Kidney Sections Research Articles

COMPARATIVE TRANSCRIPTOMIC ANALYSIS IDENTIFIES EPITHELIAL-MESENCHYMAL TRANSITION AND INFLAMMATION AS COMMON PATHOGENETIC MECHANISMS IN HYPERTENSIVE AND DIABETIC NEPHROPATHY

Objective: Type 2 diabetic (T2DN) and hypertensive nephrosclerosis (HN) are the main causes of chronic kidney disease (CKD). To obtain novel insights into pathogenetic mechanisms, potentially suggesting common therapeutic procedures, we comparatively nalysed transcriptomes of kidney biopsies obtained from patients with T2DN or HN and control archived formalin-fixed and paraffin-embedded (FFPE) kidney biopsies. Design and method: FFPE kidney biopsies from adult patients were selected from the Norwegian Kidney Biopsy Registry. Specimens from patients with HN, T2DN and normal-appearing control renal biopsies (Ctrl) (n = 6 for each group) were assessed. Total RNA was extracted from 10 μm whole kidney sections and processed for RNA sequencing, while immunohistochemistry (IHC) was performed on 3-μm tissue sections. Enzyme-Linked ImmunoSorbent Assay (ELISA) targeting the AXL receptor tyrosine kinase was performed on serum from separate cohorts with HN and T2DN/T1DN. Results: Gene expression analysis showed 769 differentially expressed genes (DEG) for T2DN and 284 HN genes (log fold change > 1, adjusted P < 0.05). Only 39 of the HN genes were uniquely DEG compared to T2DN. Cluster analysis on the RNA seq data separated diseased and normal tissues, also with a distinct overlap between HN and T2DN profiles. Detailed analysis of gene expression profiles revealed upregulation of pathways related to partial epithelial to mesenchymal transition, including extracellular matrix organisation in both diseases. AXL and vimentin expression was also enhanced at the protein level in both HN and T2DN biopsies, as detected by immunohistochemistry (IHC). AXL abundance was also increased in serum from T2DN/T1DN and HN patients. Pathways related to inflammation was also overexpressed, particularly related to TH1 and TH2 helper cell pathways. The presence of CD4 and CD8 positive T- cells were confirmed by IHC. Conclusions: Similarities of T2DN and HN proteomes suggest common pathogenetic mechanisms and the possibility to envisage targeted treatments addressing both main causes of CKD.

PodoCount: A Robust, Fully Automated, Whole-Slide Podocyte Quantification Tool

IntroductionPodocyte depletion is a histomorphologic indicator of glomerular injury and predicts clinical outcomes. Podocyte estimation methods or podometrics are semiquantitative, technically involved, and laborious. Implementation of high-throughput podometrics in experimental and clinical workflows necessitates an automated podometrics pipeline. Recognizing that computational image analysis offers a robust approach to study cell and tissue structure, we developed and validated PodoCount (a computational tool for automated podocyte quantification in immunohistochemically labeled tissues) using a diverse data set.MethodsWhole-slide images (WSIs) of tissues immunostained with a podocyte nuclear marker and periodic acid–Schiff counterstain were acquired. The data set consisted of murine whole kidney sections (n = 135) from 6 disease models and human kidney biopsy specimens from patients with diabetic nephropathy (DN) (n = 45). Within segmented glomeruli, podocytes were extracted and image analysis was applied to compute measures of podocyte depletion and nuclear morphometry. Computational performance evaluation and statistical testing were performed to validate podometric and associated image features. PodoCount was disbursed as an open-source, cloud-based computational tool.ResultsPodoCount produced highly accurate podocyte quantification when benchmarked against existing methods. Podocyte nuclear profiles were identified with 0.98 accuracy and segmented with 0.85 sensitivity and 0.99 specificity. Errors in podocyte count were bounded by 1 podocyte per glomerulus. Podocyte-specific image features were found to be significant predictors of disease state, proteinuria, and clinical outcome.ConclusionPodoCount offers high-performance podocyte quantitation in diverse murine disease models and in human kidney biopsy specimens. Resultant features offer significant correlation with associated metadata and outcome. Our cloud-based tool will provide end users with a standardized approach for automated podometrics from gigapixel-sized WSIs.

Evaluation of sub-acute toxicity profile of methanol extract of Adansonia digitata leaves in Wistar rats

This study evaluated the sub-acute toxicity profile of methanol extracts of Adansonia digitata leaves on Wistar rats. Cold maceration method was used to prepare the methanol extract of Adansonia digitata leaves (MEADL). Twenty-four (24) adult male Wistar rats were randomly divided into 4 groups of 6 rats each. Group A (control) received 5% dimethylsufoxide (DMSO) at 5 ml/kg, while groups B -D received MEADL at 100, 200 and 400 mg/kg, respectively. All treatments were administered orally and once daily for 28 consecutive days. The weight, haematological and lipid profile, antioxidant status as well as liver and kidney function tests were evaluated. The extract (200 and 400 mg/kg) caused a significant (p<0.05) reduction in the triacylglycerol and very low density lipoprotein cholesterol levels of the treated groups when compared with the control group. The platelet count of the MEADL treated groups were significantly (p < 0.05) elevated when compared with the control group. The extract did not cause significant (p > 0.05) change in the serum markers of liver and kidney function test, but produced histopathological changes in the kidney and liver section of the treated rats in a dose-dependent manner. These findings justify its use in folkloric medicine for treatment of several diseases, but caution should be exercised as it is associated with histopathological changes in the liver and kidney at high doses.

Enhanced corrosion resistance, antibacterial activity and biocompatibility of gentamicin-montmorillonite coating on Mg alloy-in vitro and in vivo studies

Implant-associate infection (IAI) is a major cause of failure of bone implant materials, and one of the significant challenges in clinical managements. A synergistic coating strategy combining montmorillonite (MMT) sustained release, adsorption of bacteria and gentamicin (GS) bactericidal is proposed herein to tackle infection issues. Surface morphology, microstructure and chemical composition of the samples were investigated using SEM, XRD, FT-IR and XPS. Electrochemical experiments and immersion experiments reveal that corrosion resistance of Mg samples with GS/MMT coatings was higher than that of bare Mg alloy substrate in DMEM solution. In vitro studies demonstrated that the GS/MMT coating had a significant inhibitory effect on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The viability of MC3T3-E1 cells was 92.7% after a co-culturing for 72 h. After a subcutaneous transplantation of 90 days, the survival rate was 100% for GS/MMT-coated Mg alloy specimens with no infection at the implantation sites and no toxic damage to liver, kidney and local muscles pathological sections. This study provides a novel method for the preparation of sustained-release antimicrobial coatings on biodegradable Mg alloys as promising candidates for orthopedic implant materials.

Spatially revealed perfluorooctane sulfonate-induced nephrotoxicity in mouse kidney using atmospheric pressure MALDI mass spectrometry imaging

Perfluorooctane sulfonate (PFOS), an emerging environmental persistent pollutant, has attracted extensive attention due to its potential nephrotoxicity. However, little is known about the spatial variations of lipid metabolism associated with PFOS exposure. In this study, atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-MALDI MSI) was used to reveal the spatial distributions of PFOS and its adverse effect on lipid metabolism directly in mouse kidney sections. We have observed that PFOS accumulated in the renal pelvis and outer cortex regions, with some found in the medulla and inner cortex regions. Hematoxylin and eosin (H&E) staining results also demonstrated that the accumulation of PFOS caused damage to the mouse kidney, which was consistent with AP-MALDI MSI results. Furthermore, a total of 42 lipids were shown to be significantly different in the spatial distribution patterns and variations between control and PFOS exposure mice groups, including the significant down-regulation of lyso-glycerophospholipids (Lyso-GPs), phosphatidic acids (PA), phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS) sphingomyelins (SM) and sulfatides (ST) in renal medulla or cortex region of mouse kidney sections, and remarkable up-regulation of cholesterol and phosphatidylinositols (PI) in the cortex regions of mouse kidney sections. The AP-MALDI MSI provides a new tool to explore spatial distributions and variations of the endogenous metabolites for the risk assessment of environmental pollutants.

The Impacts of Ultra-Slim Plus and Slim Smart on the Morphology of Kidneys and Renal Function of Albino Rats as Weight Reducing Drugs

Aims and objectives: Current study was designed to investigate the effects of two weight reducing drugs i.e. Slim Smart and Ultra-Slim Plus on kidneys and renal function. Experimental Study: This study was conducted in the Experimental Institute (Animal House) of The University of Lahore, Anatomy departments and Biochemistry department from November 2021 to May 2022. A total 25 adult male albino rat were collected and divided them into three different groups. Plain distilled water, Slim Smart and Ultra Slim plus were used in glass bottles. Under an anatomical microscope, the TA index was used to quantify interstitial fibrosis, glomerular hyalinization, vascular alterations, and tubular atrophy. Cell infiltration was computed as a percentage of inflammatory cells / HPF, and the cortical medulla ratio (C / M) was also calculated. SPSS version 20 was used to gather and analyze the raw data. One-way ANOVA was used to compare groups for quantitative variables such as renal cell infiltration. Group (mean ± standard) error was used for this analysis. Post-tacky tests were also used for cortical medullary ratios and (p< 0.05) was considered important. Results: Sections of Albino Rate kidney stained with H&E and PAS and examined histologically revealed mesangial cellular hypertrophy, thickened vascular walls, and lymphocyte infiltration. The results of group-A and group-C were significant but in group-B much inflammation was seen. A slightly significant changes (p< 0.05) were seen in serum creatinine and blood urea levels in different groups. Conclusion: Slim Smart and Ultra Slim Plus induce mesangial hyper cellularity and lymphocyte infiltration, as well as thickening of blood vessel walls. These alterations are consistent with interstitial nephritis, thus individuals with renal problems should use these medications with care. Keywords: Slim Smart, Ultra Slim Plus, Mesangial hyper cellularity, Lymphocyte infiltration, Nephrotoxicity

Tamoxifen Increased Parasite Burden and Induced a Series of Histopathological and Immunohistochemical Changes During Chronic Toxoplasmosis in Experimentally Infected Mice.

The global distribution of breast cancer and the opportunistic nature of the parasite have resulted in many patients with breast cancer becoming infected with toxoplasmosis. However, very limited information is available about the potential effects of tamoxifen on chronic toxoplasmosis and its contribution to the reactivation of the latent infection. The present study investigated the potential effects of tamoxifen on chronic toxoplasmosis in animal models (Swiss albino mice). Following induction of chronic toxoplasmosis and treatment with the drug for 14 and 28 days, the anti-parasitic effects of tamoxifen were evaluated by parasitological assessment and counting of Toxoplasma cysts. In addition, the effects of the drug on the parasite load were evaluated and quantitated using TaqMan real-time quantitative PCR followed by investigation of the major histopathological changes and immunohistochemical findings. Interestingly, tamoxifen increased the parasite burden on animals treated with the drug during 14 and 28 days as compared with the control group. The quantification of the DNA concentrations of Toxoplasma P29 gene after the treatment with the drug revealed a higher parasite load in both treated groups vs. control groups. Furthermore, treatment with tamoxifen induced a series of histopathological and immunohistochemical changes in the kidney, liver, brain, and uterus, revealing the exacerbating effect of tamoxifen against chronic toxoplasmosis. These changes were represented by the presence of multiple T. gondii tissue cysts in the lumen of proximal convoluted tubules associated with complete necrosis in their lining epithelium of the kidney section. Meanwhile, liver tissue revealed multiple T. gondii tissue cysts in hepatic parenchyma which altered the structure of hepatocytes. Moreover, clusters of intracellular tachyzoites were observed in the lining epithelium of endometrium associated with severe endometrial necrosis and appeared as diffuse nuclear pyknosis combined with sever mononuclear cellular infiltration. Brain tissues experienced the presence of hemorrhages in pia mater and multiple T. gondii tissue cysts in brain tissue. The severity of the lesions was maximized by increasing the duration of treatment. Collectively, the study concluded novel findings in relation to the potential role of tamoxifen during chronic toxoplasmosis. These findings are very important for combating the disease, particularly in immunocompromised patients which could be life-threatening.

First report of cyprinid herpesvirus 2 isolated from the golden crucian carp in China

In this study, a virus strain designated GCC2021 was isolated from the golden crucian carp (Carassius auratus red var. ♂ × Cyprinus carpio L. mirror ♀) in Jiangsu Province, China. GCC2021 was propagated in the Gibel carp brain (GiCB) cell line with a typical cytopathic effect. Healthy golden crucian carp was injected with GCC2021 virus solution intraperitoneally, and the symptoms of the artificially infected fish were consistent with those of the naturally diseased golden crucian carps. The mortality was 6.67 ± 3.33% (kidney homogenate group) and 4.44 ± 1.93% (cell culture virus group). The histopathological results indicated systemic inflammation and changes in multiple organs. Electron microscopy observation using ultra-thin sections of the diseased fish kidney revealed a number of spherical particles with capsid that were approximately 110–120 nm in diameter, and the mature virus particle was about 170–220 nm in diameter. The helicase (Hel) gene of GCC2021 was amplified using PCR and sequenced. PCR amplification showed positive results for GCC2021 in the diseased fish, artificially infected fish, and infected GiCB cells. Sequence analysis of the amplification products showed 99.68% identity with the helicase gene of CyHV-2 SY-C1 strain. Phylogenetic analysis of the CyHV-2 ORF25B non-coding region revealed that the isolate clustered with CyHV-2 1301 strain, SY strain, and SY-C1 strain. CyHV-2 copies in different tissues were determined using digital PCR. Viral loads were detected in the liver, spleen, kidney, brain, intestine, and gill, with the highest in the kidney and lowest in the brain. In this study, it was provided solid evidence that CyHV-2 could infect golden crucian carp.

Application of indocyanine green and near-infrared fluorescence imaging for the assessment of peritoneal dialysis-related complications

Background Ultrasound, computed tomographic peritoneography, methylene blue, and peritoneal scintigraphy are commonly used to identify peritoneal dialysis-related complications in clinical settings. This study aimed to investigate the diagnostic value of indocyanine green in peritoneal dialysis-related complications and to study the effect of indocyanine green on residual renal function and peritoneal function.Methods Twenty male Sprague–Dawley rats were used to establish models, including a pleural effusion model (A, n = 4), abdominal hernia model (B, n = 4), subcutaneous leakage model (C, n = 4), and control (D, n = 8). They were injected with a 20 mL mixture of peritoneal dialysate and indocyanine green at varying concentrations prepared for near-infrared fluorescence imaging. We compared the results of near-infrared-I and near-infrared-II imaging. Radiologists evaluated the image quality, morphology, and thickness of the peritoneum, and the residual renal function was assessed using haematoxylin and eosin staining.Results Lesions in each rat model group were observed by changing the body position and imaging parameters. Pathological kidney and peritoneal sections showed no changes. Meanwhile, near-infrared-I fluorescence imaging of ICG has a better signal-to-background ratio than near-infrared-II.Conclusion Near-infrared-I fluorescence imaging of ICG has a better SBR than near-infrared-II and it is sufficient for for diagnosing peritoneal dialysis-related complications and ICG has no impact on residual renal function and peritoneal function. This method has clinical application potential in promptly diagnosing peritoneal dialysis-related complications.

Identification of IQGAP1 as a SLC26A4 (Pendrin)-Binding Protein in the Kidney.

Background: Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl−/HCO3 − exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin. Methods: A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity. Results: The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4’s C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl−/HCO3 − exchanger activity. Conclusion: IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin’s interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states.

MO583: Cafeteria Diet-Induced Obesity Worsens Experimental CKD

Abstract BACKGROUND AND AIMS Obesity is an important risk factor for chronic kidney disease (CKD). It carries hemodynamic and morphologic changes in the kidney resulting in renal function reduction. CKD is associated with glomerulosclerosis and tubulointerstitial fibrosis that involve collagen deposition. The aim of the study was to evaluate the impact of obesity on kidney fibrosis in a model of 5/6th nephrectomized rats fed a cafeteria diet. METHOD Twenty four rats were fed acafeteria diet and 10 rats received a standard diet for 6 months. For each diet group, half of the rats underwent 5/6 nephrectomy (SNx) at 4 months. Expression of collagen type 1, 3, 4 was quantified by immunohistochemistry in kidney sections at 6 months. Adipocyte size was measured in epididymal adipose tissue (eWAT), and the presence of macrophages was determined by CD68 immunostaining. RESULTS Rats fed the cafeteria diet were hyperglycemic and hypertensive. Their body weight was higher than those fed the standard diet. SNx rats had increased serum creatinine levels, which were even higher in the cafeteria group (71 ± 10 µM) compared with standard diet group (54 ± 5 µM) (P < 0.05). Serum creatinine level was comparable in non-nephrectomized animals fed the cafeteria (32 ± 1 µM) or standard diet (29 ± 1 µM). Collagen 1 level in kidney tissue was increased in both SNx groups with no clear effect of the diet (respectively, 7.1 ± 0.6% and 8.9 ± 0.9% of tissue area with the standard and cafeteria diets). Renal expression of collagen 3 and 4 was significantly increased in SNx animals with cafeteria diet (8.6 ± 1.5 and 10.9 ± 1.9% tissue area, respectively) compared with control rats fed the same diet (5.2 ± 0.5 and 6.3 ± 0.6% tissue area, respectively). Adipocyte size of eWAT was significantly increased by cafeteria diet but reduced in nephrectomized rats. In the cafeteria-SNx group, CD68 immunostaining showed a significant increase in macrophage number in kidney (P = 0.01) and a consistent tendency in eWAT (Fig. 1). CONCLUSION Cafeteria diet-induced obesity worsened the 5/6 nephrectomy-induced CKD. The expression of collagens 3 and 4 as well as the number of macrophages were increased in kidneys from the cafeteria SNx group suggesting an exacerbation by pre-existing obesity of CKD-induced renal inflammation and fibrosis.

MO1019: Sigma-1 Receptor Agonists are Protective in a Rat Model of Kidney Transplantation

Abstract BACKGROUND AND AIMS End-stage renal disease affects nearly 2 million people worldwide. The disease is associated with an irreversible deterioration in renal function and can only be treated by dialysis or kidney transplantation (KTx). KTx is associated with better long-term outcomes and quality of life compared with dialysis, but the shortage of donor organs is a serious and unsolved problem. Graft survival is highly dependent on the extent of cold and warm ischemic injury during Tx. We recently described the renoprotective effects of Sigma-1 receptor (S1R) agonist treatment in IRI. Thus, our aim was to develop a novel preservation solution that, with the addition of S1R agonist compounds, minimizes ischemic damage in order to improve the condition of grafts and so increase the number of organs suitable for Tx. METHOD Kidneys of male Wistar rats were perfused and placed in ice cold (i) custodiol preservation solution; custodiol containing S1R agonists, (ii) fluvoxamine or (iii) SA-4503 for 2 h, then autotransplanted and sacrificed 24 h after reperfusion. Sham-operated rats served as controls. In a second experiment, kidneys of wild-type and S1R knockout mice were perfused and placed in an ice-cold preservation solution containing an original, selective S1R agonist compound (VCC) for 24 h of cold ischemia and tissue samples were collected. Renal function parameters were determined. Renal expression of tubular injury markers (Kim-1, Ngal) and inflammatory cytokines (Il-1α, Il-6, Tnf-α, Mcp-1) were measured. Periodic acid-Schiff staining was performed on kidney tissue sections to evaluate structural changes. CD45 immunostaining was performed on kidney sections to determine the extent of leukocyte infiltration. DNA fragmentation resulted by apoptotic events in the kidney was evaluated by TUNEL-assay. RESULTS S1R agonists mitigated renal functional impairment and tubular dilatation following Tx. Expression of early and sensitive tubular injury markers was markedly less elevated in S1R agonist-treated kidneys. S1R agonists alleviated renal apoptosis as shown on TUNEL-stained kidney sections. Decreased numbers of CD45 + leukocytes and decreased inflammatory cytokine expressions confirmed the anti-inflammatory effect of S1R agonists. The S1R agonist VCC compound mitigated cold ischemic structural kidney damage in wild-type but not in S1R KO mice, which confirms the protective role of the receptor. CONCLUSION The addition of S1R agonists to the preservation solution during Tx improves graft function and alleviates structural damage, thus improving long-term outcomes. S1R agonists reduce graft injury during cold storage, therefore the number of transplantable donor organs can be increased. FUNDING OTKA PD-131 637; FK-124 491; 2020–4.1.1.-TKP2020-6 183 069 269; 2020–4.1.1.-TKP2020-6 183 169 273; KDP-2020/1 019 145.

MO012: Development of an Accurate Automated Segmentation Algorithm to Measure Total Kidney Volume in ADPKD Suitable for Clinical Application (The Cystvas Study)

Abstract BACKGROUND AND AIMS A major barrier to the routine adoption of total kidney volume (TKV) as a clinical biomarker of disease for autosomal dominant polycystic disease (ADPKD) is the significant human operator time required even by experienced analysts (typically, 45–90 min per patient). Several groups have investigated automated and semi-automated kidney segmentation methods to either reduce or eliminate the human interaction required. However, such tools have mostly been developed using data from single centers, which may not translate well to other centers. To date, there has been little attempt to develop or validate algorithms using multi-center and multi-scanner data. Here, we report an automated segmentation tool capable of high performance across different patient populations and scanner sequences using 1.5 T MRI data from four centers (the CYSTic consortium). The algorithm was subsequently tested in a separate clinical cohort to assess its likely performance during routine clinical use. METHOD All 1.5 T studies from the CYSTic trial were downloaded (acquired from Siemens Avanto, GE Optima and Siemens Aera, using different sequences). Cases with poor image quality or with sections of kidney missing from the field of view were excluded. A single, experienced operator selected the most appropriate image series for segmentation and manually segmented each patient's kidneys using a commercial software program (MIM Encore). There were 454 kidneys segmented from 227 scans. These data were used for algorithm training and validation. In addition, 48 routine clinical scans from the Sheffield 3D Lab were extracted from the archives along with their original segmentations (performed by six different analysts), to use as a test set. None of the patients in the clinical test set were included in the training set. An ensemble U-net algorithm was created using the nnUNet approach Isensee et al. (nnU-Net: a self-configuring method for deep learning-based biomedical image segmentation. Nat Meth 2021; 18(2): 203–211), whereby the CYSTic data were used in a 5-fold cross-validation, with stratification across the four centers (i.e. each center contributed 80% of the available data to algorithm training in each fold). Algorithm training proceeded according to the standard heuristic nnUnet functions, using a 3D architecture, for 100 epochs. Segmented kidneys were split into left and right sides during post-processing, through analysis of the position of the center of gravity of segmented regions. Once trained, the five algorithms from cross-validation were applied in an ensemble to the clinical test cohort. RESULTS In both cross-validation and clinical testing phases, the median DICE score was 0.96 for each kidney (IQR of 0.95–0.97 in cross-validation on both sides, And 0.95–0.97 on the left side for clinical testing and 0.96 for the right). The median total kidney volume error was −0.46% (−2.02 to 1.27) for the left side in cross-validation and −0.82% (−2.55 to 0.86) for the right. In the clinical testing phase, the median volume errors were −1.8% (−3.69 to 1.29), left and −1.79% (−3.95 to 0.65), right. The mean time taken to manually segment kidneys in the CYSTIc dataset was 54 min per scan (SD of 31 min). Use of the algorithm as a first pass segmentation, with subsequent checking and editing by an operator, would significantly reduce human input time to a few minutes per case CONCLUSION Our new algorithm demonstrates high accuracy compared to the gold standard of manual TKV segmentation and performs well in a wide range of patients with ADPKD imaged using different scanners at several European centers. Its high performance in a real-world clinical dataset demonstrates that such tools can provide a reliable means of measuring TKV in routine practice and reduces the previous barrier of analyst time and experience.

MO448: Progressive Tubular Injury Caused by High Phosphate Intake is Associated With Activation of STAT3/KIM-1 Signalling and Macrophage Recruitment in Mice

Abstract BACKGROUND AND AIMS Dietary phosphate intake greatly exceeds the recommended daily allowance in the Western population. Elevated phosphate levels are linked to increased cardiovascular and all-cause mortality, impaired bone health and premature ageing. The renal effects of chronic high dietary phosphate intake for healthy individuals are still not clear. METHOD Male C57BL/6 mice received a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD) for 1 to 6 months. We collected blood, urine and kidneys to investigate phosphate metabolism and progression of kidney injury. In vitro analysis for the toxic effects of phosphate or the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor (FGF) 23 was done in HK-2 humane proximal tubule (PT) cells. RESULTS HPD in mice caused hyperphosphataemia despite increased phosphaturia. Both intact FGF23 and PTH plasma levels were significantly increased in mice on HPD at 1 month onward. Mice on HPD showed significantly increased creatinine levels and albuminuria. Histopathological analysis of the kidneys revealed increased PT injury in mice receiving HPD compared to NPD controls from 2 months onward followed by a rapid progression of organ damage from month 5 to 6. Picrosirius red staining of kidney sections showed a progressively increased accumulation of collagen fibers in renal cortex of mice on HPD, which was significant from 4 months onward and accompanied by enhanced expression of transforming growth factor-β1, collagen 1 and fibronectin. The significant induction of the tubular injury marker Havcr1, encodes for Kim-1, started already after 1 month of feeding HPD, progressed over time and was strongest after 6 months. Immunofluorescence staining revealed Kim-1 localization to damaged PT, which was most prominent after 3 and 6 months of HPD. Renal Kim-1 protein levels were positively associated with tubular injury score and tubulointerstitial fibrosis. Histological staining and quantification revealed significantly increased number of pStat3+ cells in PTs of HPD-fed mice at months 1, 2, 5 and 6, indicating activated Stat3 signaling. The number of pStat3+ cells was positively associated with Kim-1 synthesis. Inflammation cytokine array with kidney tissue lysates showed, among others, enhanced expression of monocyte chemoattractant protein (MCP)-1 and macrophage colony-stimulating factor (MCSF) in HPD-fed mice. The mRNA expression of Csf1 (encodes for MCSF) was upregulated in HPD-fed mice and most pronounced after 6 months. Ccl2 mRNA expression (encodes for MCP-1) was significantly enhanced after 3, 5 and 6 months of HPD compared to NPD. Immunofluorescence staining showed a strong and specific induction of MCP-1 in PT epithelial cells due to HPD. The HPD-induced Ccl2 expression was positively associated with Kim-1 expression indicating an interaction between pStat3/Kim-1 signalling pathway and MCP-1. Immunohistochemical staining and flow cytometry analysis revealed a significantly increased recruitment of F4/80+ macrophages in renal tissue of HPD-fed mice after 3 and 6 months. The enhanced renal macrophage recruitment in the entire HPD cohort was associated with increased MCP-1 protein synthesis and increased tubular injury score. In vitro experiments using HK-2 cells showed that stimulation with FGF23 or high phosphate, but not PTH, induced the phosphorylation of STAT3. Interestingly, only treatment with high phosphate significantly upregulated HAVCR1 and CCL2 expression. CONCLUSION Chronic oral phosphate load in mice induced progressive inflammatory kidney injury dominated by macrophages. The direct toxic effects of phosphate are mediated by Stat3/Kim-1 signalling activation in PT cells. Our data support the hypothesis that kidney injury induced by widespread high dietary phosphate intake may cause a global health problem and underline the urgent need for clinical studies on this issue.

MO282: Sigma-1 Receptor Agonists are Protective in Renal Ischemia/Reperfusion Injury

Abstract BACKGROUND AND AIMS Renal ischemia/reperfusion injury (IRI)-induced acute kidney injury is associated with high mortality and morbidity and effective therapies are lacking. We previously showed that Sigma-1 receptor (S1R) agonist fluvoxamine treatment is protective against renal IRI (Hosszu et al, 2017). Here in a rat model, we studied the renoprotective effect of specific, high affinity S1R agonists focusing on the S1R-Akt-eNOS signaling pathway and renal vasoregulation. METHOD 8-week old male Wistar rats (n = 8/group) were subjected to 50 min unilateral ischemia with contralateral nephrectomy, kidney tissue was harvested 24 h after reperfusion. 30 min prior to the ischemic insult rats were treated i.p. as follows: (i) isotonic saline as vehicle; (ii) highly specific, high affinity S1R agonist SA-4503; or (iii) SA-4503 + S1R antagonist NE100. Sham-operated rats were used as controls. Renal functional parameters (BUN, serum creatinine, serum aspartate aminotransferase, kidney injury molecule 1 (Kim1), neutrophil gelatinase-associated lipocalin (Ngal) were evaluated. Structural damage was assessed on PAS-stained kidney sections. Renal S1R, phospho-Akt and phospho-eNOS, Hif-1α protein levels and serum nitric oxide (NO) concentration were measured. In vitro experiments were performed on HK2 human proximal tubular epithelial cells treated with a 10 µM dose of S1R agonist SA-4503 or PRE-084. RESULTS Impaired kidney function and structural damage after I/R were ameliorated by SA-4503. Expressions of early and sensitive tubular injury markers Kim1 and Ngal were markedly less elevated in SA-4503-treated rats. This recovery was blocked with the addition of the S1R antagonist NE100. S1R, phospho-Akt and phospho-eNOS protein levels were significantly elevated in the kidneys of SA-4503 treated rats. Vasodilator nitric oxide concentration was reduced in the kidney after IRI, but returned to control levels in SA-4503-treated rats. Treatment with S1R agonists SA-4503 and PRE-084 increased the NO production of HK-2 cells. CONCLUSION The specific and high affinity S1R agonist SA-4503 acts directly on proximal tubular cells by activating the S1R-NOS system. Thereby SA-4503 is renoprotective by increasing vasodilative NO production and thus improving post-ischemic renal perfusion. Based on our data activation of S1R and downstream signaling pathways could provide a novel therapeutic option in renal IRI. Founding OTKA PD-131 637; FK-124 491; 2020–4.1.1.-TKP2020-6 183 069 269; 2020–4.1.1.-TKP2020-6 183 169 273; KDP-2020/1 019 145

MO624: The Relationship Between Renal Tubular Epithelial Cell Necroptosis and The Severity of Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Diabetic nephropathy (DN), a common complication of diabetic patients, usually contributes to end-stage renal failure. Accidental cell death (ACD) and regulated cell death (RCD) cause renal cell loss and deteriorate renal function during the progression of DN. MLKL is the executor of necroptosis, a major form of RCD. The multimer of phosphorylated MLKL (p-MLKL) gather at the cell membrane and form the leaking pores, performing the last step of necroptosis. This study aims to study the relationship of p-MLKL expression and the disease severity in DN patients, providing promising treatment target. METHOD A total of 30 patients who diagnosed with DN clinical-pathologically in the Peking University First Hospital from January 1998 to August 2018 were enrolled (excluding DN with primary or secondary glomerular diseases, DN combined with thrombotic microangiopathies and small vasculitis). p-MLKL were stained on renal biopsy kidney sections by immunohistochemistry. Image-Pro Plus 6.0 software (IPP) was used to analyze the expression of p-MLKL on 10 randomly taken images for each patient. The relationship between p-MLKL expression and clinical-pathological parameters was explored. Clinical parameters of plasma creatinine, peak plasma creatinine during renal biopsy period, estimated glomerular filtration rate (eGFR) were used. DN pathological changes contained: (i) glomerular lesions, (ii) arteriole lesions (iii) tubular lesions and (iv) tubules chronic interstitial lesions. RESULTS Compared with normal control, the positive expression of p-MLKL could be detected in the renal tubular interstitium of patients with DN. However, no p-MLKL positive signal was seen in the glomeruli. Patients with p-MLKL positive signals accounted for 73.33% (22/30) of the enrolled patients. Among the 30 patients, the value of p-MLKL kidney tissue IOD (integrated optical density) was positively correlated with peak serum creatinine (r = 0.373, P = 0.042), the pathological score of spherical sclerosis (r = 0.364, P = 0.048) and tubular brush border (TBB) loss (r = 0.363, P = 0.049). CONCLUSION The expression of necroptosis marker p-MLKL was significantly correlated with the clinical-pathological changes of DN patients. Its role in the progression of DN deserves further exploration.

Sexual dimorphisms in renal mitochondrial bioenergetics

BackgroundStudies have shown that men have higher incidences of end‐stage renal disease than women at every point across the lifespan. Animal models for various renal diseases have similar occurrence with males being affected more frequently. Recent efforts have been heavily focused on delineating the mechanisms behind these sex‐based differences from various angles, with the mitochondria being highlighted as a target for sex difference‐related renal pathologies. The goal of this study is to investigate the sexual dimorphisms of renal mitochondria and their dependency on renal hemodynamics before disease onset.MethodsMitochondria were isolated from PBS‐flushed kidneys of Sprague Dawley male (SDM) and female (SDF) rats at 11 weeks of age using differential centrifugation. Renal cortex (SDMC vs SDFC) and medulla (SDMMvs SDFM) were separated for individual measurements. Mitochondrial membrane potential and hydrogen peroxide production were measured in spectrofluorimetry using the fluorescent dyes TMRM and Amplex Red, respectively. A portion of mitochondria were submitted to a histology core for electron microscopy. Additionally, mitochondrial oxygen consumption rate (OCR) was also assessed using Seahorse assay. Lipid peroxide radical formation was detected using electron paramagnetic resonance (EPR) with in vivo spin trapping. As a measure of renal function, GFR was assessed using FITC‐Inulin in conscious animals.ResultsElectron microscopy revealed similar yield of mainly round‐shaped isolated mitochondria with retained cristae and membrane structures in all groups. SDFM showed a higher membrane potential than SDMM (238.1±12.4 vs 162.0±8.8 au, p<0.001; respectively). Both SDFC and SDFM had higher hydrogen peroxide production in comparison to their male counterparts (cortex: 99443.7±3061.7 vs 69943.4±1708.6 au/protein, p<0.001; respectively. medulla: 113366.7±5430.7 vs 60613±2297.1 au/protein, p<0.001; respectively). Overall female mitochondria exhibited decreases in their basal, ATP‐linked, leaked OCRs as well as spare capacity compared to males (spare capacity: cortex: 5.9±0.6 vs 14.3±1.3, p<0.001; respectively. medulla: 4.9±0.6 vs 11.6±0.7 pmol/min/protein, p<0.001; respectively). EPR showed similar lipid peroxide radical levels in both sexes and sections of kidney. In the next part of the study, we explored if renal mitochondrial bioenergetics correlated with the GFR of the animal. Our data show the following GFR values for male and female rats as normalized to two kidney weights (2KW) and 2KW/ total body weight (2KW/TBW), respectively: 1.4±0.2 vs 0.8±0.1 mL/min/2KW, p=0.1416 and 0.5±0.1 vs 0.2±0.02 mL/min/2KW/TBW, p=0.0143. We did not observe any correlation between mitochondrial membrane potential and ROS production (measured in the isolated renal cortical mitochondria) with GFR in male or female rats (n= 5 in each group).ConclusionsOverall, we show here that female renal mitochondria exhibit lower oxygen consumption, higher ROS emission, and membrane potential vs males; however, lipid peroxidation is similar. Currently, we do not see a correlation between mitochondrial bioenergetics and GFR. More studies designed with higher statistical power are required to further elucidate the relationship between renal blood flow and mitochondrial bioenergetics.

Sensitivity to Iron‐Mediated Pro‐Inflammatory Signaling in NZBWF1 Mice, a Model of Systemic Lupus Erythematosus

IntroductionThe autoimmune disease systemic lupus erythematosus predominantly affects women and can lead to lupus nephritis (LN) as a common and severe complication in approximately 50% of patients. The F1 hybrid of New Zealand Black (NZB) X New Zealand White (NZW) mice (NZBWF1) is a well‐established mouse model to study LN. Our previous data indicated that the onset of albuminuria in female 34‐week NZBWF1 mice was associated with renal iron accumulation, and iron may contribute to renal injury. This study tests the hypothesis that 34‐week NZBWF1 lupus mice are sensitized to iron's injurious and pro‐inflammatory effects compared to 8‐week normal NZBWF1 mice. As a means to differentiate effects of age as opposed to development of lupus, parallel experiments were performed in 8 and 34‐week‐old NZW mice as healthy controls.Methods and ResultsFemale 8‐week (pre‐autoimmune) and 34‐week NZBWF1 (autoimmune, pre‐albuminuric) mice together with age‐matched healthy control NZW mice underwent 24 h urine collection to confirm non‐albuminuric status. Mice were then were injected i.v. with a single dose of iron‐sucrose (2 mg elemental iron) or vehicle (saline) and sacrificed 24 h later. Plasma blood urea nitrogen (BUN) was measured to determine renal health, SRY‐Box Transcription Factor 9 (SOX‐9)‐ positive cells were quantified in kidney sections as an early tubular injury marker, and plasma monocyte chemoattractant protein‐1 (MCP‐1) was measured by ELISA as an inflammatory marker. Lupus (NZBWF1) and control mice (NZW) were compared within strains by age (8‐week vs. 34‐week) and treatment (Vehicle vs. Treatment). All statistical analyses were done by 2‐way ANOVA (n=7‐12/group), and post‐hoc testing was done using Bonferroni. Iron‐sucrose treatment significantly increased plasma MCP‐1 concentrations in the NZBWF1 and the control NZW mice (PTreatment < 0.01). There was no significant effect of age on this effect for NZW mice (PTreatment*Age = 0.2); however, in NZBWF1 mice, the iron‐sucrose induced increase in MCP‐1 was significantly greater in 34‐week compared to 8‐week mice (PTreatment*Age < 0.05; P<0.05 by posthoc test). BUN was significantly increased with age in both strains (PAge<0.05), but there was no significant effect of iron‐sucrose (PTreatment>0.05). The numbers of SOX‐9+ tubular cells increased in the 34‐week NZBWF1 mice compared to 8‐week mice (Page<0.05), consistent with early stages of renal injury, but iron sucrose treatment did not show any significant effect (PTreatment= 0.9).ConclusionOur data suggest that at the autoimmune but pre‐albuminuric stage, 34‐week NZBWF1 mice show increased pro‐inflammatory responsiveness to iron‐mediated stress. Further studies to determine whether the enhanced pro‐inflammatory effects of iron contribute to pathology in lupus nephritis are ongoing.

Immunohistochemical localization of receptor protein tyrosine phosphatase γ in mouse renal proximal tubule, thick ascending limb and distal convoluted tubule

Most normal physiological processes require that both intracellular (i) and extracellular (o) pH be maintained within a tight range. pHo is contingent on arterial (a) pH, which is determined by the ratio of the arterial carbon dioxide to the arterial bicarbonate concentration, [CO2]a/[HCO3−]a. Pulmonary ventilation controls [CO2]a. The kidney controls [HCO3−]a by adjusting HCO3− transport rates (JHCO3) across the basal membranes of particular nephron epithelia into the blood to maintain normal pHa, or compensate for an acid‐base insult. In an effort to identify the sensor proteins for pHa, [CO2]a, or [HCO3−]a, our laboratory previously reported that, in isolated proximal tubules (PTs) from receptor protein tyrosine phosphatase γ (RPTPγ) knockout (KO) mice, JHCO3 is insensitive to changes in the basolateral [CO2] or [HCO3−]. Furthermore, RPTPγ‐KO mice have a deficiency in defending against a whole‐body metabolic acidosis. These data implicate RPTPγ as a major [CO2] or [HCO3−] sensor. RPTPγ possesses an extracellular catalytically inactive carbonic‐anhydrase‐like domain (CALD) and fibronectin type III (FNIII) domain, a single transmembrane helix, and intracellular domains D1 and D2. Dimerization of RPTPγ presumably allows the inhibitory D2 of one monomer to inhibit the otherwise catalytically active D1 of the other monomer. Thus, the dimerization/monomerization state controls downstream signaling. We hypothesize that the binding of CO2 or HCO3− to the CALD alters the CALD conformation, thereby transmitting signals that control the dimerization status of RPTPγ and thereby regulate downstream effects on JHCO3. We hypothesize that RPTPγ is localized in nephron segments important for regulating HCO3− transport/reabsorption, in the basal membranes that face the blood. To determine the location of RPTPγ in wild‐type mouse kidney cryosections, we use a chicken IgY anti‐RPTPγ antibody (Ab) raised against an epitope within the FNIII domain, and co‐stain with rabbit polyclonal Abs against the Na/HCO3 cotransporter (NBCe1‐A) as a PT marker, the Na/K/Cl cotransporter 2 (NKCC2) as a thick ascending limb (TAL) marker, and the Na/Cl cotransporter (NCC) as a distal convoluted tubule (DCT) marker. Imaging sections by confocal microscopy, we observe in 3 WT mice that RPTPγ localizes primarily to the PT basal membrane. However, 54% of PTs (NBCe1‐A positive) also exhibit apical RPTPγ staining of comparable intensity to the RPTPγ staining observed in the basal membrane. We also observe both apical and basal RPTPγ staining in Bowman’s capsules. We stained additional sections from the same animals with a rabbit polyclonal primary Ab that targets the RPTPγ CALD domain. In these sections, co‐stained with the anti‐FNIII Ab, we similarly observe both apical and basal staining in PTs. We observe RPTPγ staining in a minority of TALs, and at much lower intensity than observed in the PT. We observe minimal to no RPTPγ staining in the DCT. We validated the specificity of the RPTPγ Abs on RPTPγ‐KO kidney sections, which exhibit low background levels of fluorescence for both the anti‐FNIII and anti‐CALD Abs. In conclusion, these data confirm RPTPγ expression in PT basal membranes, but also describe the first examples of apical RPTPγ expression in renal epithelia.

FOXI1 Promotes Expression of V‐ATPase and GPR116 in M‐1 Cells

G protein‐coupled receptors (GPCRs) are a diverse family of integral membrane proteins that have significant roles in numerous physiological systems. We previously reported that GPR116 (ADGRF5), an adhesion‐class GPCR, is a critical regulator of vacuolar‐type H+‐ATPase (V‐ATPase) surface expression in A‐type intercalated cells (AICs) in mouse kidney cortical collecting ducts. The V‐ATPase is a multi‐subunit proton pump that localizes to the plasma membrane in specialized acid‐secreting epithelial cells. FOXI1 is a transcription factor that regulates V‐ATPase expression in ICs and other mitochondria‐rich cells. Recently, FOXI1 was identified as a key regulator of CFTR‐rich pulmonary ionocytes which express both V‐ATPase and GPR116. Therefore, we hypothesized that FOXI1 is a transcription factor in AICs that upregulates V‐ATPase as well as GPR116. To test this hypothesis, we cloned FOXI1 from whole mouse kidney into a pME18s expression vector (pFOXI1). Transfection with pFOXI1 significantly increased GPR116 expression (qPCR) in M‐1 mouse cortical collecting duct cells (n=3, ΔCt ±SEM: untransfected = 13.8±0.6, pFoxi1 = 10.7±0.2, p<0.05), but not in HEK293T cells (n=3, not significant). RNA‐scope hybridization on murine kidney sections and pFOXI1‐transfected M‐1 cells revealed colocalization of FOXI1 and GPR116 mRNA in the same cell. Treating pFOXI1‐transfected M‐1 cells with a synthetic agonist peptide for GPR116 results in an increase of [Ca2+]iin a subset of cells (n=3, ΔF340/380 ±SEM: pFoxi1 = 0.296±0.017). Internal calcium mobilization is never seen with the agonist peptide in untransfected control cells, indicating that pFOXI1 is sufficient for the production of physiologically responsive GPR116 on the cell surface (n=3, ΔF340/380±SEM: untransfected = 0 ±0.0009). Moreover, only M‐1 cells transfected with pFOXI1 express GPR110 (ADGRF1), an adhesion‐GPCR with unknown function in the kidney, as well as three V‐ATPase subunits, ATP6V1B1, ATP6V1G3, ATP6V0D2, indicating a larger transcriptional program initiated by pFOXI1 (n=3, untransfected: Ct ~ 40 for all genes, pFoxi1 ΔCt±SEM: ADGRF1 = 13.2±0.21, ATP6V1B1 = 11.4±0.3, ATP6V1G3 = 10±0.1, ATP6V0D2 = 12.1±0.03). Additionally, these pFOXI1 cells also upregulate the expression of SLC26A4 (pendrin), a known marker of B‐type intercalated cells, suggesting that FOXI1‐driven regulation is upstream of AIC fate determination (n=3, ΔCt ± SEM: untransfected = 19.3±0.7, pFoxi1 = 16.1±0.2, p<0.01). These data reveal that FOXI1 upregulates both GPR116 and V‐ATPase in M‐1 cells, generating IC‐like cells. Finally, our study suggests that GPR116 may be a universal and genetically‐coded regulator of V‐ATPase surface expression in FOXI1‐positive cells.