Clinicopathological Relevance of PAX8 Expression Patterns in Acute Kidney Injury and Chronic Kidney Diseases.

Transcription factor PAX8, expressed during embryonic kidney development, has been previously detected in various kidney tumors. In order to investigate expression of PAX8 transcription factor in acute kidney injury (AKI) and chronic kidney diseases (CKD), immunohistochemical analysis was performed. Presence, location and extent of PAX8 expression were analyzed among 31 human kidney samples of AKI (25 autopsy cases, 5 kidney biopsies with unknown etiology and 1 AKI with confirmed myoglobin cast nephropathy), as well as in animals with induced postischemic AKI. Additionally, expression pattern was analyzed in 20 kidney biopsy samples of CKD. Our study demonstrates that various kidney diseases with chronic disease course that results in the formation of tubular atrophy and interstitial fibrosis, lead to PAX8 expression in the nuclei of proximal tubules. Furthermore, patients with PAX8 detected within the damaged proximal tubuli would be carefully monitored, since deterioration in kidney function was observed during follow-up. We also showed that myoglobin provoked acute kidney injury followed with large extent of renal damage, was associated with strong nuclear expression of PAX8 in proximal tubular cells. These results were supported and followed by data obtained in experimental model of induced postischemic acute kidney injury. Considering these findings, we can assume that PAX8 protein might be involved in regeneration process and recovery after acute kidney injury. Thus, accordingly, all investigation concerning PAX8 immunolabeling should be performed on biopsy samples of the living individuals.

Nonasthmatic Churg-Strauss syndrome superimposed on chronic pyelonephritis: a case report.

Churg-Strauss syndrome (CSS) is a granulomatous small-vessel vasculitis. Asthma is seen in the majority of patients with CSS, but atypical nonasthmatic forms of CSS are also being recognized. We herein describe a 67-year-old woman with a history of chronic pyelonephritis and drug allergy reactions who was admitted to our hospital because of worsening renal function preceded by fever, purpura, sinusitis, and a positive urine culture that confirmed a urinary infection. She was initially treated with pipemidic acid for 7 days, followed by clarithromycin for sinusitis. Laboratory tests on admission showed an absolute eosinophil count of 1750 cells/µL and serum creatinine concentration of 4.72 mg/dL. Urine and blood cultures showed no growth. Kidney biopsy revealed crescent formations with diffuse interstitial fibrosis and foci of eosinophil infiltration. An atypical form of CSS was diagnosed based on tissue eosinophilia, peripheral eosinophilia, and sinusitis. Intravenous methylprednisolone and cyclophosphamide pulse therapy together with hemodialysis treatment improved the patient's clinical condition but did not resolve the kidney damage. The onset of an atypical form of CSS in our patient manifested as symptoms and signs mimicking those of chronic pyelonephritis and drug allergy reactions. The patient's chronic kidney disease finally progressed to dialysis dependence.

Effects of Automation on Sustainability of Immunohistochemistry Laboratory.

The COVID-19 pandemic that hit the world recently caused numerous changes affecting the health system in every department. Reduced staff numbers, mostly due to illness, led to an increase in automation at every stage of laboratory work. The immunohistochemistry (IHC) laboratory conducts a high volume of slide staining every day. Therefore, we analyzed time and total costs required to obtain IHC slides in both the manual and automated way, comparing their efficiency by processing the same sample volume (48 microscope slides-the maximum capacity that an automated immunostainer-DAKO, Autostainer Link 48, Part No AS48030-can process over a single cycle). The total IHC procedure time to run 48 slides manually by one technician was 460 min, while the automated process finished a cycle within 390 min (15.22% less time). The final cost of a single manual IHC slide was 12.26 EUR and 7.69 EUR for slides labeled in the automated immunostainer, which reduced final costs by 37.27%. Thus, automation of the IHC procedure reduces the time and costs of the IHC process, contributing significantly to the sustainability of the healthcare system during the COVID-19 pandemic, overcoming insufficient human resources.

Olive leaf extract attenuates adriamycin-induced focal segmental glomerulosclerosis in spontaneously hypertensive rats via suppression of oxidative stress, hyperlipidemia, and fibrosis.

Olive (Olea europaea L.) leaf extract (OLE) possesses powerful antioxidant, antihyperlipidemic, and anti-inflammatory properties. The aim was to investigated the effects of OLE on the hyperlipidemia, antioxidant defense, heme oxygenase/biliverdin reductase (HO/BVR) pathway, inflammation, and fibrosis in spontaneously hypertensive rats with focal segmental glomerulosclerosis (FSGS, a progressive form of chronic kidney disease) induced by adriamycin (2 mg/kg, i.v., twice in a 21-day period). Daily treatment of OLE (80 mg/kg, p.o.) for 6 weeks suppressed protein oxidation and lipid peroxidation (p < .01 and p < .001, respectively), significantly increased antioxidant enzymes activities and normalized antioxidant capacity, leading to the improvement of antioxidant defense independently of the HO/BVR pathway. Furthermore, the values of triglycerides (p < .01), total, and low-density lipoprotein cholesterol (p < .05, both) were improved by OLE. OLE strongly prevented glomerulosclerosis, interstitial inflammation, and fibrosis (renal injury score, FSGS: 8 ± 0.45 vs. FSGS+OLE: 4.20 ± 1.07; p < .01), as evidenced by normalized fibronectin content (p < .001), suppressed interstitial inflammatory cells infiltration and collagen deposition, without changing cytokines expressions. OLE decreased blood pressure with a tendency to reduce urine albumin loss. These data suggest that OLE may be effective in slowing down the progression of FSGS.

PRMT1 expression in renal cell tumors- application in differential diagnosis and prognostic relevance.

BACKGROUND: Protein arginine methyltransferase-1 (PRMT1) is associated with the progression of various tumor types and the process of epithelial to mesenchymal transition (EMT). However, the expression of PRMT1 in renal cell tumors (RCT) is unknown. METHODS: We evaluated PRMT1 immunohistochemical (IHC) expression on tissue microarray (TMA) of 208 specimens of RCT, including clear cell renal cell carcinomas (ccRCC), papillary RCC type I and II (pRCC I and II), chromophobe RCC (chRCC), renal oncocytomas (RO), collecting duct carcinomas - Bellini (CDC) and multilocular cystic renal cell neoplasms of low malignant potential (MLCRN-LMP). Moreover, a subset of ccRCC, pRCC, chRCC, RO were also studied using conventional sections. PRMT1 expression in tumor tissue was compared to the IHC expression of EMT-related transcription factors (ZEB1, RUNX1, and TWIST1) and cell surface markers (ß-catenin, N- and E-cadherin). Additionally, qRT-PCR expression of PRMT1 in ccRCC, pRCC, and chRCC was evaluated and the results were compared to the mRNA PRMT1 transcript profiling data in The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) cohort. RESULTS: PRMT1 immunoreactivity was observed in the majority of ccRCC, RO, all MLCRN-LMP, but in a minority of chRCC (p = 0.044), and it was associated with low grade and low stage ccRCC (p = 0.014; p = 0.044, respectively). ZEB1 immunoreactivity was noted in all RO, in minority of chRCC and neither of MLCRN-LMP (p < 0.001). The majority of PRMT1-negative ccRCC was negative to ZEB1 and showed cytoplasmic expression of TWIST1 (p = 0.028; p < 0.001, respectively). PRMT1 positive ccRCC mostly expressed RUNX1 (p = 0.019). PRMT1 and ZEB1 expression were associated with better cancer-specific survival in patients with ccRCC (p = 0.029; p = 0.009, respectively). In multivariate analysis, ZEB1 expression was an independent prognostic factor for cancer-specific survival (hazard ratio [HR], 0.367; p = 0.026). Significant IHC heterogeneity was observed in PRMT1, ZEB1 and TWIST1 expression (p < 0.001). Homogenous loss of PRMT1 was associated with high grade and high stage ccRCC, while the homogenous loss of PRMT1 and ZEB1 was more frequent in patients who died of ccRCC (p = 0.017; p = 0.040; p = 0.044; p = 0.009, respectively). Relative mRNA-PRMT1 expression in both cohorts was down-regulated in tumor tissue compared to non-tumor parenchyma (p = 0.009). Unlike in our samples, mRNA-PRMT1 expression in the TCGA cohort was not correlated to ccRCC tumor stage or grade. PRMT1, ZEB1, and TWIST1 expression were not associated with EMT related aberrant ß-catenin expression, a gain of N-cadherin or loss of E-cadherin expression. Only RUNX1 was associated with a gain of N-cadherin (p = 0.003). CONCLUSIONS: IHC expression of PRMT1 may be characteristic for low grade and low stage ccRCC, while the homogenous loss of PRMT1 may be significant for high grade and high stage ccRCC. Both, PRMT1 and/or ZEB1 expression, could be associated with better survival of the patients with ccRCC.

Modulation of NCAM/FGFR1 signaling suppresses EMT program in human proximal tubular epithelial cells.

Neural cell adhesion molecule (NCAM) and fibroblast growth factor receptor 1 (FGFR1) cross-talk have been involved in epithelial-to-mesenchymal transition (EMT) process during carcinogenesis. Since EMT also contributes to maladaptive repair and parenchymal damage during renal fibrosis, we became encouraged to explore the role of NCAM/FGFR1 signaling as initiating or driving forces of EMT program in cultured human proximal tubular epithelial cells (TECs). TECs stimulated with TGF-ß1 (10ng/mL) was used as an established in vitro EMT model. TGF-ß1 downstream effectors were detected in vitro, as well as in 50 biopsies of different human kidney diseases to explore their in vivo correlation. NCAM/FGFR1 signaling and its modulation by FGFR1 inhibitor PD173074 (100nM) were analyzed by light microscopy, immunolabeling, qRT-PCR and scratch assays. Morphological changes associated with EMT appeared 48h after TGF-ß1 treatment and was clearly apparent after 72 hours, followed by loss of CDH1 (encoding E-Cadherin) and transcriptional induction of SNAI1 (SNAIL), SNAI2 (SLUG), TWIST1, MMP2, MMP9, CDH2 (N-Cadherin), ITGA5 (integrin-α5), ITGB1 (integrin-ß1), ACTA2 (α-SMA) and S100A4 (FSP1). Moreover, at the early stage of EMT program (24 hours upon TGF-ß1 exposure), transcriptional induction of several NCAM isoforms along with FGFR1 was observed, implicating a mechanistic link between NCAM/FGFR1 signaling and induction of EMT. These assumptions were further supported by the inhibition of the EMT program after specific blocking of FGFR1 signaling by PD173074. Finally, there was evidence for an in vivo TGF-ß1 pathway activation in diseased human kidneys and correlation with impaired renal excretory functions. Collectively, NCAM/FGFR1 signaling appears to be involved in the initial phase of TGF-ß1 initiated EMT which can be effectively suppressed by application of FGFR inhibitor.

Effects of Single and Combined Losartan and Tempol Treatments on Oxidative Stress, Kidney Structure and Function in Spontaneously Hypertensive Rats with Early Course of Proteinuric Nephropathy.

Oxidative stress has been widely implicated in both hypertension and chronic kidney disease (CKD). Hypertension is a major risk factor for CKD progression. In the present study we have investigated the effects of chronic single tempol (membrane-permeable radical scavenger) or losartan (angiotensin II type 1 receptor blocker) treatment, and their combination on systemic oxidative status (plasma thiobarbituric acid-reactive substances (pTBARS) production, plasma antioxidant capacity (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, pABTS), erythrocyte antioxidant enzymes activities) and kidney oxidative stress (kTBARS, kABTS, kidney antioxidant enzymes activities), kidney function and structure in spontaneously hypertensive rats (SHR) with the early course of adriamycin-induced nephropathy. Adult SHR were divided into five groups. The control group received vehicle, while the other groups received adriamycin (2 mg/kg, i.v.) twice in a 21-day interval, followed by vehicle, losartan (L,10 mg/kg/day), tempol (T,100 mg/kg/day) or combined T+L treatment (by gavage) during a six-week period. Adriamycin significantly increased proteinuria, plasma lipid peroxidation, kidney protein oxidation, nitrite excretion, matrix metalloproteinase-1 (MMP-1) protein expression and nestin immunostaining in the kidney. Also, it decreased kidney antioxidant defense, kidney NADPH oxidase 4 (kNox4) protein expression and abolished anti-inflammatory response due to significant reduction of kidney NADPH oxidase 2 (kNox2) protein expression in SHR. All treatments reduced protein-to-creatinine ratio (marker of proteinuria), pTBARS production, kidney protein carbonylation, nitrite excretion, increased antioxidant capacity and restored kidney nestin expression similar to control. Both single treatments significantly improved systemic and kidney antioxidant defense, bioavailability of renal nitric oxide, reduced kMMP-1 protein expression and renal injury, thus retarded CKD progression. Losartan improved blood pressure, as well as tubular injury and restored anti-inflammatory defense by reverting kNox2 expression to the control level. Interestingly, tempol was more successful in reducing systemic oxidative stress, proteinuria, kMMP-1 and glomerulosclerosis. However, combined treatment failed to overcome the beneficial effects of single treatments in slowing down the progression of ADR-induced nephropathy in SHR.

Granulomatous interstitial nephritis associated with influenza A: H1N1 infection--A case report.

INTRODUCTION: The causes of acute tubulointerstitial nephritis can be grouped into four broad categories: medications, infections, immunologic diseases, or idiopathic processes. Here we report a 17-year-old female who developed acute kidney injury (AKI) due to granulomatous interstitial nephritis (GIN) associated with influenza A: H1N1 infection. CASE OUTLINE: The illness presented after two weeks of respiratory tract infection, skin rash and hypermenorrhea. On admission the patient was febrile, with bilateral pedal edema, macular skin rash, and auscultatory finding that suggested pneumonia. Laboratory investigations showed normocytic anemia, azotemia, hematuria and proteinuria. Renal ultrasound was normal. Antinuclear antibodies, antineutrophil cytoplasmic antibodies, lupus anticoagulant, antiphospholipid antibodies were negative with normal complement. Urine cultures including analysis for Mycobacterium tuberculosis were negative. The diagnosis of influenza A: H1N1 infection was made by positive serology. A kidney biopsy showed interstitial nephritis with peritubular granulomas. Glomeruli were normal. Staining for immunoglobulins A, M, G, and F was negative. The girl was treated with oseltamivir phosphate (Tamiflu; Genentech, Inc., South San Francisco, CA, USA) for five days, as well as with tapered prednisone after a starting dose of 2 mg/kg. The treatment resulted in a complete remission during two years of follow-up. CONCLUSION: We present a severe but reversible case of GIN and AKI associated with influenza A: H1N1 infection. Although a causal effect cannot be confirmed, this case suggests that influenza A: H1N1 should be considered in the differential diagnosis of GIN manifested with AKI in children.

Angiotensin 2 type 1 receptor blockade different affects postishemic kidney injury in normotensive and hypertensive rats.

Many studies demonstrated that angiotensin 2 type 1 receptor (AT1R) blockade accelerates renal recovery in post-ischaemic kidney but there are many controversies related to its net effect on kidney structure and function. During the past years, our research group was trying to define the pathophysiological significance of the renin-angiotensin system on post-ischemic acute renal failure (ARF) development in normotensive Wistar as well as hypertensive rats (SHR). This review mostly summarizes our experience in that field. Our previous studies in normotensive rats revealed that AT1R blockade, except slightly renal vascular resistance improvement, had no other obvious beneficial effects, and therefore implies angiotensin 2 (Ang-2) overexpression as non-dominant on kidney reperfusion injuries development. Similarly it was observed in Wistar rats with induced mild (L-NAME, 3 mg/kg b.w.) nitric oxide (NO) deficiency. Expectably, in strong induced (L-NAME, 10 mg/kg b.w.) NO deficiency associated with ARF, massive tubular injuries indicate harmful effects of AT1R blockade, implying strongly disturbed glomerular filtration and suggesting special precaution related to AT1R blockers usage. Opposite to previous, by our opinion, AT1R antagonism promises new advance in treatment of essentially hypertensive subjects who develop ARF. Increased glomerular filtration, diminished oxidative stress, and most importantly improved tubular structure in postishemic SHR treated with AT1R blocker losartan, implicate Ang-2 over production as potently agent in the kidney ischemic injury, partly trough generation of reactive oxygen species. These data contribute understanding the pathogenesis of this devastating illness in hypertensive surroundings.

Immunolocalization of the TASK2 Potassium Channel in Frog Kidney.

TASK2 (K2P5. 1, KCNK5) is a two-pore domain K⁺ channel belonging to the TALK subgroup of the K2P family of proteins. TASK2 expression has been reported in a variety of cells and tissues ranging from kidney to immune cells and including specific neurons, its proposed functions spanning from involvement in the regulation of cell volume to control of excitability. The purpose of this study was to determine the tubule location ofthe TASK2 K⁺ channel protein in frog kidney applying polyclonal antibody against the carboxyl terminus of human TASK2 (KCNK5) protein. Immunohistochemical analysis revealed that TASK2 is expressed on distal tubules and proximal epithelial cells. TASK2 is strongly expressed predominantly on the luminal part ofthe proximal epithelial cells and slightly cytoplasmatic staining is expressed. Distal tubules showed diffuse cytoplasmatic staining as well as slight staining on the apical parts ofthe cells. These findings suggest that the TASK2 K⁺ channel has cell-specific roles in renal potassium ion transport.