Immunohistochemical and electronmicroscopic features of mesenchymal-to-epithelial transition in human developing, postnatal and nephrotic podocytes.

Differentiation of human podocytes starts with mesenchymal-to-epithelial transition (MET) of the metanephric mesenchyme into the S-shaped nephrons. During further development, differentiating podocytes regain mesenchyme-like cell characteristics by epithelial-to-mesenchymal transition (EMT), leading to formation of the terminally differentiated, non-dividing cell. Both MET and EMT processes involve changes in content and organization of cytoskeletal and actin filaments, accompanied by the increased glomerular vascularization. Here, we analyze and compare normal human developing, postnatal and nephrotic podocytes and glomeruli, using immunohistochemical and double immunofluorescent methods for detection of markers of cytoskeletal filaments (nestin, cytokeratin 10-CK10, vimentin and α-SMA), vasculogenesis (CD31 and VEGF) and podocyte function (receptor for advanced glycation end products, RAGE). In addition, electron microscopy is used to detect ultrastructural changes of the podocytes. Early metanephric cup mesenchyme expresses all investigated markers except α-SMA, which characterizes only surface mesenchymal cells. In differentiating podocytes and cells of Bowman's capsule (parietal podocytes) nestin decreases, vimentin increases, while CK10 gradually disappears. Increase in α-SMA is associated with blood vessels development, appearance of podocyte pedicles and slit diaphragm and loss of intercellular connections (zonulae adherentes). Increase in CD31 characterizes vascular glomerular tufts development, while decrease in RAGE expression accompanies normal podocyte differentiation. In congenital nephrotic syndrome of the Finnish type, dedifferentiated podocytes display changes in cytoskeletal filaments and depletion of podocyte pedicles, while glomerular vascular supply is diminished. Our data also suggest high potential of metanephric mesenchyme and parietal podocytes in possible regeneration of the damaged podocytes.

[C1Q NEPHROPATHY: CASE REPORTS AND LITERATURE REVIEW]. / C1Q-NEFROPATIJA: PRIKAZI BOLESNIKA I PREGLED LITERATURE.

C1q nephropathy is considered a form of glomerulonephritis, defined by histological findings of dominant Clq immune deposits in renal biopsy. It is a rare disease, most often manifested in children and young adults. The most common clinical manifestation of the disease is nephrotic syndrome, but other renal syndromes could also be found. The cause of the disease is not known, but the immune pathogenesis could be assumed. Often, resistance to glucocorticoid or other immunosuppressive therapy is present, potentially leading to chronic renal insufficiency. We present ten patients with renal biopsy and clinical findings of Clq nephropathy. None of the patients had clinical or serological manifestations of systemic lupus. All patients had normal findings of C3 and C4 components of complement, as well as normal ANF, anti-dsD-NA and ANCA antibodies.

Mechanism of cystogenesis in nephrotic kidneys: a histopathological study.

BACKGROUND: Nephrotic syndrome (NS) is pathological condition characterized by heavy proteinuria. Our study investigates hypothesis that change in cell proliferation of proximal tubules influences primary cilia structure and function and promotes cystogenesis in congenital nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS). METHODS: CNF kidneys were analyzed genetically. Proliferation (Ki-67), apoptosis (caspase-3), and primary cilia (α-tubulin) length and structure were analyzed immunohistochemically and ultrastructurally in healthy, CNF and FSGS kidneys. Cyst diameters were measured and correlated with proliferation index. RESULTS: Proximal tubules cells of healthy kidneys did not proliferate. In nephrotic kidneys, tubules with apparently normal diameter covered by cuboidal/columnar epithelium (PTNC) contained 81.54% of proliferating cells in CNF and 36.18% in FSGS, while cysts covered with columnar epithelium (CC) contained 37.52% of proliferating cells in CNF and 45.23% in FSGS. The largest cysts, covered with squamous epithelium (CS) had 11.54% of proliferating cells in CNF and 13.76% in FSGS. Increase in cysts diameter correlated with changes in proliferation index, tubular cells shape, primary cilia formation and appearance of apoptotic cells. CONCLUSIONS: We present a novel histopathological data on the structure and possible changes in function of tubular cell in NS kidneys during cystogenesis. We suggest existence of common principles of cystogenesis in CNF and FSGS kidneys, including serious disturbances of tubular cells proliferation and apoptosis, and faulty primary cilia signaling leading to deterioration of proteinuria in NS kidneys.

A Case of Unilateral Otologic Symptoms as Initial Manifestations of Granulomatosis With Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a chronic and systematic autoimmune condition characterized by granuloma formation and necrotizing vasculitis of small to medium-sized vessels. GPA initially presents itself as respiratory and renal symptoms. Although temporal bone involvement is not uncommon, an otologic disorder is rarely the initial symptom. We present a case of a 36-year-old man who presented with unilateral ear pain, hearing loss, and facial palsy. After a series of diagnostics and temporal bone and chest imaging, he was diagnosed with GPA with multiorgan involvement. Cyclophosphamide and methylprednisolone relieved the patient's ear pain and partially improved his hearing, facial palsy, and overall clinical condition. Although uncommon, systemic GPA may cause initial otologic symptoms and should not be dismissed as a possible cause of an otologic disease resistant to standard therapy.

Expression of Connexins 37, 40 and 45, Pannexin 1 and Vimentin in Laryngeal Squamous Cell Carcinomas.

Approximately 60% of patients with squamous cell carcinoma (LSCC) have regional occult metastatic disease/distant metastases at the time of diagnosis, putting them at higher risk for disease progression. Therefore, biomarkers are needed for early prognostic purpose. The aim of this study was to analyze the expression pattern of connexins (Cx) 37, 40 and 45, pannexin1 (Panx1) and vimentin in LSCC and correlate with tumor grade (G) and outcome. METHODS: Thirty-four patients who underwent (hemi-)laryngectomy and regional lymphadenectomy due to LSCC from 2017 to 2018 in University Hospital Split, Croatia, were studied. Samples of tumor tissue and adjacent normal mucosa embedded in paraffin blocks were stained using the immunofluorescence method and were semi-quantitatively analyzed. RESULTS: The expression of Cx37, Cx40, and Panx1 differed between cancer and adjacent normal mucosa and between histological grades, being the highest in well-differentiated (G1) cancer and low/absent in poorly differentiated (G3) cancer (all p < 0.05). The expression of vimentin was the highest in G3 cancer. Expression of Cx45 was generally weak/absent, with no significant difference between cancer and the controls or between grades. Lower Panx1 and higher vimentin expression were found to be prognostic factors for regional metastatic disease. Lower Cx37 and 40 expressions were present in patients with disease recurrence after the three-year follow-up period. CONCLUSION: Cx37 and Cx40, Panx1, and vimentin have the potential to be used as prognostic biomarkers for LSCC.

Her-2/neu assessment for gastric carcinoma: validation of scoring system.

BACKGROUND/AIMS: Gastric cancer is the second leading cause of cancer mortality in the world. Amplification of HER-2/neu oncogene has become an important biomarker for identifying patients who respond to HER-2 targeting therapy. A number of studies have analyzed HER-2/neu overexpression in gastric carcinoma, and the rate of HER2 positivity is variable, ranging from 6% to 35%. METHODOLOGY: In our study HER-2/neu expression was assessed on 73 samples of primary gastric cancer, using immunohistochemistry. For 19 patients preoperative biopsy samples and resected specimens were available. Additionally, internal ring study was performed to estimate intraobserver variability of IHC scoring among pathologists at our department. RESULTS: HER-2/neu overexpression was found in 10 (13.6%) of the tested samples, and it was more common in intestinal (22.5%) than the diffuse type (3.7%). Not one of the 6 analyzed mixed type tumors showed HER-2/neu expression. For the paired samples (preoperative biopsy samples and resected specimens) the concordance rate for HER-2/neu expression was 94.7%. CONCLUSIONS: According to high concordance rate in paired samples we consider it appropriate to evaluate HER2 expression on biopsy specimens, especially in unresectable cases, and to re-evaluate it on resected specimens if available, due to high heterogeneity of a gastric cancer.

Prognostic value of connective tissue growth factor and c-Myb expression in IgA nephropathy and Henoch-Schönlein purpura-A pilot immunohistochemical study.

AIM: Adverse and advanced prognostic signs in IgA nephropathy (IgAN) are interstitial fibrosis and tubular atrophy, but early predictors of bad outcome are still lacking. We investigated expression of connective tissue growth factor (CTGF) and c-Myb in renal biopsies of IgAN and Henoch-Schönlein purpura (HSP), because these gene products are indirectly included in fibrosis and epithelial-mesenchymal transition (EMT). METHODS: The sample included 23 patients and 8 controls who underwent nephrectomy due to renal cancer. The slides cut from the paraffin blocks were prepared for standard indirect immunoflourescence, using antibodies to CTGF and c-Myb. Ten high-power non-overlapping fields were photographed on Olympus IX51 microscope. Average percent of positive tubular cells, as well as number of positive cells per glomerulus were calculated. RESULTS: The cytoplasmic tubular CTGF expression was higher in IgAN/HSP than in controls (P < 0.001), whereas no difference was found in glomeruli (P = 0.437). The nuclear c-Myb expresssion in glomeruli and tubules was higher in IgAN/HSP than in controls (P < 0.05). In the follow-up, decline in renal function correlated with glomerular and tubular c-Myb, as well as tubular CTGF expression (all P < 0.05). CONCLUSION: Our results proposed c-Myb and CTGF as novel, early and sensitive markers of chronic kidney disease and worse renal outcome, but larger series are needed.

Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development.

AIM: Present study analyses the co-localisation of RIP5 with FGFR1, FGFR2 and HIP2 in the developing kidney, as RIP5 is a major determinant of urinary tract development, downstream of FGF-signaling. METHODS: Paraffin embedded human kidney tissues of 16 conceptuses between the 6th-22th developmental week were analysed using double-immunofluorescence method with RIP5/FGFR1/FGFR2 and HIP2 markers. Quantification of positive cells were performed using Kruskal-Wallis test. RESULTS: In the 6th week of kidney development RIP5 (89.6%) and HIP2 (39.6%) are strongly expressed in the metanephric mesenchyme. FGFR1 shows moderate/strong expression in the developing nephrons (87.3%) and collecting ducts (70.5%) (p < 0.05). RIP5/FGFR1 co-localized at the marginal zone and the ureteric bud with predominant FGFR1 expression. FGFR2 (26.1%) shows similar expression pattern as FGFR1 (70.5%) in the same kidney structures. RIP5/FGFR2 co-localized at the marginal zone and the collecting ducts (predominant expression of FGFR2). HIP2 is strongly expressed in collecting ducts (96.7%), and co-localized with RIP5. In 10th week, RIP5 expression decrease (74.2%), while the pattern of expression of RIP5 and FGFR1 in collecting ducts (33.4% and 91.9%) and developing nephrons (21.9% and 32.4%) (p < 0.05) is similar to that in the 6th developmental week. Ureter is moderately expressing RIP5 while FGFR1 is strongly expressed in the ureteric wall. FGFR2 is strongly expressed in the collecting ducts (84.3%) and ureter. HIP2 have 81.1% positive cells in the collecting duct. RIP5/FGFR1 co-localize in collecting ducts and Henley's loop. CONCLUSIONS: The expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the human kidney development might indicate their important roles in metanephric development and ureteric muscle layer differentiation through FGF signaling pathways.

Expression of DENDRIN in several glomerular diseases and correlation to pathological parameters and renal failure - preliminary study.

BACKGROUND: In glomerular injury dendrin translocates from the slit diaphragm to the podocyte nucleus, inducing apoptosis. We analyzed dendrin expression in IgA glomerulonephritis and Henoch Schönlein purpura (IgAN/HSP) versus in podocytopathies minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and compared it to pathohistological findings and renal function at the time of biopsy and the last follow-up. METHODS: Twenty males and 13 females with median of age 35 years (min-max: 3-76) who underwent percutaneous renal biopsy and had diagnosis of glomerular disease (GD) were included in this retrospective study. Fifteen patients had IgAN/HSP and eighteen podocytopathy. Control group consisted of ten patients who underwent nephrectomy due to renal cancer. Dendrin expression pattern (membranous, dual, nuclear or negative), number of dendrin positive nuclei and proportion of dendrin negative glomeruli were analyzed. RESULTS: In GD and the control group significant differences in number of dendrin positive nuclei and proportion of dendrin negative glomeruli were found (P = 0.004 and P = 0.003, respectively). Number of dendrin positive nuclei was higher in podocytopathies than in IgAN/HSP, 3.90 versus 1.67 (P = 0.028). Proportion of dendrin negative glomeruli correlated to higher rates of interstitial fibrosis (P = 0.038), tubular atrophy (P = 0.011) and globally sclerotic glomeruli (P = 0.008). Dual and nuclear dendrin expression pattern were connected with lower rate of interstitial fibrosis and tubular atrophy than negative dendrin expression pattern (P = 0.024 and P = 0.017, respectively). Proportion of dendrin negative glomeruli correlated with lower creatinine clearance (CC) at the time of biopsy and the last follow-up (P = 0.010 and P < 0.001, respectively). Dendrin expression pattern correlated to CC at the last follow-up (P = 0.009), being lower in patients with negative than nuclear or dual dendrin expression (P = 0.034 and P = 0.004, respectively). CONCLUSION: In this pilot study the number of dendrin positive nuclei was higher in podocytopathies than in inflammatory GD. Negative dendrin expression pattern correlated to chronic tubulointerstitial changes and lower CC, which needs to be confirmed in a larger series.

Cathepsin K expression in melanoma is associated with metastases.

INTRODUCTION: Melanoma of the skin shows a tendency to metastasize via lymph or blood secreting matrix metalloproteinases and cathepsins, which enable penetration through the dermis. Cathepsin K acts in cytoplasm of atypical melanocytes and completely cleaves internalized collagen. MATERIALS AND METHODS: Expression of cathepsin K was analyzed immunohistochemically in 45 melanomas and correlated to morphological and clinical parameters. RESULTS: During six years follow up, 13 patients developed lymph node metastases and three of them distant metastases. Positive expression of cathepsin K was found in 19 cases. In univariate regression analysis histological type, pagetoid spread, mitotic activity and cathepsin K expression were significantly connected to metastases. Cathepsin K was significantly associated to histologic type, ulceration, pagetoid spread and mitotic rate. In multiple logistic regression adjusted to these variables, cathepsin K was an independent predictor in occurrence of metastases (P=0.015). Median to the occurrence of metastases was 40 months in patients with cathepsin K positive expression and 71 months in patients with cathepsin K negative expression (P<0.001). CONCLUSIONS: In this preliminary study positive expression of cathepsin K in melanoma of the skin is associated with other unfavorable prognostic factors. We consider cathepsin K expression in primary tumor would significantly precipitate occurrence of metastases.

Giant perianal angiomyofibroblastoma--a case report.

A 45-year old female had a long history of slow growing perianal tumor at the right side of her anus. Encapsulated tumour was found intraoperatively and completely excised using the Harmonic Scalpel. Tumour was well-circumscribed and relatively firm; measuring 12x6x4 cm. Histologically it was composed of oval to spindle cells with minimal nuclear atypia, set in mucous matrix with numerous thin-walled blood vessels. Immunohistochemically, expression of smooth-muscle actin and desmin, as well as estrogen and progesterone receptor were found in the tumour cells. The diagnosis of angiomyofibroblastoma was established. This rare benign tumour typically involves vulvovaginal, pelvic and perinal region. It is important to separate this neoplasm from locally invasive aggressive angiomyxoma and low grade fibromyxoid sarcoma, which can arise in the the same localisation. The patient was discharged on the third postoperative day and no recurrence was noted in 18 months follow-up.

Influence of open testicular biopsy in prepubertal rats on rats' adulthood fertility with correlation to serum levels of inhibin B and follicle stimulating hormone.

OBJECTIVE: Open testicular biopsy (OTB) is one of the options to accurately assess fertility potential of the undescended testis. The aim of the study was to investigate consequences of OTB in prepubertal rats on their adulthood fertility. METHODS: Thirty-eight prepubertal male rats were divided into three groups depending on day 20 procedure. The first group was the control group, the second sham operated and the third has left OTB. Bilateral orchiectomy was performed on day 70 to all groups, with determination of serum inhibin B and follicle stimulating hormone (FSH). Removed testes were compared according to the weight, volume, spermatogenesis, histological and apoptotic changes in both testes with differences in serum levels of inhibin B and FSH. RESULTS: Ipsilateral testicular weight, volume, and spermatogenesis reduction with a reduction of tubular number, diameter and germinative epithelium was found in OTB group. Significant increase in apoptotic index was found in biopsied testis without compensatory hypertrophy of contralateral testis. Differences of inhibin B and FSH were not statistically significant among three groups. CONCLUSION: OTB in prepubertal rats has detrimental effects on fertility in adulthood. It does not cause compensatory hypertrophy of the contralateral testis nor does it disturb serum levels of inhibin B and FSH.

IMP3 can predict aggressive behaviour of lung adenocarcinoma.

BACKGROUND: Lung cancer most often presents as an inoperable tumour and the diagnosis is usually performed on a small biopsy/cytology specimen. In the group of non small cell lung cancer - not otherwise specified, adenocarcinoma phenotype can be determined immunohistochemically using TTF-1 and Napsin A. Expression of oncofetal protein IMP3 in human cancer is associated with poor differentiation and aggressive behaviour. In the present study expression of IMP3 was correlated with expression of TTF-1 and Napsin A, histological subtype and clinical stage of lung adenocarcinoma. We were interested whether distant metastases are associated with IMP3 overexpression, regardless of the histologic subtype of adenocarcinoma. METHODS: In retrospective study, consecutive series of 105 patients with advanced lung adenocarcinoma diagnosed from 2006 to 2009 in Clinical Hospital Center Split, Croatia, were analysed. Clinical data were collected from the Pulmology Department and time of death from the Mortality Registry. Paraffin blocks of bronchoscopic biopsies were collected from the Institute of Pathology and 15 cases excluded from the analysis due to insufficient material. Expression of IMP3, Napsin A and TTF-1 were analysed by indirect enzyme immunohistochemistry. Statistical analysis was performed and P values less than 0.05 considered significant. RESULTS: Of 90 patients, 71 (78%) were males and 19 (22%) females. Median age for males was 61.5 years (min-max 43-83) and for females 61 years (min-max 44-86). Pleural effusion was found in 15 (16.6%) and distant metastases in 45 (50%) cases. According to histological subtypes, there were 34 acinar, 2 lepidic, 2 papillary and 52 solid subtypes. IMP3 overexpression was found in 63 cases (70%) and was correlated with solid subtype (P = 0.002) and negative/weak Napsin A expression (P = 0.004). Strong Napsin A expression correlated with TTF-1 expression (P = 0.003) and lower histological grades (P = 0.031). Patients with IMP3 overexpression more often had distant metastases than patients with negative IMP3, 55.5% versus 33.3% (P = 0.033). Non solid subtypes with IMP3 overexpression developed distant metastasis more common than non solid subtypes with negative IMP3, 72% versus 35% (P = 0.028). CONCLUSIONS: Expression of IMP3 correlates with solid subtype and with distant metastases regardless of histological subtype of lung adenocarcinoma. VIRTUAL SLIDES: http://www.diagnosticpathology.diagnomx.eu/vs/1966211581795258