Kidney Slices Research Articles

Selective mitochondrial antioxidant MitoTEMPO reduces renal dysfunction and systemic inflammation in experimental sepsis in rats

BackgroundExcess mitochondrial reactive oxygen species (mROS) in sepsis is associated with organ failure, in part by generating inflammation through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. We determined the impact of a mitochondrial-targeted antioxidant (MitoTEMPO) on mitochondrial dysfunction in renal proximal tubular epithelial cells, peritoneal immune cell function ex vivo, and organ dysfunction in a rat model of sepsis. MethodsThe effects of MitoTEMPO were assessed ex vivo using adenosine triphosphate and lipopolysaccharide-stimulated rat peritoneal immune cells and fresh rat kidney slices exposed to serum from septic rats. We assessed mROS production and phagocytotic capacity (flow cytometry), mitochondrial functionality (multiphoton imaging, respirometry), and NLRP3 inflammasome activation in cell culture. The effect of MitoTEMPO on organ dysfunction was evaluated in a rat model of faecal peritonitis. ResultsMitoTEMPO decreased septic serum-induced mROS (P<0.001) and maintained normal reduced nicotinamide adenine dinucleotide redox state (P=0.02) and mitochondrial membrane potential (P<0.001) in renal proximal tubular epithelial cells ex vivo. In lipopolysaccharide-stimulated peritoneal immune cells, MitoTEMPO abrogated the increase in mROS (P=0.006) and interleukin-1β (IL-1β) (P=0.03) without affecting non-mitochondrial oxygen consumption or the phagocytotic-induced respiratory burst (P>0.05). In vivo, compared with untreated septic animals, MitoTEMPO reduced systemic IL-1β (P=0.01), reduced renal oxidative stress as determined by urine isoprostane levels (P=0.04), and ameliorated renal dysfunction (reduced serum urea (P<0.001) and creatinine (P=0.05). ConclusionsReduction of mROS by a mitochondria-targeted antioxidant reduced IL-1β, and protected mitochondrial, cellular, and organ functionality after septic insults.

Local Inhibition of Indoleamine 2,3-Dioxygenase Mitigates Renal Fibrosis.

Chronic kidney disease (CKD) is a major global health concern and renal fibrosis is an integral part of the pathophysiological mechanism underlying disease progression. In CKD patients, the majority of metabolic pathways are in disarray and perturbations in enzyme activity most likely contribute to the wide variety of comorbidities observed in these patients. To illustrate, catabolism of tryptophan by indoleamine 2,3-dioxygenase (IDO) gives rise to numerous biologically active metabolites implicated in CKD progression. Here, we evaluated the effect of antagonizing IDO on renal fibrogenesis. To this end, we antagonized IDO using 1-methyl-D-tryptophan (1-MT) and BMS-98620 in TGF-β-treated murine precision-cut kidney slices (mPCKS) and in mice subjected to unilateral ureteral obstruction (UUO). The fibrotic response was evaluated on both the gene and protein level using qPCR and western blotting. Our results demonstrated that treatment with 1-MT or BMS-985205 markedly reduced TGF-β-mediated fibrosis in mPCKS, as seen by a decreased expression of collagen type 1, fibronectin, and α-smooth muscle actin. Moreover, IDO protein expression clearly increased following UUO, however, treatment of UUO mice with either 1-MT or BMS-986205 did not significantly affect the gene and protein expression of the tested fibrosis markers. However, both inhibitors significantly reduced the renal deposition of collagen in UUO mice as shown by Sirius red and trichrome staining. In conclusion, this study demonstrates that IDO antagonism effectively mitigates fibrogenesis in mPCKS and reduces renal collagen accumulation in UUO mice. These findings warrant further research into the clinical application of IDO inhibitors for the treatment of renal fibrosis.

Novel soluble guanylyl cyclase activators increase glomerular cGMP, induce vasodilation and improve blood flow in the murine kidney.

Background and PurposeGeneration of cGMP via NO‐sensitive soluble guanylyl cyclase (sGC) has been implicated in the regulation of renal functions. Chronic kidney disease (CKD) is associated with decreased NO bioavailability, increased oxidative stress and oxidation of sGC to its haem‐free form, apo‐sGC. Apo‐sGC cannot be activated by NO, resulting in impaired cGMP signalling that is associated with chronic kidney disease progression. We hypothesised that sGC activators, which activate apo‐sGC independently of NO, increase renal cGMP production under conditions of oxidative stress, thereby improving renal blood flow (RBF) and kidney function.Experimental ApproachTwo novel sGC activators, runcaciguat and BAY‐543, were tested on murine kidney. We measured cGMP levels in real time in kidney slices of cGMP sensor mice, vasodilation of pre‐constricted glomerular arterioles and RBF in isolated perfused kidneys. Experiments were performed at baseline conditions, under L‐NAME‐induced NO deficiency, and in the presence of oxidative stress induced by ODQ.Key ResultsMouse glomeruli showed NO‐induced cGMP increases. Under baseline conditions, sGC activator did not alter glomerular cGMP concentration or NO‐induced cGMP generation. In the presence of ODQ, NO‐induced glomerular cGMP signals were markedly reduced, whereas sGC activator induced strong cGMP increases. L‐NAME and ODQ pretreated isolated glomerular arterioles were strongly dilated by sGC activator. sGC activator also increased cGMP and RBF in ODQ‐perfused kidneys.Conclusion and ImplicationsGC activators increase glomerular cGMP, dilate glomerular arterioles and improve RBF under disease‐relevant oxidative stress conditions. Therefore, sGC activators represent a promising class of drugs for chronic kidney disease treatment.LINKED ARTICLESThis article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc

Abstract 2864: RET as a novel therapeutic target in breast cancer brain metastases

Abstract Brain metastases (BrM) are reported in approximately 10-30% of breast cancer patients with a poor median overall survival of 8.5 months. Current therapeutic strategies are mostly limited to surgery, chemotherapy and radiotherapy, making the study of novel druggable targets and brain-permeable small molecule inhibitors an urgent clinical need. Our lab recently reported a hybrid-capture exome RNA-sequencing study on 21 patient-matched pairs of primary breast tumors and brain metastases and identified upregulation of several receptor tyrosine kinases including RET in BrM. The ligand for RET is Glial Cell Derived Neurotrophic Factor (GDNF), which is released by astrocytes and microglia in response to neuroinflammation and brain tissue damage. Clinical BrM cell growth in ex vivo cultures was reduced by the multikinase inhibitor Cabozantinib, indicating a potential role for RET in the growth of BrM lesions. We hypothesize that the high level of brain-derived GDNF provides a permissive microenvironment for RET overexpressing breast cancer cells to form brain metastases, and will evaluate the therapeutic potential of RET targeted therapies for breast cancer BrM patients. Our preliminary data show that in RET-overexpressing (RET-OE) breast cancer cell models, 30-minute GDNF treatment induces RET phosphorylation at two distinct tyrosine residues, along with downstream signaling illustrated by ERK1/2, AKT, and STAT3 phosphorylation. This induction was effectively abrogated after shRNA-mediated RET knockdown. We employed a phospho-kinase array to identify novel downstream targets regulated by the GDNF-RET signaling axis, and found that GDNF induces phosphorylation of multiple targets (including p27 and RSK) in RET-OE cells. Interestingly, phosphorylation of multiple Src family kinases (Lck, Lyn, Fgr) was downregulated with GDNF treatment. We will further examine these downstream targets via reverse phase protein array (RPPA). GDNF triggers migration of RET-OE cell lines in both 2D wound scratch and 3D transwell migration assays. Additionally, we present results obtained through an ex vivo organotypic coculture system, in which RET-OE cells are cultured on top of 300-micron thick murine brain, kidney, or liver slices. RET overexpression increases colony formation compared with empty vector control, suggesting a role for RET signaling in the initial establishment of metastatic brain lesions. Differences in colonization between empty vector transfected and RET-OE cells remain minimal in kidney and liver sections, suggesting a role for RET specific to the brain microenvironment. We will examine RET-driven brain metastasis in vivo by employing RET-OE and RET-deficient cell lines in intracranial, intracardiac, and mammary fat pad injection. These models of BrM will be randomized to RET inhibitor or vehicle treatment to evaluate the efficacy of RET-targeted therapy in the setting of breast cancer brain metastasis. Citation Format: Simeng Liu, Geoffrey Pecar, Jagmohan Hooda, Jennifer M. Atkinson, Fangyuan Chen, Adrian V. Lee, Steffi Oesterreich. RET as a novel therapeutic target in breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2864.

MO074TILVESTAMAB, A FUNCTION-BLOCKING MONOCLONAL ANTIBODY INHIBITOR OF AXL RTK SIGNALLING, LIMITS THE ONSET OF RENAL FIBROTIC CHANGES IN HUMAN KIDNEYS EX VIVO

Abstract Background and Aims Interstitial fibrosis, characterised by the accumulation of extracellular matrix in the cortical interstitium, is directly correlated with progressive chronic kidney disease secondary to inflammatory, immunologic, obstructive or metabolic causes. An invariant histologic marker of this progression is the accumulation of fibroblasts, with the phenotypic appearance of activated myofibroblasts expressing alpha smooth muscle actin (αSMA) within intracellular contractile stress fibres. Once present, these myofibroblasts are prognostic indicators of expansion of fibrotic matrix and progressive tubular atrophy, leading towards end-stage disease. The Receptor Tyrosine Kinase AXL is involved in a range of kidney pathologies, with increased activity associated with Epithelial to Mesenchymal Transition (EMT) and tubular proliferation following podocyte loss. In mice treated with an angiotensin-converting enzyme (ACE) inhibitor, enhancement of AXL expression is localised to tubular segments within the medulla and there is evidence of parallel regulatory control of ACE and AXL. We have demonstrated enhanced expression of AXL and the mesenchymal marker, vimentin in diseased human kidney tissue secondary to diabetes or hypertension. Targeting AXL with a small-molecule inhibitor has previously been reported to attenuate fibrosis and reduce inflammation in the unilateral ureteric-outflow obstruction (UUO) model of kidney fibrosis in mice (Landolt et al., 2019). Tilvestamab is a novel function blocking humanized anti-AXL antibody. Tilvestamab blocks GAS6-mediated AXL receptor activation in fibroblasts and renal tubule epithelial cells and mediates AXL receptor internalization and degradation. In this study we aimed to further characterise AXL as a target in CKD and to investigate anti-fibrotic efficacy of tilvestamab. Method Eight weeks old male C57BL/6 mice underwent UUO operation. After 15 days, kidneys were dissociated and stained with a high dimensional single cell mass cytometry 33 markers antibody panel. Data were analysed using JMP Genomics (v.8.2). Precision Cut Kidney Slices (PCKSs) from explanted human kidney tissue were propagated in a bioreactor (Paish et al., 2019, FibroFind, UK). PCKS were incubated for 72hrs in the presence of investigational drugs. Secreted collagen1a1 were quantified by ELISA. RNA was reverse transcribed to cDNA and used in qPCRs to measure Col1a1 and αSMA. FFPE sections were stained for αSMA. High magnification images were taken of each slide and analysed for surface area covered by the stain. Results Expression pattern of AXL during development of kidney fibrosis in the UUO model was investigated using a mass cytometry antibody panel designed for identifying subpopulations of immune cells as well as cell populations of the fibrotic stroma. Two predominant cell populations were affected by ligation; the mesenchymal and the immune island. AXL was a marker characterising several of the key populations that expanded upon ligation supporting a role for AXL in kidney fibrosis pathogenesis. In an ex vivo model of human PCKS, tilvestamab dose-dependently reduced the levels of αSMA. When combined with the lower of two doses of the ACE inhibitor enalapril, the lowest dose of tilvestamab synergized to reduce αSMA levels further as well as reducing secreted Collagen 1a1. Conclusion AXL expression is induced in key cell populations during development of kidney fibrosis supporting AXL as a novel target in CKD. Tilvestamab represents a promising strategy for the pharmacologic intervention of kidney fibrosis, and the potential synergy with current reno-protective therapies warrants further exploration.

Activation of the kidney sodium chloride cotransporter by the β2-adrenergic receptor agonist salbutamol increases blood pressure

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In exvivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

Ameliorative potentials of Persea americana leaf extract on toxicants - induced oxidative assault in multiple organs of wistar albino rat

BackgroundPersea americana (PA) is a popular medicinal plant in folkloric medicines. The present study therefore investigates the ameliorative potentials of its leaves extract on carbon tetrachloride and rifampicin-induced toxicity in the liver, kidney and heart of albino rat. This was aimed at assessing the potentials of the plant in the management of liver, kidney and heart diseases.MethodologyForty (40) rats were randomly divided into eight (8) groups of five animals each. Groups I and II were administered with only distilled water and carbon tetrachloride (CCl4) respectively. Groups III and IV animals received 3.0 ml/kg bw of CCl4 and were treated with the extract at 50 mg/kg bw and 100 mg/kg respectively, while group V received 100 mg/kg bw. of silymarin orally for 14 days. Groups VI animals were administered with rifampicin (250 mg/kg bw.) only, while groups VII and VIII animals received rifampicin and were treated with 50 mg and 100 mg/kg bw of the extract respectively. Activities of creatine kinase, aspartate amino transferase, alanine amino transferase, alkaline phosphatase, superoxide dismutase, catalase as well as levels of urea, uric acid, bilirubin and malonidialdehyde (MDA) were assayed. Lipid profiles and histopathological examination of liver and kidney slices were also performed.ResultTreatment with P. americana significantly (p < 0.05) restored all deranged biochemical parameters (creatine kinase, aspartate amino transferase, alanine amino transferase, alkaline phosphatase, urea, uric acid, bilirubin, MDA, lipid profile as well as superoxide dismutase and catalase) in a dose-dependent manner. Normal hepatic and renal histoarchitecture were also restored following treatment with P. americana.ConclusionAmelioration of distorted cardiac, hepatic and renal histoarchitecture as well as restoration of lipid profile, biomarkers of liver and kidney injury and antioxidant enzymes (catalase and superoxide dismutase) affirm the potential usefulness of P. americana in the management of liver, kidney and heart diseases.

Peptide Hormone Insulin Regulates Function, Expression, and SUMOylation of Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3) plays an important role in the disposition of various anionic drugs which impacts the pharmacokinetics and pharmacodynamics of the therapeutics, thus influencing the pharmacological effects and toxicity of the drugs. In this study, we investigated the effect of insulin on the regulation of OAT3 function, expression, and SUMOylation. We demonstrated that insulin induced an increase in OAT3 transport activity through a dose- and time-dependent manner in COS-7 cells. The insulin-induced elevation in OAT3 function was blocked by PKA inhibitor H89, which correlated well with OAT3 protein expression. Moreover, both PKA activator Bt2-cAMP-induced increase and insulin-induced increase in OAT3 function were blocked by PKB inhibitor AKTi1/2. To further investigate the involvement of SUMOylation, we treated OAT3-expressing cells with insulin in presence or absence of H89 or AKTi1/2 followed by examining OAT3 SUMOylation. We showed that insulin enhanced OAT3 SUMOylation, and such enhancement was abrogated by H89 and AKTi1/2. Lastly, insulin increased OAT3 function and SUMOylation in rat kidney slice. In conclusion, our investigations demonstrated that insulin regulated OAT3 function, expression, and SUMOylation through PKA/PKB signaling pathway. Graphical abstract.

Sodium dichloroacetate attenuates the growth of B16-F10 melanoma in vitro and in vivo: an opportunity for drug repurposing.

Sodium dichloroacetate (DCA) is a metabolic regulator used to treat diabetes. Since DCA inhibits pyruvate dehydrogenase kinase, decreasing lactic acid formation, it can reverse the Warburg effect in cancer cells, promoting apoptosis. Therefore, this study aimed to investigate the potential of DCA as a drug repurposing candidate for the treatment of melanoma. For the in-vitro assay, murine B16-F10 melanoma cells were treated with 0.5, 1, 5, 10, 20 or 50 mM DCA for 3 days, analyzed with the crystal violet method. The in-vivo effect of DCA was evaluated in B16-F10 tumor-bearing C57BL/6 mice treated with different doses of DCA (0, 25, 75 or 150 mg/kg) by gavage for 10 days, followed by measurement of tumor volume. Upon necropsy, representative slices of lung, liver, kidney, spleen and intestine were collected, processed and submitted for histopathological examination. The DCA concentrations of 10, 20 and 50 mM reduced B16-F10 cell viability after 48 and 72 h of treatment, whereas 20 and 50 mM were effective after 24 h of treatment. A significant reduction in tumor growth was observed in B16-F10 melanoma bearing mice at all doses, with no change in body weight or histology. DCA attenuates the growth of B16-F10 melanoma in vitro and in vivo, without systemic toxic effects. Therefore, DCA is a candidate for drug repurposing against melanomas.

Superresolving the kidney\u2014a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney’s glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world.Graphical abstract

Optimization of the alamarBlue viability assay for rat kidney slices

Optimization of the alamarBlue viability assay for rat kidney slices

Tamoxifen attenuates renal fibrosis in human kidney slices and rats subjected to unilateral ureteral obstruction

Background and PurposeRenal fibrosis plays an important role in the development and progression of chronic kidney disease (CKD). Clinical studies have shown that CKD progresses differently in males and females, which may be related to circulating levels of sex hormones. In this study, we investigated the effect of tamoxifen (TAM), a selective estrogen receptor modulator (SERM), on renal fibrosis in male and female rats subjected to unilateral ureteral obstruction (UUO) and human precision-cut kidney slices (PCKS). Experimental ApproachFemale, ovariectomized female (OVX), and male rats were subjected to 7 days of UUO and treated with TAM by oral gavage. Moreover, we studied individual responses to TAM treatment in PCKS prepared from female and male patients. In all models, the expression of fibrosis markers was examined by western blot, qPCR, and immunohistochemistry. Key ResultsTAM decreased the expression of fibronectin, α-smooth muscle actin, and collagen-1 and -3 in female, OVX, and male rats. In addition, TAM mitigated TGF‐β-induced fibrosis in human PCKS, irrespective of sex, yet interindividual differences in treatment response were observed. Conclusion and ImplicationsTAM ameliorates renal fibrosis in males and females, although we did observe sex differences in drug response. These findings warrant further research into the clinical applicability of TAM, or other SERMs, for the personalized treatment of renal disease.

Renal injury and hepatic effects from the methylimidazolium ionic liquid M8OI in mouse

The ionic liquid 1-octyl-3-methylimidazolium (M8OI) has been found in the environment and identified as a hazard for triggering the liver disease primary biliary cholangitis (PBC). Given limited toxicity data for M8OI and other structurally-related ionic liquids, target organs for M8OI toxicity were examined. Adult male C57Bl6 mice were acutely exposed to 0–10 mg/kg body weight M8OI via 2 intraperitoneal injections (time zero and 18 h) and effects examined at 24 h. At termination, tissue histopathology, serum and urinary endpoints were examined. No overt pathological changes were observed in the heart and brain. In contrast, focal and mild to multifocal and moderate degeneration with a general trend for an increase in severity with increased dose was observed in the kidney. These changes were accompanied by a dose-dependent increased expression of Kim1 in kidney tissue, marked elevations in urinary Kim1 protein and a dose-dependent increase in serum creatinine. Hepatic changes were limited to a significant dose-dependent loss of hepatic glycogen and a mild but significant increase in portal tract inflammatory recruitment and/or fibroblastic proliferation accompanied by a focal fibrotic change. Cultured mouse tissue slices reflected these in vivo effects in that dose-dependent injury was observed in kidney slices but not in the liver. Kidney slices accumulated higher levels of M8OI than liver slices (e.g. at 10 μM, greater than 4 fold) and liver slices where markedly more active in the metabolism of M8OI. These data indicate that the kidney is a target organ for the toxic effects of M8OI accompanied by mild cholangiopathic changes in the liver after intraperitoneal administration.

Oxidation of energy substrates in tissues of largemouth bass (Micropterus salmoides).

This study tested the hypothesis that amino acids are oxidized at higher rates than glucose and palmitate for ATP production in tissues of largemouth bass (LMB, a carnivorous fish). Slices (10 to 50 mg) of liver, proximal intestine, kidney, and skeletal muscle isolated from LMB were incubated at 26°C for 2h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5mM D-glucose) containing either D-[U-14C]glucose, 2mM L-alanine plus L-[U-14C]alanine, 2mM L-aspartate plus L-[U-14C]aspartate, 2mM L-glutamate plus L-[U-14C]glutamate, 2mM L-glutamine plus L-[U-14C]glutamine, 2mM L-leucine plus L-[U-14C]leucine, or 2mM palmitate plus [U-14C]palmitate. In parallel experiments, tissues were incubated with a [U-14C]-labeled tracer and a mixture of unlabeled substrates [alanine, aspartate, glutamate, glutamine, leucine, and palmitate (2mM each) plus 5mM glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In separate experiments, O2 consumption by each tissue was measured in the presence of individual or a mixture of substrates. The activities of key metabolic enzymes were also measured. Results indicated that the liver and skeletal muscle had a limited ability to oxidize glucose and palmitate to CO2 for ATP production in the presence of individual or a mixture of substrates due to low activities of carnitine palmitoyltransferase-I, hexokinase and pyruvate dehydrogenase. In the presence of individual substrates, each amino acid was actively oxidized by all the tissues. In the presence of a mixture of substrates, glutamine and glutamate were the major metabolic fuels in the proximal intestine and kidney, as glutamine for the liver and aspartate for skeletal muscle. All the tissues had high activities of glutaminase, glutamate dehydrogenase, and transaminases. At the same extracellular concentration of amino acids (2 mM) in a mixture of energy substrates, glutamine was the major metabolic fuel for the liver of the LMB, glutamine and glutamate for the proximal intestine and kidneys, and aspartate for the skeletal muscle. Glutamine plus glutamate plus aspartate generated 60-70% of ATP in LMB tissues.

Comparative study of nanoparticle uptake and impact in murine lung, liver and kidney tissue slices

To determine responses to nanoparticles in a more comprehensive way, current efforts in nanosafety aim at combining the analysis of multiple endpoints and comparing outcomes in different models. To this end, here we used tissue slices from mice as 3D ex vivo models and performed for the first time a comparative study of uptake and impact in liver, lung, and kidney slices exposed under the same conditions to silica, carboxylated and amino-modified polystyrene. In all organs, only exposure to amino-modified polystyrene induced toxicity, with stronger effects in kidneys and lungs. Uptake and distribution studies by confocal microscopy confirmed nanoparticle uptake in all slices, and, in line with what observed in vivo, preferential accumulation in the macrophages. However, uptake levels in kidneys were minimal, despite the strong impact observed when exposed to the amino-modified polystyrene. On the contrary, nanoparticle uptake and accumulation in macrophages were particularly evident in lung slices. Thus, tissue digestion was used to recover all cells from lung slices at different exposure times and to determine by flow cytometry detailed uptake kinetics in lung macrophages and all other cells, confirming higher uptake by the macrophages. Finally, the expression levels of a panel of targets involved in inflammation and macrophage polarization were measured to determine potential effects induced in lung and liver tissue. Overall, this comparative study allowed us to determine uptake and impact of nanoparticles in real tissue and identify important differences in outcomes in the organs in which nanoparticles distribute.

Evaluation of different sterilization methods for decellularized kidney tissue

The main goal of this study was to assess the effect of different sterilization treatment for sterilization of decellularized kidney tissue. Rabbit kidneys were decellularized by the perfusion-based method using sodium dodecyl sulfate (SDS) and Triton X-100. Then, decellularized kidney slices were prepared and sterilized by an antibiotic cocktail, PAA (0.5 %, 1% and 1.5 %), 5KG γ-irradiation and 320–480 nm UV-irradiation. Histological evaluations, DNA quantification assay, MTT assay, scanning electron microscopy (SEM), mechanical test and bacterial and fungal culture tests were performed to determine the quality of decellularization and sterilization processes. The kidney slices were seeded by adipose-derived mesenchymal stem cells (ASCs) to assess the cell adhesion capability after treatment. The results of the current study indicated that PAA 0.5 % was the most efficient method to completely decontaminate rabbit decellularized kidney tissue while preserving the mechanical properties and main components of the matrix which are necessary for cell-matrix interaction and cell adhesion. The 5KG γ-irradiation was determined to be the most destructive sterilization method, with reduced the mechanical strengths as well as altered microstructure of the kidney matrix and no cell adhesion. In addition, UV-irradiation is not able to sterile the decellularized tissues. Therefore PAA 0.5 % sterilization method can be a powerful means for sterilization of biological scaffolds.

P0529THE DIFFERENCE IN DIETARY RESTRICTION-INDUCED NEPHROPROTECTIVE MECHANISMS IN YOUNG AND OLD ANIMALS

Abstract Background and Aims Acute kidney injury (AKI) is a widespread disease affecting mostly old people. Dietary restriction (DR), based on the reduction of food intake, is believed to be one of the most efficient approaches ameliorating damage in different pathological conditions including age-associated diseases. The aim of the study was to investigate the protective mechanisms of DR in the model of AKI in young and old rats. Method All experiments were made on young (3-4 months) and old (22-24 months) male rats. DR was performed by limiting the amount of food for 35% of the ad libitum (AL) daily intake. Since earlier, we showed ineffectiveness of 4-weeks DR in old rats, in this study we applied 35% DR lasting 8 weeks for old rats and 4 weeks for young rats. During DR, we registered the weight loss and measured the level of adiponectin, as this hormone is closely associated with adipose tissue metabolism. Renal ischemia/reperfusion (I/R) was used as a model of ischemic AKI. I/R was performed by clamping the left renal pedicle for 40 minutes followed by reperfusion with simultaneous contralateral nephrectomy. The severity of AKI was evaluated by measuring blood urea nitrogen (BUN), serum creatinine (SCr) and the levels of protein biomarkers of AKI (NGAL and L-FABP) in urine. Proliferation in kidney epithelium in response to I/R was analyzed by PCNA protein level in kidney tissue. We evaluated the function of mitochondria by measuring TMRE/MitoTracker Green ratio in vital kidney slices; in kidney homogenates, we also analyzed levels of Bcl-XL and Bcl-XS proteins. The production of reactive oxygen species (ROS) was evaluated by staining vital kidney slices with DCF. The content of lipid peroxidation products was measured using Image-iT Lipid Peroxidation Kit, and the level of carbonylated proteins was determined by OxyBlot Protein Oxidation Detection Kit. The activation of autophagic-lysosomal system was estimated by western blotting to LC3 II/LC3 I ratio and LAMP1 level, as well as by staining vital kidney slices with LysoTracker Green probe. Results The body weight of rats during DR dropped as far as 20% by the end of 4 weeks in young rats and 30% by the end of 8 weeks in old rats. Nevertheless, adiponectin concentration elevated during DR only in the serum of young rats. DR strongly influenced mitochondria function, in particular, elevated mitochondrial membrane potential both in kidney cells of young and old rats. DR also resulted in increasing the Bcl-XL level. We revealed the decrease of ROS and lipid peroxidation products in vital kidney slices, but only in kidneys of young rats. However, DR reduced the content of carbonyl groups more than 2 times in animals of both ages. We showed that activation of autophagy in response to DR and I/R occurred only in the kidneys of young rats, indicating deterioration of autophagy signaling in old animals. We also found that 48 h after I/R PCNA level increased 19 times in young kidney, although old rats showed only 4-fold elevation of kidney cells proliferation. Estimation of kidney injury markers (NGAL, L-FABP) in urine revealed that 2-month DR led to some protection in old rats. Nonetheless, despite all positive alterations in kidney tissue of old rats, DR was not able to ameliorate impairment of kidney function after I/R, whereas all young rats showed significant improvement of SCr and BUN levels. Conclusion Short-term DR has a significant nephroprotective effect against renal I/R in young rats. Old animals require longer periods of food restriction, after which some protective alterations are observed. We propose, protection of kidney in old and young rats is implemented through slightly different mechanisms and some of them are missing in old animals.

Predictive Value of Precision-Cut Kidney Slices as an Ex Vivo Screening Platform for Therapeutics in Human Renal Fibrosis.

Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFβ or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFβ and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.

Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells.

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

The effect of tamoxifen on gender differences in unilateral ureteral obstructed rats and human kidney slices

BACKGROUNDRenal fibrosis is regarded as the most damaging process in chronic kidney disease (CKD) and it is the most consistent predictor of irreversible loss of renal function. To date, there is no good treatment for renal fibrosis. Interestingly, females are less prone to CKD, which might be due to estrogen. We have previously shown that tamoxifen (TAM), a selective estrogen receptor modulator, plays a role for regulation of renal water and salt balance. In this study, we will investigate the effect of tamoxifen on a well‐established in vivo model using unilateral ureteral obstruction (UUO)‐induced fibrosis in female, male and ovariectomized (OVX) female rats. Furthermore, a recently develop model of human renal fibrosis was used to study multicellular pathological processes in human tissue.METHODSFibrosis was induced by 7 days of UUO. Female OVX rats had both their ovaries removed two weeks before UUO. Tamoxifen (25 mg/kg for female rats and 50 mg/kg for male rats; a higher dose of tamoxifen was lethal for female rats) was given by oral gavage starting 5 days before UUO. Tamoxifen treatment was continued for 7 days after UUO. Human precision‐cut kidney slices were exposed to TGF‐β for 48h in order to make the tissue fibrotic and afterwards treated with tamoxifen for 6h. Histological changes were examined by HE staining. Expression of α‐smooth muscle actin, fibronectin, and TGF‐β were evaluated by immunohistochemistry, quantitative PCR, and western blot analysis.RESULTSRenal fibrosis was increased after UUO in both male, female and OVX rats. Tamoxifen treatment reduced fibronectin, α‐SMA and TGF‐β gene expression in female UUO rats, but not in male and in OVX rats subjected to UUO. Yet, tamoxifen significantly reduced the protein level of fibrotic markers and the kidney weight in response to UUO in both female as well as OVX and male rats. Furthermore, our data demonstrated that sham operated female and female OVX rats had a higher plasma creatinine level compared to male rats. On the other hand, TAM treated male rats subjected to UUO had a significantly increased urea plasma level, which was not observed in female and female OVX rats. Importantly, treatment with tamoxifen has a tendency to reduce fibronectin in human kidney slices exposed to TGF‐β from women but not in men.CONCLUSIONThese findings indicate that tamoxifen reduces UUO‐induced fibrosis in both female, female OVX and male rats, but have different effects on females and males when it comes to gene levels of fibrotic markers and plasma data. Furthermore, our finding provide preclinical evidence that treatment with tamoxifen affects men and women differently and the concentration of the drug must be adapted to the gender.Support or Funding InformationThis study was kindly supported by Karen Elise Jensen Foundation, the Danish Council for Independent Research, Aarhus University Foundation and the AP Møller Foundation.