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1.
Am J Physiol Renal Physiol ; 327(1): F103-F112, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38779750

ABSTRACT

α-1-Microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme-binding, and mitochondrial protection properties. The investigational drug RMC-035, a modified therapeutic A1M protein, was assessed for biodistribution and pharmacological activity in a broad set of in vitro and in vivo experiments, supporting its clinical development. Efficacy and treatment posology were assessed in various models of kidney ischemia and reperfusion injury (IRI). Real-time glomerular filtration rate (GFR), functional renal biomarkers, tubular injury biomarkers (NGAL and KIM-1), and histopathology were evaluated. Fluorescently labeled RMC-035 was used to assess biodistribution. RMC-035 demonstrated consistent and reproducible kidney protection in rat IRI models as well as in a model of IRI imposed on renal impairment and in a mouse IRI model, where it reduced mortality. Its pharmacological activity was most pronounced with combined dosing pre- and post-ischemia and weaker with either pre- or post-ischemia dosing alone. RMC-035 rapidly distributed to the kidneys via glomerular filtration and selective luminal uptake by proximal tubular cells. IRI-induced expression of kidney heme oxygenase-1 was inhibited by RMC-035, consistent with its antioxidative properties. RMC-035 also dampened IRI-associated inflammation and improved mitochondrial function, as shown by tubular autofluorescence. Taken together, the efficacy of RMC-035 is congruent with its targeted mechanism(s) and biodistribution profile, supporting its further clinical evaluation as a novel kidney-protective therapy.NEW & NOTEWORTHY A therapeutic A1M protein variant (RMC-035) is currently in phase 2 clinical development for renal protection in patients undergoing open-chest cardiac surgery. It targets several key pathways underlying kidney injury in this patient group, including oxidative stress, heme toxicity, and mitochondrial dysfunction. RMC-035 is rapidly eliminated from plasma, distributing to kidney proximal tubules, and demonstrates dose-dependent efficacy in numerous models of ischemia-reperfusion injury, particularly when administered before ischemia. These results support its continued clinical evaluation.


Subject(s)
Alpha-Globulins , Kidney , Reperfusion Injury , Animals , Reperfusion Injury/pathology , Reperfusion Injury/metabolism , Reperfusion Injury/prevention & control , Reperfusion Injury/drug therapy , Alpha-Globulins/metabolism , Alpha-Globulins/pharmacology , Male , Kidney/drug effects , Kidney/pathology , Kidney/metabolism , Disease Models, Animal , Glomerular Filtration Rate/drug effects , Mice, Inbred C57BL , Humans , Mice , Heme Oxygenase-1/metabolism , Rats , Rats, Sprague-Dawley , Acute Kidney Injury/pathology , Acute Kidney Injury/metabolism , Acute Kidney Injury/drug therapy , Acute Kidney Injury/prevention & control , Tissue Distribution
2.
Commun Biol ; 6(1): 208, 2023 02 22.
Article in English | MEDLINE | ID: mdl-36813870

ABSTRACT

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.


Subject(s)
Diabetic Nephropathies , Podocytes , Humans , Mice , Animals , Kidney Glomerulus , Diabetic Nephropathies/metabolism , Kidney/metabolism , Albuminuria/metabolism , Mice, Transgenic , Mice, Knockout
4.
Acta Physiol (Oxf) ; 233(1): e13668, 2021 09.
Article in English | MEDLINE | ID: mdl-33900001

ABSTRACT

AIM: Hypoxia-inducible factors (HIFs) are O2 -sensitive transcription factors that regulate multiple biological processes which are essential for cellular adaptation to hypoxia. Small molecule inhibitors of HIF-prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-dependent transcriptional programs and have broad clinical potential. HIF-PHIs are currently in global late-stage clinical development for the treatment of anaemia associated with chronic kidney disease. Although the effects of hypoxia on renal haemodynamics and function have been studied in animal models and in humans living at high altitude, the effects of pharmacological HIF activation on renal haemodynamics, O2 metabolism and metabolic efficiency are not well understood. METHODS: Using a cross-sectional study design, we investigated renal haemodynamics, O2 metabolism, gene expression and NO production in healthy rats treated with different doses of HIF-PHIs roxadustat or molidustat compared to vehicle control. RESULTS: Systemic administration of roxadustat or molidustat resulted in a dose-dependent reduction in renovascular resistance (RVR). This was associated with increased glomerular filtration rate (GFR), urine flow and tubular sodium transport rate (TNa ). Although both total O2 delivery and TNa were increased, more O2 was extracted per transported sodium in rats treated with high-doses of HIF-PHIs, suggesting a reduction in metabolic efficiency. Changes in RVR and GFR were associated with increased nitric oxide (NO) generation and substantially suppressed by pharmacological inhibition of NO synthesis. CONCLUSIONS: Our data provide mechanistic insights into dose-dependent effects of short-term pharmacological HIF activation on renal haemodynamics, glomerular filtration and O2 metabolism and identify NO as a major mediator of these effects.


Subject(s)
Biological Phenomena , Renal Insufficiency, Chronic , Animals , Cross-Sectional Studies , Hypoxia-Inducible Factor-Proline Dioxygenases , Nitric Oxide , Prolyl Hydroxylases , Rats
5.
Int Immunopharmacol ; 78: 106042, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31812067

ABSTRACT

Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in αKlotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of αKlotho, a sight that may inspire novel therapeutic approaches.


Subject(s)
Acute Kidney Injury/immunology , Bone Marrow Transplantation/adverse effects , Glucuronidase/metabolism , Graft vs Host Disease/complications , Acute Kidney Injury/blood , Acute Kidney Injury/diagnosis , Acute Kidney Injury/pathology , Animals , Biomarkers/analysis , Biomarkers/metabolism , Disease Models, Animal , Down-Regulation/immunology , Female , Fibroblast Growth Factor-23 , Fibroblast Growth Factors/blood , Fibroblast Growth Factors/immunology , Graft vs Host Disease/blood , Graft vs Host Disease/immunology , Humans , Interferon-gamma/blood , Interferon-gamma/immunology , Kidney , Klotho Proteins , Lipocalin-2/analysis , Lipocalin-2/metabolism , Male , Mice , Transplantation, Homologous/adverse effects , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/immunology
6.
Kidney Int ; 92(6): 1370-1383, 2017 12.
Article in English | MEDLINE | ID: mdl-28847650

ABSTRACT

Hypoxia in the embryo is a frequent cause of intra-uterine growth retardation, low birth weight, and multiple organ defects. In the kidney, this can lead to low nephron endowment, predisposing to chronic kidney disease and arterial hypertension. A key component in cellular adaptation to hypoxia is the hypoxia-inducible factor pathway, which is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3. In the adult kidney, PHD oxygen sensors are differentially expressed in a cell type-dependent manner and control the production of erythropoietin in interstitial cells. However, the role of interstitial cell PHDs in renal development has not been examined. Here we used a genetic approach in mice to interrogate PHD function in FOXD1-expressing stroma during nephrogenesis. We demonstrate that PHD2 and PHD3 are essential for normal kidney development as the combined inactivation of stromal PHD2 and PHD3 resulted in renal failure that was associated with reduced kidney size, decreased numbers of glomeruli, and abnormal postnatal nephron formation. In contrast, nephrogenesis was normal in animals with individual PHD inactivation. We furthermore demonstrate that the defect in nephron formation in PHD2/PHD3 double mutants required intact hypoxia-inducible factor-2 signaling and was dependent on the extent of stromal hypoxia-inducible factor activation. Thus, hypoxia-inducible factor prolyl-4-hydroxylation in renal interstitial cells is critical for normal nephron formation.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/physiology , Kidney/growth & development , Procollagen-Proline Dioxygenase/physiology , Renal Insufficiency/genetics , Anemia/blood , Anemia/drug therapy , Anemia/etiology , Animals , Cell Hypoxia/physiology , Clinical Trials, Phase III as Topic , Disease Models, Animal , Enzyme Inhibitors/therapeutic use , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Humans , Hydroxylation/physiology , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Kidney/cytology , Kidney/metabolism , Kidney Diseases/complications , Kidney Diseases/drug therapy , Mice , Molecular Targeted Therapy/methods , Mutation , Organ Size/physiology , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Procollagen-Proline Dioxygenase/genetics , Renal Insufficiency/mortality , Renal Insufficiency/pathology , Stromal Cells/metabolism
7.
Nat Cell Biol ; 19(6): 639-652, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28530660

ABSTRACT

Loss-of-function (LOF) mutations in the endothelial cell (EC)-enriched gene endoglin (ENG) cause the human disease hereditary haemorrhagic telangiectasia-1, characterized by vascular malformations promoted by vascular endothelial growth factor A (VEGFA). How ENG deficiency alters EC behaviour to trigger these anomalies is not understood. Mosaic ENG deletion in the postnatal mouse rendered Eng LOF ECs insensitive to flow-mediated venous to arterial migration. Eng LOF ECs retained within arterioles acquired venous characteristics and secondary ENG-independent proliferation resulting in arteriovenous malformation (AVM). Analysis following simultaneous Eng LOF and overexpression (OE) revealed that ENG OE ECs dominate tip-cell positions and home preferentially to arteries. ENG knockdown altered VEGFA-mediated VEGFR2 kinetics and promoted AKT signalling. Blockage of PI(3)K/AKT partly normalized flow-directed migration of ENG LOF ECs in vitro and reduced the severity of AVM in vivo. This demonstrates the requirement of ENG in flow-mediated migration and modulation of VEGFR2 signalling in vascular patterning.


Subject(s)
Arteriovenous Malformations/prevention & control , Endoglin/metabolism , Endothelial Cells/metabolism , Neovascularization, Pathologic , Neovascularization, Physiologic , Signal Transduction , Telangiectasia, Hereditary Hemorrhagic/prevention & control , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Arteriovenous Malformations/genetics , Arteriovenous Malformations/metabolism , Arteriovenous Malformations/pathology , Cell Lineage , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Endoglin/deficiency , Endoglin/genetics , Endothelial Cells/pathology , Genetic Predisposition to Disease , Humans , Kinetics , Mice, Knockout , Phenotype , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Stress, Mechanical , Telangiectasia, Hereditary Hemorrhagic/genetics , Telangiectasia, Hereditary Hemorrhagic/metabolism , Telangiectasia, Hereditary Hemorrhagic/pathology , Tissue Culture Techniques , Transfection , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics
8.
J Transl Med ; 15(1): 47, 2017 02 23.
Article in English | MEDLINE | ID: mdl-28231806

ABSTRACT

BACKGROUND: Imatinib causes increased turnover of stromal collagen, reduces collagen fibril diameter, enhances extracellular fluid turnover and lowers interstitial fluid pressure (IFP) in the human colonic carcinoma KAT-4/HT-29 (KAT-4) xenograft model. METHODS: We compared the effects of imatinib on oxygen levels, vascular morphology and IFP in three experimental tumor models differing in their content of a collagenous extracellular matrix. RESULTS: Neither the KAT4 and CT-26 colonic carcinoma models, nor B16BB melanoma expressed PDGF ß-receptors in the malignant cells. KAT-4 tumors exhibited a well-developed ECM in contrast to the other two model systems. The collagen content was substantially higher in KAT-4 than in CT-26, while collagen was not detectable in B16BB tumors. The pO2 was on average 5.4, 13.9 and 19.3 mmHg in KAT-4, CT-26 and B16BB tumors, respectively. Treatment with imatinib resulted in similar pO2-levels in all three tumor models but only in KAT-4 tumors did the increase reach statistical significance. It is likely that after imatinib treatment the increase in pO2 in KAT-4 tumors is caused by increased blood flow due to reduced vascular resistance. This notion is supported by the significant reduction observed in IFP in KAT-4 tumors after imatinib treatment. Vessel area varied between 4.5 and 7% in the three tumor models and was not affected by imatinib treatment. Imatinib had no effect on the fraction of proliferating cells, whereas the fraction of apoptotic cells increased to a similar degree in all three tumor models. CONCLUSION: Our data suggest that the effects of imatinib on pO2-levels depend on a well-developed ECM and provide further support to the suggestion that imatinib acts by causing interstitial stroma cells to produce a less dense ECM, which would in turn allow for an increased blood flow. The potential of imatinib treatment to render solid tumors more accessible to conventional treatments would therefore depend on the degree of tumor desmoplasia.


Subject(s)
Colonic Neoplasms/metabolism , Extracellular Matrix/metabolism , Imatinib Mesylate/pharmacology , Neoplasms, Experimental/metabolism , Oxygen/pharmacology , Animals , Apoptosis/drug effects , Cell Line, Tumor , Collagen/metabolism , Colonic Neoplasms/blood supply , Colonic Neoplasms/pathology , Extracellular Fluid/drug effects , Extracellular Fluid/metabolism , Extracellular Matrix/drug effects , Mice, SCID , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/pathology , Pressure , Receptor, Platelet-Derived Growth Factor beta/metabolism , Stromal Cells/metabolism , Tumor Burden/drug effects , Water
9.
Clin Cancer Res ; 19(18): 5092-103, 2013 Sep 15.
Article in English | MEDLINE | ID: mdl-23864164

ABSTRACT

PURPOSE: Restoration of the p53 function in tumors is a promising therapeutic strategy due to the high potential of p53 as tumor suppressor and the fact that established tumors depend on p53 inactivation for their survival. Here, we addressed the question whether small molecule RITA can reactivate p53 in neuroblastoma and suppress the growth of neuroblastoma cells in vitro and in vivo. EXPERIMENTAL DESIGN: The ability of RITA to inhibit growth and to induce apoptosis was shown in seven neuroblastoma cell lines. Mechanistic studies were carried out to determine the p53 dependence and the molecular mechanism of RITA-induced apoptosis in neuroblastoma, using cell viability assays, RNAi silencing, co-immunoprecipitation, qPCR, and Western blotting analysis. In vivo experiments were conducted to study the effect of RITA on human neuroblastoma xenografts in mice. RESULTS: RITA induced p53-dependent apoptosis in a set of seven neuroblastoma cell lines, carrying wild-type or mutant p53; it activated p53 and triggered the expression of proapoptotic p53 target genes. Importantly, p53 activated by RITA inhibited several key oncogenes that are high-priority targets for pharmacologic anticancer strategies in neuroblastoma, including N-Myc, Aurora kinase, Mcl-1, Bcl-2, Wip-1, MDM2, and MDMX. Moreover, RITA had a strong antitumor effect in vivo. CONCLUSIONS: Reactivation of wild-type and mutant p53 resulting in the induction of proapoptotic factors along with ablation of key oncogenes by compounds such as RITA may be a highly effective strategy to treat neuroblastoma.


Subject(s)
Apoptosis/drug effects , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Oncogenes/drug effects , Piperazines/pharmacology , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Tumor Suppressor Protein p53/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Cell Proliferation/drug effects , Female , Humans , Immunoprecipitation , In Vitro Techniques , Mice , Mice, SCID , Mutation/genetics , Neuroblastoma/metabolism , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
10.
PLoS One ; 4(12): e8149, 2009 Dec 04.
Article in English | MEDLINE | ID: mdl-19997591

ABSTRACT

Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Extracellular Fluid/drug effects , Neoplasms, Experimental/drug therapy , Pressure , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Animals , Apoptosis/drug effects , Blood Volume/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Humans , Mice , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Pericytes/drug effects , Pericytes/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use
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