Obstructive nephropathy and molecular pathophysiology of renal interstitial fibrosis.

The kidneys play a key role in maintaining total body homeostasis. The complexity of this task is reflected in the unique architecture of the organ. Ureteral obstruction greatly affects renal physiology by altering hemodynamics, changing glomerular filtration and renal metabolism, and inducing architectural malformations of the kidney parenchyma, most importantly renal fibrosis. Persisting pathological changes lead to chronic kidney disease, which currently affects ∼10% of the global population and is one of the major causes of death worldwide. Studies on the consequences of ureteral obstruction date back to the 1800s. Even today, experimental unilateral ureteral obstruction (UUO) remains the standard model for tubulointerstitial fibrosis. However, the model has certain limitations when it comes to studying tubular injury and repair, as well as a limited potential for human translation. Nevertheless, ureteral obstruction has provided the scientific community with a wealth of knowledge on renal (patho)physiology. With the introduction of advanced omics techniques, the classical UUO model has remained relevant to this day and has been instrumental in understanding renal fibrosis at the molecular, genomic, and cellular levels. This review details key concepts and recent advances in the understanding of obstructive nephropathy, highlighting the pathophysiological hallmarks responsible for the functional and architectural changes induced by ureteral obstruction, with a special emphasis on renal fibrosis.

Tissue engineering strategies combining molecular targets against inflammation and fibrosis, and umbilical cord blood stem cells to improve hampered muscle and skin regeneration following cleft repair.

Cleft lip with or without cleft palate is a congenital deformity that occurs in about 1 of 700 newborns, affecting the dentition, bone, skin, muscles and mucosa in the orofacial region. A cleft can give rise to problems with maxillofacial growth, dental development, speech, and eating, and can also cause hearing impairment. Surgical repair of the lip may lead to impaired regeneration of muscle and skin, fibrosis, and scar formation. This may result in hampered facial growth and dental development affecting oral function and lip and nose esthetics. Therefore, secondary surgery to correct the scar is often indicated. We will discuss the molecular and cellular pathways involved in facial and lip myogenesis, muscle anatomy in the normal and cleft lip, and complications following surgery. The aim of this review is to outline a novel molecular and cellular strategy to improve musculature and skin regeneration and to reduce scar formation following cleft repair. Orofacial clefting can be diagnosed in the fetus through prenatal ultrasound screening and allows planning for the harvesting of umbilical cord blood stem cells upon birth. Tissue engineering techniques using these cord blood stem cells and molecular targeting of inflammation and fibrosis during surgery may promote tissue regeneration. We expect that this novel strategy improves both muscle and skin regeneration, resulting in better function and esthetics after cleft repair.

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.

Targeting Oxidative Stress for the Treatment of Liver Fibrosis.

Oxidative stress is a reflection of the imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of the antioxidant system. Excessive ROS, generated from various endogenous oxidative biochemical enzymes, interferes with the normal function of liver-specific cells and presumably plays a role in the pathogenesis of liver fibrosis. Once exposed to harmful stimuli, Kupffer cells (KC) are the main effectors responsible for the generation of ROS, which consequently affect hepatic stellate cells (HSC) and hepatocytes. ROS-activated HSC undergo a phenotypic switch and deposit an excessive amount of extracellular matrix that alters the normal liver architecture and negatively affects liver function. Additionally, ROS stimulate necrosis and apoptosis of hepatocytes, which causes liver injury and leads to the progression of end-stage liver disease. In this review, we overview the role of ROS in liver fibrosis and discuss the promising therapeutic interventions related to oxidative stress. Most importantly, novel drugs that directly target the molecular pathways responsible for ROS generation, namely, mitochondrial dysfunction inhibitors, endoplasmic reticulum stress inhibitors, NADPH oxidase (NOX) inhibitors, and Toll-like receptor (TLR)-affecting agents, are reviewed in detail. In addition, challenges for targeting oxidative stress in the management of liver fibrosis are discussed.

Regional Differences in Human Intestinal Drug Metabolism.

The intestines are key for the absorption of nutrients and water as well as drug metabolism, and it is well known that there are clear differences in the expression profile of drug metabolism enzymes along the intestinal tract. Yet only a few studies have thoroughly investigated regional differences in human intestinal drug metabolism. In this study, we evaluated phase I and phase II metabolism in matched human ileum and colon precision-cut intestinal slices (PCIS). To this end, human PCIS were incubated for 3 hours with testosterone and 7-hydroxycoumarin (7-HC) to examine phase I and phase II metabolism, respectively. Metabolite formation was assessed by high-performance liquid chromatography analysis. Our results demonstrated that androstenedione, 6ß-hydroxytestosterone, 2ß-hydroxytestosterone, and 7-HC sulfate were predominantly formed in the ileum, while 15α-hydroxytestosterone and 7-HC glucuronide were mainly produced in the colon. Moreover, we also observed sex differences in phase II metabolite formation, which appeared to be higher in men compared with women. Taken together, we demonstrated that phase I metabolism predominantly occurs in ileum PCIS, while phase II metabolism mostly takes place in colon PCIS. Moreover, we revealed that human PCIS can be used to study both regional and sex differences in intestinal metabolism.

In vitro and ex vivo anti-fibrotic effects of LY2109761, a small molecule inhibitor against TGF-ß.

Fibrosis is a pathophysiological state characterized by the excessive formation/deposition of fibrous extracellular matrix. Transforming growth factor-beta (TGF-ß) is a central profibrotic mediator, and targeting TGF-ß is a promising strategy in the development of drugs for the treatment of fibrosis. Therefore, the effect of LY2109761, a small molecule inhibitor against TGF-ß with targets beyond TGF-ß signaling, on fibrogenesis was elucidated in vitro (HepG2 cells and LX-2 cells) and ex vivo (human and rat precision-cut liver slices). Our results displayed an anti-fibrotic effect of LY2109761, as it markedly down-regulated gene and protein expression of collagen type 1, as well as gene expression of the inhibitor of metalloproteinases 1. This effect on fibrosis markers was partially mediated by targeting TGF-ß signaling, seeing that LY2109761 inhibited TGF-ß1 gene expression and SMAD2 protein phosphorylation. Interestingly, particularly at a high concentration, LY2109761 decreased SMAD1 protein phosphorylation and gene expression of the inhibitor of DNA binding 1, which appeared to be TGF-ß-independent effects. In conclusion, LY2109761 exhibited preclinical anti-fibrotic effects via both TGF-ß-dependent and -independent pathways. These results illustrate that small molecule inhibitors directed against TGF-ß could possibly influence numerous signaling pathways and thereby mitigate fibrogenesis.

Hyperuricemia influences tryptophan metabolism via inhibition of multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP).

Hyperuricemia is related to a variety of pathologies, including chronic kidney disease (CKD). However, the pathophysiological mechanisms underlying disease development are not yet fully elucidated. Here, we studied the effect of hyperuricemia on tryptophan metabolism and the potential role herein of two important uric acid efflux transporters, multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP). Hyperuricemia was induced in mice by treatment with the uricase inhibitor oxonic acid, confirmed by the presence of urate crystals in the urine of treated animals. A transport assay, using membrane vesicles of cells overexpressing the transporters, revealed that uric acid inhibited substrate-specific transport by BCRP at clinically relevant concentrations (calculated IC50 value: 365±13µM), as was previously reported for MRP4. Moreover, we identified kynurenic acid as a novel substrate for MRP4 and BCRP. This finding was corroborated by increased plasma levels of kynurenic acid observed in Mrp4(-/-) (107±19nM; P=0.145) and Bcrp(-/-) mice (133±10nM; P=0.0007) compared to wild type animals (71±11nM). Hyperuricemia was associated with >1.5 fold increase in plasma kynurenine levels in all strains. Moreover, hyperuricemia led to elevated plasma kynurenic acid levels (128±13nM, P=0.005) in wild type mice but did not further increase kynurenic acid levels in knockout mice. Based on our results, we postulate that elevated uric acid levels hamper MRP4 and BCRP functioning, thereby promoting the retention of other potentially toxic substrates, including kynurenic acid, which could contribute to the development of CKD.

Standardized Protocol for the Preparation of Precision-Cut Kidney Slices: A Translational Model of Renal Fibrosis.

Renal fibrosis is a hallmark of progressive renal diseases. To date, there is a lack of effective therapeutics for the treatment of renal fibrosis, in part due to the scarcity of clinically relevant translational disease models. Since the early 1920s, hand-cut tissue slices have been used as a means to better understand organ (patho)physiology in a variety of scientific fields. From that time, the equipment and methodology for the preparation of tissue slices has continuously improved, thereby expanding the applicability of the model. Nowadays, precision-cut kidney slices (PCKS) have been demonstrated to be an extremely valuable translation model for renal (patho)physiology, bridging the gap between preclinical and clinical research. A key feature of PCKS is that the slices contain all cell types and acellular components of the whole organ in the original configuration while preserving cell-cell and cell-matrix interactions. In this chapter, we describe how to prepare PCKS and how the model can be implemented in fibrosis research.

Transcutaneous measurement of renal function in two rodent models of obstructive nephropathy.

OBJECTIVE: Glomerular filtration rate (GFR) is a key indicator of renal function. In both clinical practice and pre-clinical research, serum levels of endogenous filtration markers, such as creatinine, are often used to estimate GFR. However, these markers often do not reflect minor changes in renal function. In this study, we therefore set out to evaluate the applicability of transcutaneous GFR (tGFR) measurements to monitor the changes in renal function, as compared to plasma creatinine (pCreatinine), in two models of obstructive nephropathy, namely unilateral ureteral obstruction (UUO) or bilateral ureteral obstruction followed by release (BUO-R) in male Wistar rats. RESULTS: UUO animals showed a significant reduction in tGFR compared to baseline; whereas pCreatinine levels were not significantly changed. In BUO animals, tGFR drops 24 h post BUO and remains lower upon release of the obstruction until day 11. Concomitantly, pCreatinine levels were also increased 24 h after obstruction and 24 h post release, however after 4 days, pCreatinine returned to baseline levels. In conclusion, this study revealed that the tGFR method is superior at detecting minor changes in renal function as compared to pCreatinine measurements.

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

OBJECTIVE: Renal 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. RESULTS: Using Western blotting and gene expression arrays, we show that transforming growth factor-ß (TGF-ß) induced fibrosis in human PCKS. In addition, our results demonstrated that butaprost, SC-19220 and tamoxifen - all putative anti-fibrotic compounds - altered TGF-ß-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-ß/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.

Prostaglandin E2 receptors as therapeutic targets in renal fibrosis.

Prostaglandin E2 (PGE2), a lipid mediator produced by the cyclooxygenase enzyme system, is the main prostaglandin in the kidney. PGE2 is involved in various physiological and pathophysiological processes in the kidney, including renal hemodynamics, water and salt balance, and renal fibrosis-a key pathological feature of progressive kidney diseases. PGE2 functions by binding to four G-protein-coupled EP receptors (EP1 to EP4), which stimulate different intracellular signaling pathways. The intrarenal distribution of the four EP receptors as well as the different downstream signaling pathways associated with each receptor give rise to the distinct functional consequence of activating each receptor subtype. This review summarizes the current data on the renal expression of the four EP receptors and delineates the role of each receptor in renal fibrosis.

EP1 receptor antagonism mitigates early and late stage renal fibrosis.

AIM: Renal 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. METHODS: The 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. RESULTS: EP1 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-ß-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-ß-induced FN expression in MDCK cells and HRFs. CONCLUSION: This 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-ß-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-ß-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.

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

BACKGROUND AND PURPOSE: Renal 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 APPROACH: Female, 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 RESULTS: TAM 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-ß-induced fibrosis in human PCKS, irrespective of sex, yet interindividual differences in treatment response were observed. CONCLUSION AND IMPLICATIONS: TAM 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.

Estrogen regulates aquaporin-2 expression in the kidney.

Estrogens are primarily identified as sex hormones that, for a long time, have been known as important regulators of female reproductive physiology. However, our understanding of the role of estrogens has changed over the past years. It is now well accepted that estrogens are also involved in other physiological and pathological processes in both genders. This is due to the fact that estrogen can act both local as well as on a systemic level. Next to its role in reproductive physiology, there is accumulating evidence that estrogen influences multiple systems involved in water homeostasis. This chapter will delineate the regulatory effects of estrogen on the water channel aquaporin-2 (AQP2) found in the renal collecting duct. We will first provide an introduction to estrogen, the estrogen receptors and their role in renal physiology as well as describe the effect of selective estrogen receptor modulators (SERMs) on the kidney. Subsequently, we will focus on how estrogen and SERMs influence water balance and regulate AQP2 expression in principal cells of the collecting duct. Finally, we will describe how estrogen regulates AQP2 functionality in other organ systems.

Phenformin Attenuates Renal Injury in Unilateral Ureteral Obstructed Mice without Affecting Immune Cell Infiltration.

Phenformin and metformin are antihyperglycemic drugs that belong to the class of biguanides. Previously, we demonstrated that metformin elicits renoprotective effects in unilateral ureteral obstructed mice by reducing the infiltration of immune cells into the kidney. Since phenformin is a more potent drug as compared to metformin, we investigated the renoprotective properties of phenformin. We studied the efficacy of both drugs using mice that underwent unilateral ureteral obstruction. Renal damage was evaluated on RNA and protein level by qPCR, Western blotting, and immunohistochemistry. Moreover, we studied immune cell infiltration using flow cytometry. Both biguanides significantly reduced UUO-induced kidney injury, as illustrated by a reduction in KIM-1 protein expression. In addition, both metformin and phenformin impacted the gene expression of several inflammatory markers but to a different extent. Moreover, in contrast to metformin, phenformin did not impact immune cell infiltration into UUO kidneys. In conclusion, we demonstrated that phenformin has similar renoprotective effects as metformin, but the mechanism of action differs, and phenformin is more potent. The beneficial effects of phenformin are probably due to inhibition of the STAT3 pathway and mitochondrial complex I. Further research is needed to unveil the therapeutic potential of phenformin for the treatment of renal injury, either at low, non-toxic concentrations or as part of a combination therapy.

Host microbiota dictates the proinflammatory impact of LPS in the murine liver.

Gut microbiota can impact liver disease development via the gut-liver axis. Liver inflammation is a shared pathological event in various liver diseases and gut microbiota might influence this pathological process. In this study, we studied the influence of gut microbiota on the inflammatory response of the liver to lipopolysaccharide (LPS). The inflammatory response to LPS (1-10 µg/ml) of livers of specific-pathogen-free (SPF) or germ-free (GF) mice was evaluated ex vivo, using precision-cut liver slices (PCLS). LPS induced a more pronounced inflammatory response in GF PCLS than in SPF PCLS. Baseline TNF-α gene expression was significantly higher in GF slices as compared to SPF slices. LPS treatment induced TNF-α, IL-1ß, IL-6 and iNOS expression in both SPF and GF PCLS, but the increase was more intense in GF slices. The anti-inflammatory markers SOCS3 and IRAK-M gene expression was significantly higher in GF PCLS than SPF PCLS at 24h with 1 µg/ml LPS treatment, and IL-10 was not differently expressed in GF PCLS than SPF PCLS. In addition, TLR-4 mRNA, but not protein, at basal level was higher in GF slices than in SPF slices. Taken together, this study shows that, in mice, the host microbiota attenuates the pro-inflammatory impact of LPS in the liver, indicating a positive role of the gut microbiota on the immune homeostasis of the liver.

Exploring organ-specific features of fibrogenesis using murine precision-cut tissue slices.

Fibrosis is the hallmark of pathologic tissue remodelling in most chronic diseases. Despite advances in our understanding of the mechanisms of fibrosis, it remains uncured. Fibrogenic processes share conserved core cellular and molecular pathways across organs. In this study, we aimed to elucidate shared and organ-specific features of fibrosis using murine precision-cut tissue slices (PCTS) prepared from small intestine, liver and kidneys. PCTS displayed substantial differences in their baseline gene expression profiles: 70% of the extracellular matrix (ECM)-related genes were differentially expressed across the organs. Culture for 48 h induced significant changes in ECM regulation and triggered the onset of fibrogenesis in all PCTS in organ-specific manner. TGFß signalling was activated during 48 h culture in all PCTS. However, the degree of its involvement varied: both canonical and non-canonical TGFß pathways were activated in liver and kidney slices, while only canonical, Smad-dependent, cascade was involved in intestinal slices. The treatment with galunisertib blocked the TGFßRI/SMAD2 signalling in all PCTS, but attenuated culture-induced dysregulation of ECM homeostasis and mitigated the onset of fibrogenesis with organ-specificity. In conclusion, regardless the many common features in pathophysiology of organ fibrosis, PCTS displayed diversity in culture-induced responses and in response to the treatment with TGFßRI kinase inhibitor galunisertib, even though it targets a core fibrosis pathway. A clear understanding of the common and organ-specific features of fibrosis is the basis for developing novel antifibrotic therapies.