Kidney Specimens Research Articles

Deep Learning Tubulometrics to Stratify Tubular Injury in Whole-Slide Kidney Specimens

Deep Learning Tubulometrics to Stratify Tubular Injury in Whole-Slide Kidney Specimens

Silymarin ameliorates diazinon-induced subacute nephrotoxicity in rats via the Keap1-Nrf2/heme oxygenase-1 signaling pathway.

The goal of the current study was to clarify the potential molecular mechanism underlying the protective effects of silymarin (SIL) administration against diazinon-induced subacute nephrotoxicity, with a special emphasis on the role of the Kelch-like-associated protein-1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway in minimizing the oxidative stress induced by diazinon (DZN). Five equal groups of thirty adult male Wistar rats were created at random. Group 1 (G1) was maintained under typical control conditions and administered saline intragastrically (I/G) once daily for 4 weeks; G2 was administered olive oil I/G for 4 weeks; G3 was I/G administered silymarin daily for 4 weeks; G4 was I/G administered diazinon daily for 4 weeks. G5 was I/G administered silymarin daily 1 h before the I/G administration of the diazinon for 4 weeks. Blood samples were collected at the end of the experiment for the determination of complete blood cell count, and kidney function tests. Kidney specimens were collected for the evaluation of the oxidative markers, mRNA gene expression, protein markers, and histopathological examination. SIL reduced the renal dysfunction caused by DZN by restoring urea and creatinine levels, as well as oxidative indicators. Although the expression of Keap-1 was also elevated, overexpression of Nrf2 also enhanced the expression of HO-1, a crucial target enzyme of Nrf2. SIL is hypothesized to potentially aid in the prevention and management of nephrotoxicity caused by DZN.

Specific antigen-based stratification of membranous nephropathy in patients after haematopoietic stem cell allotransplantation - a case series and literature review.

Nephrotic syndrome (NS) is a rare complication that can occur after haematopoietic stem cell transplantation (HSCT). In patients with membranous nephropathy (MN) who have undergone allogeneic HSCT, a new antigen called protocadherin FAT1 has been identified. Our objective is to present a case series of MN patients after HSCT with a novel antigen-based stratification. Patients who developed full-blown NS due to MN after an HSCT were enrolled in the University Hospital Centre Zagreb study. The first two patients were treated with an HSCT for acute myeloid leukaemia, and both developed NS after cessation of graft versus host disease (GVHD) prophylaxis. The first patient had reduced kidney function, while the second had completely preserved function. Kidney biopsy showed MN with only subepithelial deposits. A thorough examination revealed that there was no secondary cause of the disease. The patients achieved complete remission after undergoing immunosuppression treatment. The third patient underwent HSCT for acute lymphoblastic leukaemia. He developed both acute and chronic GVHD and also experienced avascular hip necrosis. After sixteen years, the patient developed NS with preserved kidney function. The kidney specimen showed membranous nephropathy (MN) with mesangial and subepithelial deposits. Extensive research was conducted, but no secondary cause for the MN was detected. All three cases tested negative for anti-PLA2R antibodies. Biopsy tissue samples were analysed using laser microdissection and tandem mass spectrometry of glomeruli for the detection of different specific antigens. Patients one and two tested positive for FAT1, whereas patient three tested positive for PCSK6. MN can develop at various time intervals after HSCT. Specific antigen testing can help establish the relationship between MN and HSCT. In the future, serum testing for anti-FAT1 antibodies in HSCT patients could be significant in diagnosing FAT1-associated MN, similar to how anti-PLA2R antibodies are significant in diagnosing PLA2R-associated MN.

Sodium selenite attenuates inflammatory response and oxidative stress injury by regulating the Nrf2/ARE pathway in contrast-induced acute kidney injury in rats

BackgroundContrast-induced acute kidney injury (CI-AKI) is an acute renal complication that occurs after intravascular contrast agent administration. Sodium selenite (SS) is an inorganic source of Se and has potent antioxidant properties. This study intends to examine its anti-inflammatory and antioxidant effects in CI-AKI.MethodsA rat CI-AKI model was established with the pretreatment of SS (0.35 mg/kg). Hematoxylin-eosin staining was employed for histopathological analysis of rat kidney specimens. Biochemical analysis was conducted for renal function detection. Tissue levels of oxidative stress-related markers were estimated. Reverse transcription-quantitative polymerase chain reaction revealed the mRNA levels of proinflammatory cytokines. Western blotting showed the Nrf2 signaling-related protein expression in the rat kidney.ResultsSS administration alleviated the renal pathological changes and reduced the serum levels of serum creatinine, blood urea nitrogen, neutrophil gelatinase-associated lipocalin, cystatin C, and urinary level of kidney injury molecule-1 in CI-AKI rats. SS attenuated oxidative stress and inflammatory response in CI-AKI rat kidney tissues. SS activated the Nrf2 signaling transduction in the renal tissues of rats with CI-AKI.ConclusionSS ameliorates CI-AKI in rats by reducing oxidative stress and inflammation via the Nrf2 signaling.

A Comparative Histological Study Between the Protective and the Therapeutic Effect of Probiotic on Cisplatin Induced Nephrotoxicity in Adult Male Albino Rat

Abstract Introduction Although cisplatin (CP) is one of the most effective chemotherapies, it is associated with significant nephrotoxicity (Acute kidney injury). Lactobacillus plantarum is often used as a probiotic on its own for its beneficial properties and anti-inflammatory activity. Aim to compare the protective and therapeutic role of probiotics on the microscopic structure of the kidney in a model of cisplatin-induced nephrotoxicity. Material and Methods thirty-five adult male albino rats weighing (200-220gm) were divided into 4 groups. Group I (control group), Group II (Cisplatin group): rats received intraperitoneal injection of CP (7.5 mg/kg) for 3 days. Group III (protective group): Rats received 1 mL of probiotic orally for 7 days. From the fifth day, the rats received intraperitoneal injections of CP (7.5mg/kg) for 3 days. Group IV (Therapeutic group): Rats received intraperitoneal injection of CP for 3 days. From the fourth day, the rats received probiotic L. plantarum orally at a dose of (1 mL/day) for 14 days. The concentration of probiotics in group III and group IV was 109 Colony forming units (CFU)/mL/100 g of body weight. Kidney specimens were proceeded for light microscopic examination, as well as Morphometrical and statistical analyses. Results H&E-stained sections of Group II showed marked renal affection in which most glomeruli showed atrophy. Renal tubules were mostly distorted, necrotic, and degenerative with interstitial hemorrhage, congestion, and cellular infiltration. PAS-stained sections showed marked increased reaction in glomerular and tubular Basement membranes with an apparent loss of Brush borders of most of the PCTs. Masson`s trichrome-stained sections showed a marked increase in collagen fibers in renal interstitia mostly around blood vessels. Immunohistochemically stained sections showed a marked increase in positive cytoplasmic reaction to caspase -3 antibodies mostly in renal tubules. On the other hand, the renal structure of The protective group showed marked improvement which appeared nearly comparable to that of the control group while the therapeutic group showed minimal improvement. Conclusion The protective effect of probiotics on cisplatin-induced Acute Kidney Injury (Nephrotoxicity) was markedly better than the therapeutic effect as detected by microscopic, morphometric, and statistical studies.

Renal alterations in cats (Felis catus) housed in shelters and affected by systemic AA-amyloidosis: Clinicopathological data, histopathology, and ultrastructural features.

AA-amyloidosis is frequent in shelter cats, and chronic kidney disease is the foremost cause of death. The aims were to describe kidney laboratory and microscopic findings in shelter cats with AA-amyloidosis. Cats were included if kidney specimens were collected post-mortem and laboratory data were available within 6 months before death. Renal lesions were evaluated with optical and electron microscopy. Mass spectrometry was used to characterize amyloid. Nine domestic short-hair cats were included; 4 females and 5 males with a median age of 8 years (range = 2-13). All cats had blood analyses and urinalyses available. Serum creatinine concentrations were increased in 6 cats and symmetric dimethylarginine was increased in all of the cats. All of the cats had proteinuria. Eight of 9 cats had amyloid in the medulla, and 9 had amyloid in the cortex (glomeruli). All cats had amyloid in the interstitium. Six cats had concurrent interstitial nephritis and 1 had membranoproliferative glomerulonephritis. All cats had extrarenal amyloid deposits. Amyloid was AA in each case. In conclusion, renal deposition of amyloid occurs in both cortex and medulla in shelter cats and is associated with azotemia and proteinuria. Renal involvement of systemic AA-amyloidosis should be considered in shelter cats with chronic kidney disease. The cat represents a natural model of renal AA-amyloidosis.

#1524 SKLB023 alleviates kidney inflammation and apoptosis via inhibiting TLR4-mediated NF-κB p65 and MAPK signaling pathways in septic AKI mice

Abstract Background and Aims Sepsis is a severe and complex clinical syndrome resulting from the host's inflammatory response to infection. Acute kidney injury (AKI) is one of the major complications of sepsis with high morbidity and mortality, namely sepsis-associated acute kidney injury (S-AKI). Despite current advances, effective drugs for treatment of S-AKI are scarce. SKLB023 is a small molecule compound designed based on 5-benzylidene-thiazolidine-2,4-dione, which has a potent anti-inflammatory effect with favorable efficacy in rheumatoid arthritis. This study aims to evaluate the efficacy of SKLB023 in treating S-AKI and to explore the underlying mechanisms. Method S-AKI was induced by cecal ligation puncture (CLP) and intraperitoneal injection of LPS (10 mg/kg) in male C57BL/6 mice. SKLB023 (25 mg/kg, 50 mg/kg) was administrated by gavage three days in advance and 30 minutes earlier on the day of modeling. Mice death per group was recorded for 100 hours after CLP to evaluate the effect of SKLB023 on the survival rate of S-AKI mice. Other mice were sacrificed 16 hours after model establishment, blood and kidney specimens were collected. Renal function was measured by the detection of serum creatinine (SCr) and blood urea nitrogen (BUN). Kidney pathological injury was evaluated by the expression of kidney injury markers and the assessment of PAS/HE stainings. In vitro experiments were performed with LPS stimulation of TCMK-1 cells and SKLB023 intervention, and the doses were determined by CCK-8 assay. Possible molecular mechanisms involved in SKLB023 against S-AKI were screened by bioinformatics analysis and further validated in vivo and vitro. RT-PCR, western blot, immunofluorescence, and immunohistochemistry were applied to detect renal inflammation and apoptosis. To determine the role of TLR4, TLR4 siRNA and TLR4-KO mice were applied in S-AKI model, and TLR4 expression and the activities of NF-κB p65 and MAPK signaling pathway were assessed. One-way ANOVA was performed for comparison between groups, and logrank analysis was used for comparison of survival curve. P < 0.05 was considered as statistically significant. Results The S-AKI model was successfully established by CLP and LPS injection with obvious renal dysfunction and kidney pathological damage. The mice administrated with SKLB023 had a higher survival rate, lower levels of Scr and BUN, and improved kidney pathological injury than the mice in the CLP group. Transcriptomic analysis identified TLR4-mediated downstream signaling pathways as the potential mechanism of SKLB023 and further experiments in vivo and in vitro proved that SKLB023 regulated the expression of TLR4 and the activities of NF-κB p65 and MAPK signaling pathway in S-AKI mice and LPS-stimulated TCMK-1 cells. Moreover, TLR4 inhibited the expression of inflammatory cytokines including TNF-α, IL-1β, IL-6, iNOS, COX-2, HMGB-1 and alleviated kidney apoptosis in S-AKI mice and LPS-treated TCMK-1 cells. Additionally, knockout of TLR4 remarkably improved inflammation, apotosis, and kidney injury in LPS-treated mice and TCMK-1 cells. Conclusion SKLB023 confers renoprotective effects in S-AKI by modulating inflammation and apoptosis. TLR4 is a key target in S-AKI, and SKLB023 inhibits inflammation and apoptosis by inhibiting the activities of TLR4 mediated NF-κB p65 and MAPK pathways. SKLB023 may be proposed as one of the potential preventive and therapeutic agents for S-AKI.

#2023 The role of neutrophil extracellular traps in cisplatin-induced acute kidney injury

Abstract Background and Aims Acute kidney injury (AKI) is a public health problem that seriously endangers human health. The exact pathogenesis of AKI has not been fully elucidated. Previous studies have shown that the immune system plays a key role in the inflammatory response of AKI in the acute phase, especially innate immunity. Recent discoveries have highlighted that neutrophils, in particular the neutrophil extracellular traps (NETs), have central roles in the occurrence and development of AKI. However, the specific role and mechanism of NETs in AKI of different etiologies are remain unclear. This study aimed to investigate the role of NETs in the development of cisplatin-induced AKI and provide potential therapeutic targets for AKI treatment. Method Wild-type (WT) and NETs-deficient (PAD4-/-) mouse models of AKI were constructed by intraperitoneal injection of cisplatin. Mice were weighed at 24, 48, and 72 hours after modeling, and sacrificed at 72 hours. Serum and kidney specimens were collected for renal function analysis, pathological, immunohistochemical staining, and molecular biology experiments. Mouse bone marrow neutrophils were isolated from healthy mouse. NETs were induced in vitro by adding phorbol 12 myristate 13-acetate (PMA). Primary renal tubular epithelial cells (TECs) of mouse kidney were extracted, and the cells were collected after 24 h of cisplatin and cisplatin combined with NETs intervention, respectively. The inflammatory indicators such as TNF-α, IL-1β, MCP-1, and renal injury indicators such as KIM-1 and Ngal were detected by RT-PCR. Results Compared with the WT-cisplatin group, the PAD4-/- -cisplatin group showed a reduced degree of weight loss after modeling, lower serum creatinine and urea nitrogen levels, and significantly improved renal function. The PAD4-/- -cisplatin group showed a significant decrease in the inflammatory indicators TNF-α, IL-1β, IL-6, and renal injury indicators KIM-1 and Ngal. Cisplatin intervention for 6 hours could induce the generation of NETs. Compared with the cisplatin intervention group, the expression of inflammatory indicators TNF-α, IL-1β, and MCP-1 were significantly higher in the cisplatin combined with NETs intervention TECs group. Conclusion Cisplatin directly induces neutrophils to generate NETs, which can exacerbate cisplatin-induced injury to TECs in vitro. The inhibition of NETs by PAD4-/- improves cisplatin-induced AKI. The inhibition of NETs may be a novel target for the treatment of cisplatin AKI.

#2641 MiR-200b induces ZEB1 mediated upregulation of Matriptase and affects expression of podocin

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and a predominant reason for renal failure. Evidence from previous research indicates that abnormal expression of microRNAs (miRs) links closely to the occurrence and progression of DKD. Methods Expression of miRs in urine samples from DKD patients and in cultured renal cells stressed with transforming growth factor beta 1 (TGF-β1) was evaluated by TaqMan assay. The emerging miR of interest and its downstream targets were analyzed in human renal sections, in cultured human podocytes and in zebrafish. Results Cell free urine screening showed increased abundance of miR-200b in samples from patients with DKD when compared to healthy controls. In situ hybridization confirmed expression of miR-200b in kidney specimens from DKD patients, but was not detected in healthy renal parenchyma. Transcriptome profiling of cultured human podocytes overexpressing miR-200b identified this miR as a regulator of the ZEB1 mediated matriptase (ST14)/ podocin pathway and found TGF- β1 stress to be upstream activators of miR-200b expression in these cells. Immunofluorescence analysis of kidney sections from DKD patients showed increased expression of ST14 and decreased expression of podocin when compared to healthy controls. Furthermore, injection of a miR-200b mimic in zebrafish larvae caused an upregulation of ST14 and was accompanied by phenotypic changes such as edema, proteinuria and podocyte effacement. Conclusion Our results show that miR-200b is expressed in glomeruli and found at elevated levels in urine of DKD patients. Overexpression of miR-200b in cultured podocytes and in zebrafish induces upregulation of matriptase, a serine protease previously shown to cleave podocin. The observed increase in matriptase and decrease in podocin levels in glomeruli from DKD patients, together with the effect of a miR-200b mimic on proteinuria and ultrastructure of the pronephros in zebrafish positions miR-200b as a key regulator that modulates matriptase overactivation in DKD and warrants further studies.

#439 Nephron number determination in whole slide mice kidney specimens using a deep learning assisted disector/fractionator approach

Abstract Background and Aims Nephron number highly varies between different species and within species, ranging in humans from 200 000 to 2 000 000 nephrons per kidney. Low nephron numbers can promote early onset of end-stage kidney disease. Nephron assessment is highly needed for better diagnosis and more targeted treatment. However, its assessment in vivo is not established in clinical practice yet. Pioneer studies suggest nephron number estimation in vivo through a combination of glomerular densities in kidney biopsies together with medical imaging. In contrast, post-mortem nephron assessment either using optical clearing or by exhaustive kidney sectioning is well studied but remains laborious. Here, we propose a new semi-automated nephron counting approach using serial PAS-stained whole slide kidney sections, deep learning segmentation and slide registration. Method Mice were sacrificed, kidneys harvested, measured, halved in the coronary plane and then opposingly embedded in paraffin. Subsequently, the entire kidney was sectioned with 10 µm thickness per slide. Tissue was stained with PAS and slides were digitalized using Aperio GT 450 DX scanner (Leica, Wetzlar, Germany) (Fig. a). Deep learning segmentation of glomeruli was performed in the first and every 10th (reference) section and subsequent (look-up) section using Tensorflow. Obtained segmentation results were manually quality controlled and adjusted, if necessary, in QuPath (Fig. b). Final segmentations of the look-up slides were registered to the reference slides in ImageJ using manual landmarks with the ImageJ BigWarp plugin (Fig. c). Glomeruli being present either in the look-up slide, or in the reference slide were counted as disector particles (Q-). Kidney areas on every examined slide were measured. Total number of glomeruli and total kidney area were determined as previously described (Fig. d). Results A kidney from a 12-week-old mouse suffering chronic kidney disease (mouse line Alb-creERT2 (ki/ki); Glut9 (fl/fl)) was used. Kidney weight was 162 mg, length 11 mm, width 6 mm, depth 4 mm and kidney volume 260 mm³. In total the kidney was sectioned in 95 slices. 20 reference sections and its respective look-up sections were stained and digitalized. In total 5981 glomeruli were segmented in 40 kidney slices using the deep learning algorithm. Glomerular segmentation quality was verified by two independent observers (mean dice score 0.79). Inaccuracies, if any, where manually corrected. For image registration in average 18.1 landmarks per slide were created within the ImageJ BigWarp plugin. Registration took in average 5 minutes per slide. Glomeruli without match (disector particles, Q-) were automatically counted. Number of disector particles (Q-) ranged from 32 to 244 per slide. Total nephron number was 11.085 and kidney area 210 mm³. The differences to the manual measured volume (260 mm³) might be explained by discard of small kidney fractions for the used sectioning technique. Conclusion We report, to our knowledge, the first deep learning assisted disector/fractionator approach to determine nephron number in whole slide kidney specimens. This method requires no physical disector setup, is scalable, cheap, time efficient, and unbiased. The method was so far validated in a mouse model of chronic kidney disease. The method will further be validated on different animal models. Additionally, occurrence of atubular glomeruli will be considered in nephron number determination. Furthermore, the integration of GFR measurements might allow the calculation of mean single nephron GFR and might together with automated glomerular area measurements unravel pathophysiologic processes in kidney ageing and disease.

Early growth response protein 2 promotes partial epithelial-mesenchymal transition by phosphorylating Smad3 during renal fibrosis

Chronic kidney disease (CKD) is a serious health problem worldwide, which ultimately leads to end-stage renal disease (ESRD). Renal fibrosis is the common pathway and major pathological manifestation for various CKD proceeding to ESRD. However, the underlying mechanisms and effective therapies are still ambiguous. Early growth response 2 (EGR2) is reportedly involved in organ formation and cell differentiation. To determine the role of EGR2 in renal fibrosis, we respectively confirmed the increased expression of EGR2 in kidney specimens from both CKD patients and mice with location in proximal tubules. Genetic deletion of EGR2 attenuated obstructive nephropathy while EGR2 overexpression further promoted renal fibrosis in mice subjected to unilateral ureteral obstruction (UUO) due to extracellular matrix (ECM) deposition mediating by partial epithelial-mesenchymal transition (EMT) as well as imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs). We found that EGR2 played a critical role in Smad3 phosphorylation, and inhibition of EGR2 reduced partial EMT leading to blockade of ECM accumulation in cultured human kidney 2 cells (HK2) treated with transforming growth factor β1 (TGF-β1). In addition, the transcription co-stimulator signal transducer and activator of transcription 3 (STAT3) phosphorylation was confirmed to regulate the transcription level of EGR2 in TGF-β1-induced HK2 cells. In conclusion, this study demonstrated that EGR2 played a pathogenic role in renal fibrosis by a p-STAT3-EGR2-p-Smad3 axis. Thus, targeting EGR2 could be a promising strategy for CKD treatment.

A Novel Monoallelic ALG5 Variant Causing Late-Onset ADPKD and Tubulointerstitial Fibrosis

IntroductionMonoallelic variants in the ALG5 gene encoding asparagine-linked glycosylation protein 5 homolog (ALG5) have been recently shown to disrupt polycystin-1 maturation and trafficking via underglycosylation, causing an autosomal dominant polycystic kidney disease-like (ADPKD-like) phenotype and interstitial fibrosis. In this report we present clinical, genetic, histopathologic, protein structure and functional correlates of a new ALG5 variant, p.R79W, that we identified in two distant genetically related Irish families displaying an atypical late-onset ADPKD phenotype combined with tubulointerstitial damage. MethodsWhole exome and targeted sequencing were used for segregation analysis of available relatives. This was followed by immunohistochemistry examinations of kidney biopsies, and targeted (UMOD, MUC1) and untargeted plasma proteome and N-glycomic studies. ResultsWe identified a monoallelic ALG5 variant [GRCh37 (NM_013338.5): g.37569565G>A, c.235C>T; p.R79W] that co-segregates in 23 individuals, of whom 18 were clinically affected. We detected abnormal localization of ALG5 in the Golgi apparatus of renal tubular cells in patients’ kidney specimens. Further, we detected the pathological accumulation of uromodulin, an N-glycosylated GPI-anchored protein, in the endoplasmic reticulum, but not mucin-1, an O- and N-glycosylated protein. Biochemical investigation revealed decreased plasma and urinary uromodulin levels in clinically affected individuals. Proteomic and glycoproteomic profiling revealed the dysregulation of chronic kidney disease-associated proteins. ConclusionALG5 dysfunction adversely affects maturation and trafficking of N-glycosylated and GPI anchored protein uromodulin, leading to structural and functional changes in the kidney. Our findings confirm ALG5 as a cause of late-onset ADPKD and provide additional insight into the molecular mechanisms of ADPKD-ALG5.

DETEKSI YERSINIA PESTIS DAN PATOGEN ZOONOTIK LAINNYA PADA TIKUS DI DAERAH FOKUS PES DESA KAYUKEBEK KABUPATEN PASURUAN

Rats are known as carriers and transmissions of zoonotic diseases. About 60 types of zoonotic diseases that rats can transmit to humans. Some zoonotic diseases are Plague, Leptospirosis, Orthohantavirus infection, and Hepatitis E virus. This research aims to detect the bacteria Yersinia pestis, Leptospira, Hepatitis E virus, and Orthohantavirus in rats in Kayu Kebek Village, Pasuruan Regency. The research design used in this study is descriptive with a cross-sectional approach. The rats were captured for three days, two nights in the house, the neighborhood around the house, and the forest. The captured mice were identified as specimens of blood, spleen, kidneys, and liver. Detection of Yersinia pestis, Leptospira, and Hepatitis E is performed using PCR, while the detection of Orthohantavirus is performed using the ELISA method. A total of 45 mice were trapped, consisting of 2 genera and three species. The zoonotic pathogens detected were Leptospira and hepatitis E. Leptospira viruses were detected in Rattus tanezumi, Rattus tiomanicus, and Niviventer fluvescent. Hepatitis E virus is detected only in R. tanezumi. Transmission of leptospirosis and hepatitis E has the potential to occur in Kayu Kebek Village. Rat control is needed to prevent the transmission of leptospirosis and hepatitis E virus infection, and other zoonotic diseases transmitted by rats.

Abstract 3283: Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib resistance

Abstract Introduction: Despite numerous available treatments, metastatic renal cell carcinoma (mRCC) remains highly lethal, with over 90% of patients succumbing within 5 years. Sorafenib (SF), a second-line treatment for mRCC, exhibits modest efficacy, and the underlying molecular basis for its failure is elusive. Hyaluronic acid (HA), implicated in promoting cell proliferation, invasion, and motility through CD44 and RHAMM receptors, represents a potential player in SF resistance. Hymecromone (HC), a non-toxic dietary supplement, is known to inhibit HA synthesis and downregulate CD44 and RHAMM mRNA levels. This study investigates the elusive mechanisms of SF resistance and evaluates the effectiveness of the HC+SF combination as a targeted therapy for advanced RCC, providing insights into a novel and potentially impactful treatment approach. Methods: HA levels were measured in normal and tumor kidney specimens using ELISA-like assay and IHC. Similarly, HAS3 and UGT1A9 (A9) levels and SF glucuronidation were measured by qPCR and a glucuronidation assay using tumor microsomes. Response to SF+HC combination was examined in A9 and HAS3 knockdown and overexpression cell lines (transfectants) and primary RCC spheroids by proliferation, apoptosis, motility, and invasion assays. Target analysis was performed by immunoblot. Treatment efficacy was evaluated in subcutaneous and orthotopic xenograft mouse models, with bioluminescence imaging. Results: SF failure in mRCC was linked due to its glucuronidation and inactivation by A9. Tumor microsomes from patients who developed mRCC glucuronidated SF 5-fold higher than from non-mRCC patients. A9 levels were elevated (8-10 folds) in mRCC patients’ tumor along with HA and HAS3 (5-10 folds) as compared to non-mRCC patients and normal kidney tissues. HC treatment downregulated A9 promoter activity and inhibited SF-glucuronidation in RCC cells. SF (5uM) and HC (20, 40ug/ml) combination inhibited RCC cell and primary tumor spheroid proliferation (>90%), motility/invasion (>80%), and induced apoptosis (5-fold) and downregulated HAS3 expression (>80%), and HA synthesis in RCC cells. While A9 and HAS3 transfectants both were resistant to SF+HC treatment, A9 and HAS3 - KO transfectants were sensitive to SF. Endothelial cells co-cultured with either A9 or HAS3 transfectants were resistant to SF+HC treatment. SF+HC upregulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not in A9 or HAS3 transfectants. HC (100 or 200mg/kg) and SF (30mg/kg) oral combination abrogated Caki-1 tumor growth (>80%), without serum/tissue toxicity. HC+SF treatment decreased A9, HA and HAS3 levels, Ki67 index and microvessel density. Both A9 and HAS3 overexpressing tumors were resistant to treatment. Conclusion: This study demonstrates the mechanism of SF failure in mRCC and proposes a safe, oral adjunct combination for effective treatment. Citation Format: Anuj Kumar Sharma, Karina Aguilar, Chandramukhi S. Panda, Vinata B. Lokeshwar. Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3283.

Abstract 1163: Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment

Abstract Immunotherapy (IO) has improved the survival of patients with clear cell renal cell carcinoma (ccRCC); however, most patients will not have a durable complete response and will die from their disease. Our understanding of the development of IO resistance for ccRCC is very limited. We employed spatial transcriptomics (ST) with single cell resolution on patients with IO naïve tumors and IO exposed tumors to characterize spatial properties and differences between these two populations. Fourteen matched tumor and adjacent stromal fields of view (FOV) from surgically resected primary kidney specimens were analyzed using CosMx Spatial Molecular Imager (SMI). Non-tumor cell phenotyping was performed using the Kidney Cell Atlas. A generalized linear model was developed to identify malignant tumor cells in kidney tissues and validated by a pathologist. Differential cell abundance, clustering, and gene expression for each cell phenotype were compared between the two groups. Spatial gene set enrichment (GSEA) of modified hallmark gene sets were also compared. CD8+ T cells were more abundant in IO exposed vs IO naïve tumor FOVs. YES1 expression in the malignant tumor cells was significantly higher in IO exposed samples compared to IO naïve samples (false discovery rate [FDR] = 0.084). Spatial GSEA showed significant clustering of many gene sets (cells of high enrichment closer together than expected by chance), most notably epithelial-mesenchymal transition (EMT) pathway. Next, a post-hoc analysis of genes belonging to known ligand-receptor pairs in the EMT pathway was completed using bivariate Moran’s I to estimate spatial autocorrelation. We found significantly higher spatial autocorrelation of ITGAV-COL4A1 in the stroma of IO exposed FOVs than IO naïve FOVs (FDR = 0.008). Cell types with the highest expression of ITGAV and COL4A1 were found to be fibroblasts, endothelial, and tumor cells. Protein validation with multiplex immunofluorescence (mIF) confirmed cells positively stained for anti-integrin alpha-V were more abundance on stromal FOVs (p < 0.001) in IO exposed samples. Fibroblast (SMA+) cells in stromal FOVs were more likely to be positively stained for anti-integrin alpha-V (p < 0.001) in IO exposed than IO naïve FOVs. ITGAV protein is involved in migration from primary to metastatic disease in several cancers, and its inhibition increases T cell killing indicating ccRCC endothelial and tumor cells may be using this signaling for immune evasion and disease progression. Further, collagen and cancer associated fibroblasts (CAFs) play a role in tumor growth. To our knowledge, this is the first study to leverage single-cell resolution ST to identify potential ways by which ccRCC tumors become resistant to IO therapy. Citation Format: Alex C. Soupir, Mitchell T. Hayes, Taylor C. Peak, Oscar E. Ospina, Nicholas H. Chakiryan, Anders E. Berglund, Paul A. Stewart, Jonathan Nguyen, Carlos M. Moran-Segura, Natasha L. Francis, Paola M. Ramos-Echevarria, Jad Chahoud, Roger Li, Kenneth Y. Tsai, Jodi A. Balasi, Yamila Caraballo-Perez, Jasreman Dhillon, Lindsey A. Martinez, Warren E. Gloria, Nathan Schurman, Sean Kim, Mark Gregory, James J. Mule, Brooke L. Fridley, Brandon J. Manley. Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1163.

Prolonged Formalin Exposure on Liver and Kidney Function Associated with TP53 Gene Expression in Quail Birds (Coturnix coturnix)

Background: This study was the first to highlight the deleterious effect of formalin gas on gene expression of the tumor protein TP53 in the digestive and urinary system. This experiment was conducted according to animal ethics and regulations of the College of veterinary medicine/the University of Al-Qadisiyah. Method: Three groups were exposed to two hours of formalin gas twice daily, two hours in the morning and evening, for 30 days, and the fourth group was the control with the same environmental conditions except for formalin. Then, quails were killed on different days (10, 20, and 30 days), and Liver, kidney, and blood specimens were collected for gene expression, histological, and biochemical studies. Result: displayed that formalin gas caused by disturbances in liver and kidney functions, in addition to the great pathological liver and kidney changes, showed acute hepatitis and nephritis. 5% of the formalin gas significantly increased the expression of TP53in the liver and kidney regularly with a period of exposure, Conclusion: that P53 might be considered a suitable biomarker of molecular for formalin impaction in humans and animals especially the birds.

Nephroprotective effect of bone marrow mesenchymal stem cells on cisplatin induced kidney injury in albino rats

isplatin is a chemotherapeutic drug used in the treatment of a variety of cancers, with a known side effect of nephrotoxicity. Using stem cell therapy represents a distinctive and encouraging approach to remediate damaged organs. The administration of bone marrow mesenchymal stem cells (BMMSCs) has the potential to mitigate the adverse effects of cisplatin nephrotoxicity, thus helping with both functional and histological recuperation. Twenty-four mature male albino rats were divided into four groups. 1 ml of normal saline was injected intraperitoneally (I.P.) into the control group. Cisplatin was injected once (6 mg/kg I.P.) into the cisplatin group. 0.5 ml of culture media with 5 x 106 BMMSCs was injected i.p. with 6 mg/kg I.P. cisplatin in the BMMSC group. The withdrawal group received no treatment after cisplatin injections. At different times, groups were sacrificed. Kidney specimens were made for histology and immunohistochemistry. Morphometric and statistical studies were done. Blood urea and serum creatinine were evaluated before sacrifice. There were statistically significant differences between the studied groups regarding markers of incidence of acute tubular necrosis and recovery, suggesting that cisplatin therapy caused acute tubular necrosis, whereas BMMSCs improved renal function markers, including blood urea and serum creatinine levels and tissue restoration. Stem cell rats also showed cluster of differentiation 44 (CD44) in cells near tubules, helping injured kidneys regenerate tubular cells. The use of bone-marrow-derived mesenchymal stem cells (BMMSCs) mitigated the nephrotoxic effects of cisplatin, thus showing a restorative effect on both functional and histological parts.

Renal regenerative capacity related to stem cell reserve in nephrectomized rats.

On the new era of stem cell therapy, the present experimental study was conducted to investigate renal regenerative capacity related to kidney stem cell reserve in different nephrectomy (Nx) models. Three- and eight-week-old rats (n = 168) were randomly divided into four groups to include control and three Nx subgroups (1/6 Nx, 1/2 Nx, and 5/6 Nx) (Fig.1). On post-Nx days 15, 30 and 60, kidney specimens were obtained to determine renal regenerative capacity. The specimens were examined with immunofluorescence. CD90/CD105 and Ki-67 expressions were determined as stem cell and cellular proliferation markers, respectively. Fig.1 Intraoperative photographs showing three different types of nephrectomies (unilateral total Nx has not been shown in 5/6 Nx group) RESULTS: CD90 and CD105 expressions were stronger in glomeruli, but Ki-67 expressions were present only in tubuli. When all Nx types and post-Nx days were considered, both 3- and 8-week-old rats undergone 5/6 Nx had the highest glomerular CD90 and CD105 double expressions. While the expressions gradually increased toward the day 60 in 3-weeks old rats, 8-week-old rats had almost stable double expressions. The strongest tubular Ki-67 expressions were seen in 5/6 Nx groups of both in 3- and 8-week-old rats. The expressions were strongest on day 15 and then gradually decreased. Ipsilateral 1/6 Nx groups had stronger Ki-67 expression than contralateral ones in both age groups. Kidneys may pose a regenerative response to tissue/volume loss through its own CD90- and CD105-related stem cell reserve which mainly takes place in glomeruli and seems to have some interactions with Ki-67-related tubular proliferative process. This response supports that kidney stem cells may have a potential to overcome tissue/volume loss-related damage.

A Case Report of Pulmonary Alveolar Proteinosis with Associated Opportunistic Infection of Pneumocystis jirovecii and Molluscum Contagiosum

Abstract Pulmonary alveolar proteinosis (PAP) is an idiopathic rare diffuse pulmonary disease, first described in 1958 by Rosen et al. Its estimated prevalence is about 1 in 3.7–6.9 × 106 with a male: female ratio of 1:1–2:1. Majority of the patient’s age ranges between 20 and 50 years. PAP on microscopy is characterized by the presence of massive insoluble, amorphous, phospholipid-rich protein deposits in the bronchial and alveolar cavities. Most patients with acquired PAP present with cough and exertional dyspnea. It has been studied that there is increased risk of superinfection in PAP with opportunistic organisms like pneumocystis and vice versa. Definitive diagnosis of Pneumocystis jirovecii pneumonia rests on the demonstration of the organism within the alveoli by special stains like Grocott Methenamine Silver stain. Molluscum contagiosum (MC) is a common superficial skin infection caused by the poxvirus. MC is characterized by painless papules commonly seen in children and immunocompromised individuals. Here, we present a 34-year-old female who had complaints of severe difficulty in breathing and was brought dead to our hospital. On external examination, she had multiple warts over chest, abdomen, and over genitalia. Internal examination was unremarkable. Specimens of kidney, lung, and skin biopsy of genital warts sent for histopathological examination revealed acute tubular necrosis, P. jirovecii with PAP, and MC respectively.

Efficacy of cryodehydration technique in preserving the gross and histoarchitectural details of goat visceral and musculoskeletal specimens.

This study sought to determine the effectiveness of the cryodehydration technique in preserving the morphologic and morphometric attributes of the anatomical specimens of goats. Different anatomical parts of a goat, i.e., heart, lungs, spleen, liver, kidney, and musculoskeletal specimens, were collected and fixed in 15% formalin. Later on, the fixed specimens were cryodehydrated by fast freezing (burning process) and repeated freezing-thawing sessions, followed by wood glue coating. Finally, the macroscopic (i.e., weight, color, texture, odor, and durability) and microscopic characteristics (by routine hematoxylin and eosin staining) of the cryodehydrated specimens were studied. The resultant specimens produced excellent color and texture and were lightweight (60%-80% weight loss), soft, dry, odorless, durable, and easy to handle. The histoarchitectural details of the heart and skeletal muscle were well preserved, while some distinctive alterations were observed in the parenchymatous organs, i.e., breach in cellular integrity, loss of cell cytoplasm, loss of cytoplasmic and nuclear clarity, increased sinusoidal space, dilatation of the renal tubules, and reduction in glomerular size. Nevertheless, the basic histoarchitecture of each specimen was yet to be distinctly identifiable. The current study findings suggest that the cryodehydration technique can preserve gross anatomical features as well as histoarchitectural details and can be an effective tool for facilitating veterinary education and research.