Precision-cut Kidney Slices Research Articles

Optimization of long-term incubation of precision-cut kidney slices

Precision-cut kidney slices (PCKS) provide a powerful model to close the gap between in vivo and in vitro research. Publications by various authors favor different incubation conditions, media, and antibiotics, that have not yet been compared in a standardized manner. After preparation, rat-PCKS were incubated in a total of nine combinations of incubation media and antibiotics for four days. We found that a combination of DMEM/F-12 and gentamicin showed the highest levels of viability. Utilizing both qualitative and quantitative methods, we observed stable levels of cellular viability for 10 days when incubated in the most suitable medium combination of DMEM and gentamicin. Additionally, a calcein acetoxymethyl/ethidium homodimer-1 based live/dead staining, analysis of total protein content and lactate dehydrogenase (LDH) were explored to assess both short- and long-term tissue viability. PCKS showed a significant decrease in total protein content, leveling off at around 60% over the duration of 10 days. To be able to evaluate viability irrespective of decreases in total protein detected, we chose to utilize the alamarBlue Cell Viability Assay. Quantifying both intra- and extracellular activity of LDH, while using different concentrations of ethanol as a positive control, we explored enzyme content as a parameter for cell membrane damage and cytotoxicity in PCKS. Overall, we showed that PCKS are suitable for both short- and long-term observation by optimizing incubation parameters, with numerous possibilities for other assays and methods in future studies.

#1945 Comparative analysis of kidney transplantation modeled using precision-cut kidney slices and kidney transplantation in pigs

Abstract Background and Aims Kidney transplants are at risk for so far unavoidable ischemia-reperfusion injury. Several experimental kidney transplantation models are available to study this injury, but all have their own limitations. Here, we describe precision-cut kidney slices (PCKS) as a novel model of kidney ischemia-reperfusion injury in comparison with pig and human kidney transplantation. Method Following bilateral nephrectomy in pigs, we applied warm ischemia (1 h), cold ischemia (20 h) and a reperfusion period (4 h) to one whole kidney undergoing transplantation to a recipient pig and, in parallel, established PCKS undergoing ischemia and modeled reperfusion. Tissues were analyzed using standard histopathology assessment by two blinded pathologists, and bulk RNAseq with gene set enrichment analysis with comparison to human kidney biopsy gene sets obtained from Gene Expression Omnibus. Results Histopathological assessment revealed the presence of some but not all morphological features of tubular injury in PCKS as seen in pig kidney transplantation. RNAseq demonstrated that the majority of changes occurred after reperfusion only, with a partial overlap between PCKS and kidney transplantation, with some differences in transcriptional response attributable to systemic inflammatory responses and immune cell migration. Comparison of PCKS and pig kidney transplantation with RNAseq data from human kidney biopsies by gene set enrichment analysis revealed that both PCKS and pig kidney transplantation reproduced the post-reperfusion pattern of human kidney transplantation. In contrast, only post-cold ischemia PCKS and pig kidney partially resembled the gene set of human acute kidney injury. Conclusion Overall, the present study established that a PCKS protocol can model kidney transplantation and its reperfusion-related damage on a histological and a transcriptomic level. PCKS may thus expand the toolbox for developing novel therapeutic strategies against ischemia-reperfusion injury.

Effects of organophosphates on precision-cut kidney slices

Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 µM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from the control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms, including inflammatory response, oxidative stress or activation of apoptosis to further understand the nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme-inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.

#1290 Adverse proinflammatory responses in the kidney after FGF23 administration in three murine kidney disease models

Abstract Background and Aims The hormone FGF23 is a biomarker associated with morbidity and mortality in individuals with and without kidney disease, but the role of FGF23 excess as a pathogenic factor is incompletely understood. Here, we investigated renal FGF23 signaling in kidney disease models. Method This study used three independent disease models: i) Male C57BL/6 mice (n = 16) were treated with Freud's adjuvant and nephrotoxic serum (NTS) to induce anti-glomerular basement membrane (anti-GBM) disease. ii) Female BALB/c mice (n = 8) were treated with adriamycin (doxorubicin) 10.5 mg/kg to induce focal and segmental glomerulosclerosis (FSGS). iii) Male DBA/2J mice (n = 20) were fed a diet containing adenine 0.2%. Control mice of the respective same strain and sex received vehicle injections. Mice of the anti-GBM and FSGS models and controls received intravenous injections of recombinant FGF23 1 µg or vehicle for six consecutive days (anti-GBM) or once (FSGS), with dissection 24 h after the last injection. Mice of the adenine model were dissected after 15 weeks. Following dissection, ex vivo precision-cut kidney slices (PCKS) were obtained from n = 4 mice and maintained for 24 at 37°C, 95% O2 and 5% CO2, followed by a 1 h treatment with recombinant FGF23 200 ng/ml or vehicle. Glomerular filtration rate (GFR) or renal retention parameters in serum and albuminuria were assessed. Kidneys underwent histological assessment using hematoxylin & eosin or Masson's trichrome. Renal RNA was isolated for bulk RNAseq. Differential gene expression was assessed to determine the effect of FGF23 treatment in disease-free controls, in each disease model, and the interaction between FGF23 X disease effect for the anti-GBM and FSGS groups. Gene set enrichment analysis was performed with a focus on the immune system. Results Anti-GBM mice showed a 45 ± 27% GFR decline (mean ± SD) compared to baseline, whereas controls showed an GFR increase of 11 ± 36% (p < 0.01). Kidney histology revealed an interstitial infiltrate. The FSGS model showed elevated serum creatinine and albuminuria, and hypercellularity in some glomeruli. Mice of the adenine model showed progressively increased blood urea nitrogen concentrations starting after 2 weeks, up to a 3-fold increase at week 14. Histology revealed interstitial fibrosis and tubular atrophy. Bulk RNAseq revealed proinflammatory transcriptional signatures after FGF23 treatment in all disease models, regardless of which timepoint or model: In the anti-GBM model, Hallmark gene sets of inflammatory response, interferon-α response, interferon-β response and allograft rejection were significantly increased after 6 days of FGF23 treatment, whereas disease-free controls showed no significantly changed immune gene sets after 6 days of FGF23. In FSGS mice, none of the Hallmark gene sets were changed after 1 FGF23 injection, but top individual gene expression affected by 1 FGF23 injection were an increased proinflammatory transcription factor Cebpb and an increased DNA damage transcript Dclre1b. Finally, the PCKS of adenine model revealed no significant Hallmark gene sets, but the two genes with differential expression were a proinflammatory macrophage/fibroblast chemokine Ccl4 (increased) and an immunoglobulin heavy variable Ighv2-2 (decreased). Conclusion FGF23-driven patterns of proinflammatory gene expression were observed in three experimental models of kidney disease, both in vivo and ex vivo assessment of FGF23 signaling, thus excluding effects of FGF23 on other organs. As these findings were limited to transcriptional analyses, the present data warrant further confirmation and exploration of FGF23 as a pathogenic agent in kidney disease.

Dexamethasone and Enalaprilat but Not SB525334 Exhibit Anti-Inflammatory Properties in Precision-Cut Kidney Slices (PCKSs) Obtained from Mice with Adriamycin-Induced Nephropathy

Dexamethasone and Enalaprilat but Not SB525334 Exhibit Anti-Inflammatory Properties in Precision-Cut Kidney Slices (PCKSs) Obtained from Mice with Adriamycin-Induced Nephropathy

Slice of Life: Porcine Kidney Slices for Testing Antifibrotic Drugs in a Transplant Setting

Circulatory death donor (DCD) kidneys are increasingly used to enlarge the donor pool. These kidneys undergo ischemia-reperfusion injury, frequently leading to renal fibrosis. Transforming growth factor beta 1 (TGF-β1) and matrix metalloproteases have been identified as central mediators of fibrosis and inhibition of these targets could attenuate fibrosis. We studied whether galunisertib, doxycycline, taurine, and febuxostat alleviated fibrosis in precision-cut kidney slices (PCKS). PCKS were prepared from porcine kidneys that were exposed to 30 min of warm ischemia followed by 3 h of oxygenated hypothermic machine perfusion. We subsequently incubated PCKS for 48 h at 37 °C with the described compounds. To further elucidate the antifibrotic effects of galunisertib, we cultured PCKS with TGF-β1. We first screened the effects of the compounds without TGF-β1. Most significant effects were observed for galunisertib which lowered the expression of ACTA2, TGFB1, FN2, and SERPINE1. We then investigated the effects of galunisertib in fibrotic PCKS incubated with TGF-β1. TGF-β1 significantly increased expression of TGFB1, FN1, SERPINE1, and SERPINH1. Galunisertib, however, attenuated the expression of all fibrosis-related genes. Galunisertib appears to be a promising antifibrotic compound requiring further research in a preclinical model and may ultimately be administered during machine perfusion as an antifibrotic treatment in a transplant setting.

An animal-free preclinical drug screening platform based on human precision-cut kidney slices

ObjectiveRenal fibrosis is one of the main pathophysiological processes underlying the progression of chronic kidney disease and kidney allograft failure. In the past decades, overwhelming efforts have been undertaken to find druggable targets for the treatment of renal fibrosis, mainly using cell- and animal models. However, the latter often do not adequately reflect human pathogenesis, obtained results differ per strain within a given species, and the models are associated with considerable discomfort for the animals. Therefore, the objective of this study is to implement the 3Rs in renal fibrosis research by establishing an animal-free drug screening platform for renal fibrosis based on human precision-cut kidney slices (PCKS) and by limiting the use of reagents that are associated with significant animal welfare concerns.ResultsUsing Western blotting and gene expression arrays, we show that transforming growth factor-β (TGF-β) induced fibrosis in human PCKS. In addition, our results demonstrated that butaprost, SC-19220 and tamoxifen – all putative anti-fibrotic compounds – altered TGF-β-induced pro-fibrotic gene expression in human PCKS. Moreover, we observed that all compounds modulated fairly distinct sets of genes, however they all impacted TGF-β/SMAD signaling. In conclusion, this study revealed that it is feasible to use an animal-free approach to test drug efficacy and elucidate mechanisms of action.

Soluble Klotho protects against glomerular injury through regulation of ER stress response

αKlotho (Klotho) has well established renoprotective effects; however, the molecular pathways mediating its glomerular protection remain incompletely understood. Recent studies have reported that Klotho is expressed in podocytes and protects glomeruli through auto- and paracrine effects. Here, we examined renal expression of Klotho in detail and explored its protective effects in podocyte-specific Klotho knockout mice, and by overexpressing human Klotho in podocytes and hepatocytes. We demonstrate that Klotho is not significantly expressed in podocytes, and transgenic mice with either a targeted deletion or overexpression of Klotho in podocytes lack a glomerular phenotype and have no altered susceptibility to glomerular injury. In contrast, mice with hepatocyte-specific overexpression of Klotho have high circulating levels of soluble Klotho, and when challenged with nephrotoxic serum have less albuminuria and less severe kidney injury compared to wildtype mice. RNA-seq analysis suggests an adaptive response to increased endoplasmic reticulum stress as a putative mechanism of action. To evaluate the clinical relevance of our findings, the results were validated in patients with diabetic nephropathy, and in precision cut kidney slices from human nephrectomies. Together, our data reveal that the glomeruloprotective effects of Klotho is mediated via endocrine actions, which increases its therapeutic potential for patients with glomerular diseases.

Decellularization of precision-cut kidney slices—application of physical and chemical methods

The extracellular matrix (ECM) obtained by decellularization provides scaffolds with the natural complex architecture and biochemical composition of the target organ. Whole kidney decellularization by perfusion uses the vasculature to remove cells leaving a scaffold that can be recellularized with patient-specific cells. However, decellularization and recellularization are highly complex processes that require intensive optimization of various parameters. In pursuit of this, a huge number of animals must be sacrificed. Therefore, we used precision-cut kidney slices (PCKS) as a source of natural scaffolds, which were decellularized by immersion in chemical reagents allowing the examination of more parameters with less animals. However, chemical reagents have a damaging effect on the structure and components of the ECM. Therefore, this study aimed at investigating the effects of physical treatment methods on the effectiveness of PCKS decellularization by immersion in chemical reagents (CHEM). PCKS were treated physically before or during immersion in chemicals (CHEM) with high hydrostatic pressure (HHP), freezing–thawing cycles (FTC) or in an ultrasonic bath system (UBS). Biochemical and DNA quantification as well as structural evaluation with conventional histology and scanning electron microscopy (SEM) were performed. Compared to decellularization by CHEM alone, FTC treatment prior to CHEM was the most effective in reducing DNA while also preserving glycosaminoglycan (GAG) content. Moreover, while UBS resulted in a comparable reduction of DNA, it was the least effective in retaining GAGs. In contrast, despite the pretreatment with HHP with pressures up to 200 MPa, it was the least effective in DNA removal. Histological scoring showed that HHP scaffolds received the best score followed by UBS, FTC and CHEM scaffolds. However further analysis with SEM demonstrated a higher deterioration of the ultrastructure in UBS scaffolds. Altogether, pretreatment with FTC prior to CHEM resulted in a better balance between DNA removal and structural preservation.

247.11: The Effect of Different Nutrients on Mitochondrial Function During Long Term Incubation of Precision-Cut Kidney Slices

247.11: The Effect of Different Nutrients on Mitochondrial Function During Long Term Incubation of Precision-Cut Kidney Slices

MO063: A New Tool for Preclinical Research and Drug Discovery: Extracellular Matrix Remodeling Quantification in Human Precision-Cut Kidney Slices

Abstract BACKGROUND AND AIMS Accumulation of extracellular matrix (ECM) proteins is a hallmark of renal fibrosis, leading to altered tissue homeostasis, kidney failure and ultimately death. Current treatment strategies are ineffective in halting renal fibrogenesis, and drug discovery is hampered by the lack of clinically relevant biomarkers. Thus, there is an urgent need for early and pharmacodynamic biomarkers of renal fibrosis to support the development of new therapeutic treatments. In this study, we explored the use of non-invasive biomarkers of ECM turnover in human precision-cut kidney slices (PCKS): we observed the release of these markers over time in the slice supernatants and measured the changes following treatment with a prostaglandin E2 (PGE2) EP1 receptor antagonist. METHOD PCKS were prepared from healthy and fibrotic human kidney tissue obtained from patients treated at Aarhus University Hospital, Skejby. The slices were cultured in 48-well plates in medium (300 µl/well) and incubated for up to 72 hours to stimulate the development of renal fibrosis. The medium was collected at hours 0, 24, 48 and 72. Biomarkers of collagen type I (PRO-C1), III (PRO-C3), fibronectin (FN; FBN-C) and α-SMA formation and MMP-mediated degradation of collagen type I (C1M), III (C3M) and IV (Tumstatin; TUM) were measured in the medium by ELISA assays developed at Nordic Bioscience. mRNA expression levels of COL1A1, FN and α-SMA were measured by qPCR at all time points. Viability was assessed by measuring the ATP content of the slices at all time points. The anti-fibrotic effect of SC-19 220—a PGE2 EP1 receptor antagonist—was studied in fibrotic PCKS at hours 24 and 48 after treatment. RESULTS Fibrosis developed spontaneously in human kidney slices, as shown by increasing levels of PRO-C1 (P < 0.0001), PRO-C3 (P < 0.0001), FBN-C (P < 0.0001) and α-SMA (P < 0.0001) over time. Levels of C1M (not significant) and C3M (P < 0.01) did not increase to the same extent, and levels of TUM decreased over time (P < 0.001), indicating that this model reflects the process of fibrogenesis over fibrolysis. In addition, protein levels of PRO-C1 and FBN-C correlated significantly with mRNA levels of COL1A1 (r = 0.92, P = 0.0088) and FN (r = 0.86, P = 0.0277), respectively. Treatment with SC-19 220 reduced the gene expression of COL1A1, FN and α-SMA, and at a protein level it decreased levels of PRO-C1, PRO-C3 and FBN-C. PCKS retained constant ATP levels up to 72 hours. CONCLUSION In conclusion, we demonstrated that quantification of ECM remodeling in human PCKS via noninvasive biomarkers can be a valuable preclinical tool to study renal fibrogenesis and the effect of anti-fibrotic treatments. This study highlights that the EP1 receptor is a promising target for halting the production of ECM proteins. Since this is a human model, results may be translated to clinical care with higher confidence.

Exploring Porcine Precision-Cut Kidney Slices as a Model for Transplant-Related Ischemia-Reperfusion Injury

Marginal donor kidneys are more likely to develop ischemia-reperfusion injury (IRI), resulting in inferior long-term outcomes. Perfusion techniques are used to attenuate IRI and improve graft quality. However, machine perfusion is still in its infancy, and more research is required for optimal conditions and potential repairing therapies. Experimental machine perfusion using porcine kidneys is a great way to investigate transplant-related IRI, but these experiments are costly and time-consuming. Therefore, an intermediate model to study IRI would be of great value. We developed a precision-cut kidney slice (PCKS) model that resembles ischemia-reperfusion and provides opportunities for studying multiple interventions simultaneously. Porcine kidneys were procured from a local slaughterhouse, exposed to 30 min of warm ischemia, and cold preserved. Subsequently, PCKS were prepared and incubated under various conditions. Adenosine triphosphate (ATP) levels and histological tissue integrity were assessed for renal viability and injury. Slicing did not influence tissue viability, and PCKS remained viable up to 72 h incubation with significantly increased ATP levels. Hypothermic and normothermic incubation led to significantly higher ATP levels than baseline. William’s medium E supplemented with Ciprofloxacin (and Amphotericin-B) provided the most beneficial condition for incubation of porcine PCKS. The porcine PCKS model can be used for studying transplant IRI.

EP1 receptor antagonism mitigates early and late stage renal fibrosis.

AimRenal fibrosis is a major driver of chronic kidney disease, yet current treatment strategies are ineffective in attenuating fibrogenesis. The cyclooxygenase/prostaglandin system plays a key role in renal injury and holds great promise as a therapeutic target. Here, we used a translational approach to evaluate the role of the PGE2‐EP1 receptor in the pathogenesis of renal fibrosis in several models of kidney injury, including human (fibrotic) kidney slices.MethodsThe anti‐fibrotic efficacy of a selective EP1 receptor antagonist (SC‐19220) was studied in mice subjected to unilateral ureteral obstruction (UUO), healthy and fibrotic human precision‐cut kidney slices (PCKS), Madin‐Darby Canine Kidney (MDCK) cells and primary human renal fibroblasts (HRFs). Fibrosis was evaluated on gene and protein level using qPCR, western blot and immunostaining.ResultsEP1 receptor inhibition diminished fibrosis in UUO mice, illustrated by a decreased protein expression of fibronectin (FN) and α‐smooth muscle actin (αSMA) and a reduction in collagen deposition. Moreover, treatment of healthy human PCKS with SC‐19220 reduced TGF‐β‐induced fibrosis as shown by decreased expression of collagen 1A1, FN and αSMA as well as reduced collagen deposition. Similar observations were made using fibrotic human PCKS. In addition, SC‐19220 reduced TGF‐β‐induced FN expression in MDCK cells and HRFs.ConclusionThis study highlights the EP1 receptor as a promising target for preventing both the onset and late stage of renal fibrosis. Moreover, we provide strong evidence that the effect of SC‐19220 may translate to clinical care since its effects were observed in UUO mice, cells and human kidney slices.

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.

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.

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.

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.

Inhibition of tyrosine kinase receptor signaling attenuates fibrogenesis in an ex vivo model of human renal fibrosis.

Poor translation from animal studies to human clinical trials is one of the main hurdles in the development of new drugs. Here, we used precision-cut kidney slices (PCKS) as a translational model to study renal fibrosis and to investigate whether inhibition of tyrosine kinase receptors, with the selective inhibitor nintedanib, can halt fibrosis in murine and human PCKS. We used renal tissue of murine and human origins to obtain PCKS. Control slices and slices treated with nintedanib were studied to assess viability, activation of tyrosine kinase receptors, cell proliferation, collagen type I accumulation, and gene and protein regulation. During culture, PCKS spontaneously develop a fibrotic response that resembles in vivo fibrogenesis. Nintedanib blocked culture-induced phosphorylation of platelet-derived growth factor receptor and vascular endothelial growth factor receptor. Furthermore, nintedanib inhibited cell proliferation and reduced collagen type I accumulation and expression of fibrosis-related genes in healthy murine and human PCKS. Modulation of extracellular matrix homeostasis was achieved already at 0.1 μM, whereas high concentrations (1 and 5 μM) elicited possible nonselective effects. In PCKS from human diseased renal tissue, nintedanib showed limited capacity to reverse established fibrosis. In conclusion, nintedanib attenuated the onset of fibrosis in both murine and human PCKS by inhibiting the phosphorylation of tyrosine kinase receptors; however, the reversal of established fibrosis was not achieved.

The nucleic acid binding protein YB-1–controlled expression of CXCL-1 modulates kidney damage in liver fibrosis

Acute kidney injury is a common complication of advanced liver disease and increased mortality of these patients. Here, we analyzed the role of Y-box protein-1 (YB-1), a nucleic acid binding protein, in the bile duct ligation model of liver fibrosis and monitored liver and subsequent kidney damage. Following bile duct ligation, both serum levels of liver enzymes and expression of hepatic extracellular matrix components such as type I collagen were significantly reduced in mice with half-maximal YB-1 expression (Yb1+/-) as compared to their wild-type littermates. By contrast, expression of the chemokine CXCL1 was significantly augmented in these Yb1+/- mice. YB-1 was identified as a potent transcriptional repressor of the Cxcl1 gene. Precision-cut kidney slices from Yb1+/- mice revealed higher expression of the CXCL1 receptor CXCR2 as well as enhanced responsivity to CXCL1 compared to those from wild-type mice. Increased CXCL1 content in Yb1+/- mice led to pronounced bile duct ligation-induced damage of the kidneys monitored as parameters of tubular epithelial injury and immune cell infiltration. Pharmacological blockade of CXCR2 as well as application of an inhibitory anti-CXCL1 antibody significantly mitigated early systemic effects on the kidneys following bile duct ligation whereas it had only a modest impact on hepatic inflammation and function. Thus, our analyses provide direct evidence that YB-1 crucially contributes to hepatic fibrosis and modulates liver-kidney crosstalk by maintaining tight control over chemokine CXCL1 expression.

Protective effects of phenolic acids on mercury-induced DNA damage in precision-cut kidney slices.

Objective(s):Precision-cut tissue slices are considered an organotypic 3D model widely used in biomedical research. The comet assay is an important screening test for early genotoxicity risk assessment that is mainly applied on in vitro models. The aim of the present study was to provide a 3D organ system for determination of genotoxicity using a modified method of the comet assay since the stromal components from the original tissue make this technique complicated.Materials and Methods:A modified comet assay technique was validated using precision-cut hamster kidney slices to analyze the antigenotoxic effect of the phenolic compounds caffeic acid, chlorogenic acid, and rosmarinic acid in tissue slices incubated with 15 µM HgCl2. Cytotoxicity of the phenolic compounds was studied in Vero cells, and by morphologic analysis in tissue slices co-incubated with HgCl2 and phenolic compounds. Results:A modification of the comet assay allows obtaining better and clear comet profiles for analysis. Non-cytotoxic concentrations of phenolic acids protected kidney tissue slices against mercury-induced DNA damage, and at the same time, were not nephrotoxic. The highest protection was provided by 3 µg/ml caffeic acid, although 6 µg/ml rosmarinic and 9 µg/ml chlorogenic acids also exhibited protective effects.Conclusion:This is the first time that a modification of the comet assay technique is reported as a tool to visualize the comets from kidney tissue slices in a clear and simple way. The phenolic compounds tested in this study provided protection against mercury-induced genotoxic damage in precision-cut kidney slices.