSGS was much higher than in patients with MCD. In a way, the expression of +\u0026thinsp;3 was detected in one third of the patients with FSGS group, while this level of expression was not reported at all in the patients of the opposite group. In a general view, tracking and determining the expression level of both markers was very helpful for distinguishing between the two types of podocytopathy. In this way, we saw a significant expression of these markers in FSGS compared to MCD. This finding has been reported in previous studies. Similar investigations have shown that the increase in the number of podocytes with simultaneous expression of PAX8 marker in animal FSGS model has been detected and confirmed. Also, the occurrence of CD44 markers has been an important marker to show the activity of PECs in FSGS disease [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A study by Okamoto et al. showed an increase in CD44 expression in glomeruli involved in FSGS, while the amount of CD44\u0026thinsp;+\u0026thinsp;cells indicated the degree of podocyte damage in FSGS[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In a parallel study, it was shown that CD44\u0026thinsp;+\u0026thinsp;was significantly increased in FSGS compared to MCD, and this method can be used to differentiate early FSGS from MCD [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFSGS is the most common primary glomerulopathy leading to end-stage kidney disease. This disease is characterized by focal and segmental destruction and fading of glomerular capillaries structure along with an increase in matrix thickness. This disease is also accompanied by extensive destruction of podocytes, and if there is a significant improvement in number of these cells, the occurrence of glomerulosclerosis can be fully expected [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In present study, the increase in CD44 expression in patients with FSGS was probably a sign of podocytes loss and the activation of PEC. In a study by Husain et al., it was reported that in FSGS, increased expression of CD44 was observed in all cases, implying loss of podocytes and simultaneous activation of PEC[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CD44 positivity in PEC in FSGS patients is associated with worse prognosis and more impaired renal function[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Roca and his colleagues reported that FSGS lesions were observed in 83% of CD44 positive patients. They stated that increased expression of CD44 had a significant relationship with resistance to steroid therapy and worse prognosis, which can be an early sign of FSGS [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In another study, it was reported that CD44 activated PEC can be an indicator of primary podocyte damage in the development of FSGS and is a good marker for the early diagnosis of CNI-induced nephrotoxicity[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Based on previous studies, it was proven that active PEC cells, in addition to being the precursors of podocyte cells, play an effective role in creating glomerular sclerotic lesions, which is due to the role of active PEC cells in extracellular matrix and production of fibrosis and finally sclerotic lesions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFindings have shown that the PAX2 gene plays an effective role in kidney organogenesis and mutation in this gene is an important cause of kidney disorders of unknown cause[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Based on previous studies, PAX8 is a very sensitive marker and can identify microsclerosis and precursor lesions and is a very good substitute for PAS dye (8). According to our findings, the sensitivity of PAX8 marker to detect sclerotic lesions in FSGS is 100% in total of two biopsies, which shows the necessity of re-biopsy in patients suspected of FSGS based on other findings. In the study of Smeet et al., PEC markers including PAX8 and CD44 were present in 87% of patient's biopsies diagnosed with primary FSGS in capillary tufts, while no PEC activation markers were found in MCD patients (8). In another study, FSGS was diagnosed in 83.3% of PAX8 positive patients, while all PAX8 negative patients were diagnosed with MCD. A number of FSGS patients who were negative for PAX8 in the first biopsy were stained with PAX8 marker in the second biopsy, and finally all patients with FSGS were positive for PAX8 marker in at least one of the two biopsies performed[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn present study, the sensitivity and specificity of PAX8 marker for FSGS diagnosis were 97.6% and 66.7%, respectively. And the sensitivity and specificity of CD44 were 73.8% and 94.4%, respectively. Of course, the specificity of PAX8 marker is probably higher than mentioned number. Because according to clinical findings and electron microscopy, a significant number of patients who were diagnosed with MCD in first biopsy probably had sclerotic lesions in subsequent follow-ups. Also, the negativity of PAX8 marker in one of the samples diagnosed with FSGS is probably due to the fact that repeated cuts on tissue led to removal of glomerulus sclerosis in IHC staining. As in similar studies, the cause of non-positive PEC markers in a small number of FSGS cases has been attributed to the focal and segmental nature of the lesions[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Because in order to find these lesions, it is necessary to examine multiple sections, while we only stained one section, and this section was taken for light microscopy after preparing the previous sections, which raises the possibility of removing the involved area. As it was seen in a study that re-biopsy made the PAX8 marker positive in FSGS patients, which was negative in the first biopsy, and finally, the examination of both biopsies showed that 100% of FSGS patients have positive PAX8 marker at least once [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn total, the two main findings in this study include the first high sensitivity of PEC markers, especially PAX8, in the diagnosis of FSGS lesions, which suggests the role of these cells in the pathogenesis of the disease. The second finding is that these markers were positive in a number of samples that were diagnosed with MCD in the initial examination with light microscopy, which suggests the need to follow up these patients in terms of the possibility of diagnosing FSGS in subsequent samplings.\u003c/p\u003e \u003cp\u003eOne of the limitations of this study was the lack of complete follow-up of the patients, which was effective in the definitive diagnosis of the MCD group. In fact, a significant number of people in this group may have sclerotic lesions in subsequent biopsies and fall into the FSGS group. If the presence of electron microscope changes in some of these patients increases the possibility of undiagnosed sclerotic lesions.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003ePAX8 and CD44 are effective in diagnosing suspected cases of FSGS and distinguishing it from MCD. The high level and intensity of PAX8 and CD44 expression in PEC cells in Bowman's capsule and capillary tuft in patients with FSGS show the high sensitivity of these markers in diagnosis of FSGS and the low expression in MCD can play an important role in distinguish between types of podocytopathies. It is suggested to use this marker to differentiate MCD from FSGS in cases of diagnostic doubt, especially in the early stages of FSGS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFocal Segmental GlomeruloSclerosis (FSGS)\u003c/p\u003e\n\u003cp\u003eMinimal Change Disease (MCD)\u003c/p\u003e\n\u003cp\u003eInterstitial Fibrosis and Tubular Atrophy (IFTA)\u003c/p\u003e\n\u003cp\u003eParietal Epithelial Cell (PEC)\u003c/p\u003e\n\u003cp\u003eGlomerular Basement Membrane (GBM)\u003c/p\u003e\n\u003cp\u003ediaminobenzene (DAB)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eapproval and consent to participate:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, all provisions of the Helsinki regulations were considered. Informed consent was obtained from all the people when the file was filed by the medical records archive for the possible use of the file information for research purposes.\u003c/p\u003e\n\u003cp\u003eThe design of the study was approved by ethics committee\u0026nbsp;of research of Tehran University of Medical Sciences\u0026nbsp;code: IR.TUMS.IKHC.REC.1399.383\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no financial support for the research, authorship and publication of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors' contributions:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamaneh Salarvand: Conceived and\u0026nbsp;designed the analysis, performed the histological examination of the kidney, reporting the slides, IHC and EM reporting\u003c/p\u003e\n\u003cp\u003eFatemeh Nili: Conceived and designed the analysis, performed the histological examination of the kidney, reporting the slides, IHC and EM reporting\u003c/p\u003e\n\u003cp\u003eFarshid Dehkhoda: analyzed and interpreted the patient data, Performed the analysis, wrote the paper\u003c/p\u003e\n\u003cp\u003eAlireza Abdollahi: Conceived and designed the analysis, collected data, contributed data\u003c/p\u003e\n\u003cp\u003eAzin Alemzadeh: Collected the data, contributed data, Performed the analysis\u003c/p\u003e\n\u003cp\u003eMaryam Abedi: collected data, contributed data and analysis tools, contributed in the histological examination of the kidney, Wrote the paper\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHarris DC, Davies SJ, Finkelstein FO, Jha V, Donner J-A, Abraham G, et al. Increasing access to integrated ESKD care as part of universal health coverage. \u003cem\u003eKidney international\u003c/em\u003e. 2019;95(4):S1-S33.\u003c/li\u003e\n\u003cli\u003eBikbov B, Purcell C, Levey A, Smith M, Abdoli A, Abebe M, et al. GBD Chronic Kidney Disease Collaboration: Global, regional, and national burden of chronic kidney disease, 1990-2017: A systematic analysis for the Global Burden of Disease Study 2017. \u003cem\u003eLancet\u003c/em\u003e. 2020;395(709-733):32061315.\u003c/li\u003e\n\u003cli\u003eReiser J, Altintas MM. Podocytes. \u003cem\u003eF1000Research\u003c/em\u003e. 2016;5.\u003c/li\u003e\n\u003cli\u003eKopp JB. Replenishment of the podocyte compartment by parietal epithelial cells. \u003cem\u003eKidney international\u003c/em\u003e. 2015;88(5):934-935.\u003c/li\u003e\n\u003cli\u003eWharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, et al. Podocyte depletion causes glomerulosclerosis: Diphtheria toxin\u0026ndash;induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. \u003cem\u003eJournal of the American Society of Nephrology\u003c/em\u003e. 2005;16(10):2941-2952.\u003c/li\u003e\n\u003cli\u003eKim S, Kim YH, Choi KH, Jeong HJ. Glomerular epithelial CD44 expression and segmental sclerosis in IgA nephropathy. \u003cem\u003eClinical and experimental nephrology\u003c/em\u003e. 2016;20:871-877.\u003c/li\u003e\n\u003cli\u003eGarin EH, Mu W, Arthur JM, Rivard CJ, Araya CE, Shimada M, et al. Urinary CD80 is elevated in minimal change disease but not in focal segmental glomerulosclerosis. \u003cem\u003eKidney International\u003c/em\u003e. 2010;78(3):296-302.\u003c/li\u003e\n\u003cli\u003eSmeets B, Stucker F, Wetzels J, Brocheriou I, Ronco P, Gr\u0026ouml;ne H-J, et al. Detection of activated parietal epithelial cells on the glomerular tuft distinguishes early focal segmental glomerulosclerosis from minimal change disease. \u003cem\u003eThe American journal of pathology\u003c/em\u003e. 2014;184(12):3239-3248.\u003c/li\u003e\n\u003cli\u003eYamazaki T, Sasaki S, Okamoto T, Sato Y, Hayashi A, Ariga T. Up-regulation of CD74 expression in parietal epithelial cells in a mouse model of focal segmental glomerulosclerosis. \u003cem\u003eNephron\u003c/em\u003e. 2016;134(4):238-252.\u003c/li\u003e\n\u003cli\u003eFatima H, Moeller MJ, Smeets B, Yang H-C, D\u0026rsquo;Agati VD, Alpers CE, et al. Parietal epithelial cell activation marker in early recurrence of FSGS in the transplant. \u003cem\u003eClinical journal of the American Society of Nephrology: CJASN\u003c/em\u003e. 2012;7(11):1852.\u003c/li\u003e\n\u003cli\u003eZhong J, Zuo Y, Ma J, Fogo AB, Jolicoeur P, Ichikawa I, et al. Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy. \u003cem\u003eKidney international\u003c/em\u003e. 2005;68(3):1048-1060.\u003c/li\u003e\n\u003cli\u003eHayashi A, Okamoto T, Yamazaki T, Sato Y, Takahashi T, Ariga T. CD44-positive glomerular parietal epithelial cells in a mouse model of calcineurin inhibitors-induced nephrotoxicity. \u003cem\u003eNephron\u003c/em\u003e. 2019;142(1):71-81.\u003c/li\u003e\n\u003cli\u003eStenger M. Calculating H-Score-The ASCO Post. \u003cem\u003eThe ASCO POST\u003c/em\u003e. 2015.\u003c/li\u003e\n\u003cli\u003eSakamoto K, Ueno T, Kobayashi N, Hara S, Takashima Y, Pastan I, et al. The direction and role of phenotypic transition between podocytes and parietal epithelial cells in focal segmental glomerulosclerosis. \u003cem\u003eAmerican Journal of Physiology-Renal Physiology\u003c/em\u003e. 2014;306(1):F98-F104.\u003c/li\u003e\n\u003cli\u003eOkamoto T, Sasaki S, Yamazaki T, Sato Y, Ito H, Ariga T. Prevalence of CD44-positive glomerular parietal epithelial cells reflects podocyte injury in adriamycin nephropathy. \u003cem\u003eNephron Experimental Nephrology\u003c/em\u003e. 2014;124(3-4):11-18.\u003c/li\u003e\n\u003cli\u003eHusain S, Ginawi I, Bashir AI, Kfoury H, Al Johani TE, Hagar H, et al. Focal and segmental glomerulosclerosis in murine models: a histological and ultrastructural characterization with immunohistochemistry correlation of glomerular CD44 and WT1 expression. \u003cem\u003eUltrastructural Pathology\u003c/em\u003e. 2018;42(5):430-439.\u003c/li\u003e\n\u003cli\u003eFroes BP, de Almeida Ara\u0026uacute;jo S, Bambirra EA, Oliveira EA, Simoes e Silva AC, Pinheiro SVB. Is CD44 in glomerular parietal epithelial cells a pathological marker of renal function deterioration in primary focal segmental glomerulosclerosis? \u003cem\u003ePediatric Nephrology\u003c/em\u003e. 2017;32:2165-2169.\u003c/li\u003e\n\u003cli\u003eRoca N, Jatem E, Abo A, Santacana M, Cruz A, Madrid \u0026Aacute;, et al. CD44-negative parietal\u0026ndash;epithelial cell staining in minimal change disease: association with clinical features, response to corticosteroids and kidney outcome. \u003cem\u003eClinical Kidney Journal\u003c/em\u003e. 2022;15(3):545-552.\u003c/li\u003e\n\u003cli\u003eMuntean C, Chirtes C, Baczoni B, Banescu C. PAX2 Gene Mutation in Pediatric Renal Disorders\u0026mdash;A Narrative Review. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e. 2023;24(16):12737.\u003c/li\u003e\n\u003cli\u003eSuzuki T, Kohatsu K, Han W, Watanabe S, Yahagi K, Nakata M, et al. Morphological features of minimal change disease and focal segmental glomerulosclerosis using repeat biopsy and parietal epithelial cell marker. \u003cem\u003eKidney Diseases\u003c/em\u003e. 2020;6(2):119-124.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"kidney: Nephrotic syndrome, Podocytes, parietal epithelial cells, Focal Segmental Glomerulosclerosis, minimal change disease","lastPublishedDoi":"10.21203/rs.3.rs-3822572/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3822572/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression evaluation of specific markers PAX8 and CD44 on kidney podocyte cells or their progenitor cells can be very helpful in diagnosing and differentiating between types of podocytopathies. In present study, the positivity of immunohistochemical markers CD44 and PAX8 in parietal epithelial cells (PECs) was evaluated to diagnose suspected cases of Focal Segmental Glomerulosclerosis (FSGS) and differentiate it from minimal change disease (MCD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis cross-sectional study was conducted on patients who underwent kidney biopsy due to nephrotic syndrome. 42 FSGS samples and 36 MCD samples were selected and biopsies were evaluated for CD44 and PAX8 markers. Suitable blocks for immunohistochemical staining that had enough tissue were selected and evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression frequency of PAX8 marker in group with FSGS was estimated as 97.6% and in group with MCD as 52.8%, which was significantly higher in FSGS group. Also, regarding the expression of CD44, in two groups with FSGS and MCD, respectively, 1 + cases equal to 26.2% and 94.4%, 2 + cases equal to 40.5% and 5.6%, and 3 + cases equal to 33.3% and 0.0%, which indicated the higher intensity of CD44 expression in FSGS group compared to MCD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of CD44 in FSGS was influenced by patients age and a direct and significant relationship was observed. The increase in amount and intensity of two markers PAX8 and CD44 expression in PEC cells in patients with FSGS shows high sensitivity of these markers in diagnosis of FSGS and the decrease in expression level in MCD can play an important role in differentiating between types of podocytopathy disorders.\u003c/p\u003e","manuscriptTitle":"Evaluation of immunohistochemical markers CD44 and PAX8 in diagnosis of Focal Segmental Glomerulosclerosis suspected cases and its differentiation from Minimal Change disease in patients with nephrotic syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 15:04:26","doi":"10.21203/rs.3.rs-3822572/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2f49c9c-b6cd-4281-9e1c-f388812cba92","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-30T05:59:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-03 15:04:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3822572","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3822572","identity":"rs-3822572","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}