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Fibroblast growth factor (FGF) 23 is a bone-derived hormone that promotes renal phosphate excretion. Serum FGF23 is increased in chronic kidney disease (CKD) and contributes to pathologic cardiac hypertrophy by activating FGF receptor (FGFR) 4 on cardiac myocytes, which might lead to the high cardiovascular mortality in CKD patients. Increases in serum FGF23 levels have also been observed following endurance exercise and in pregnancy, which are scenarios of physiologic cardiac hypertrophy as an adaptive response of the heart to increased demand. To determine whether FGF23/FGFR4 contributes to physiologic cardiac hypertrophy, we studied FGFR4 knockout mice (FGFR4 -/- ) during late pregnancy. In comparison to virgin littermates, pregnant wild-type and FGFR4 -/- mice showed increases in serum FGF23 levels and heart weight; however, the elevation in myocyte area observed in pregnant wild-type mice was abrogated in pregnant FGFR4 -/- mice. This outcome was supported by treatments of cultured cardiac myocytes with serum from fed Burmese pythons, another model of physiologic hypertrophy, where the co-treatment with an FGFR4-specific inhibitor abrogated the serum-induced increase in cell area. Interestingly, we found that in pregnant mice, the heart, and not the bone, shows elevated FGF23 expression, and that increases in serum FGF23 are not accompanied by changes in phosphate metabolism. Our study suggests that in physiologic cardiac hypertrophy, the heart produces FGF23 that contributes to hypertrophic growth of cardiac myocytes in a paracrine and FGFR4-dependent manner, and that the kidney does not respond to heart-derived FGF23.

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating (Pi-regulating) hormone produced by bone. Hereditary hypophosphatemic disorders are associated with FGF23 excess, impaired skeletal growth, and osteomalacia. Blocking FGF23 became an effective therapeutic strategy in X-linked hypophosphatemia, but testing remains limited in autosomal recessive hypophosphatemic rickets (ARHR). This study investigates the effects of Pi repletion and bone-specific deletion of Fgf23 on bone and mineral metabolism in the dentin matrix protein 1-knockout (Dmp1KO) mouse model of ARHR. At 12 weeks, Dmp1KO mice showed increased serum FGF23 and parathyroid hormone levels, hypophosphatemia, impaired growth, rickets, and osteomalacia. Six weeks of dietary Pi supplementation exacerbated FGF23 production, hyperparathyroidism, renal Pi excretion, and osteomalacia. In contrast, osteocyte-specific deletion of Fgf23 resulted in a partial correction of FGF23 excess, which was sufficient to fully restore serum Pi levels but only partially corrected the bone phenotype. In vitro, we show that FGF23 directly impaired osteoprogenitors' differentiation and that DMP1 deficiency contributed to impaired mineralization independent of FGF23 or Pi levels. In conclusion, FGF23-induced hypophosphatemia is only partially responsible for the bone defects observed in Dmp1KO mice. Our data suggest that combined DMP1 repletion and FGF23 blockade could effectively correct ARHR-associated mineral and bone disorders.

Ischemia-reperfusion injury is a major cause of acute kidney injury. Many cytokines are involved in the pathogenesis of renal ischemia-reperfusion injury. IL24 is a member of the IL10 family and has gained importance because of its apoptosis-inducing effects in tumor disease besides its immunoregulative function. Littles is known about the role of IL24 in kidney disease. Using a mouse model, we found that IL24 is upregulated in the kidney after renal ischemia-reperfusion injury and that tubular epithelial cells and infiltrating inflammatory cells are the source of IL24. Mice lacking IL24 are protected from renal injury and inflammation. Cell culture studies showed that IL24 induces apoptosis in renal tubular epithelial cells, which is accompanied by an increased endoplasmatic reticulum stress response. Moreover, IL24 induces robust expression of endogenous IL24 in tubular cells, fostering ER-stress and apoptosis. In kidney transplant recipients with delayed graft function and patients at high risk to develop acute kidney injury after cardiac surgery IL24 is upregulated in the kidney and serum. Taken together, IL24 can serve as a biomarker, plays an important mechanistic role involving both extracellular and intracellular targets, and is a promising therapeutic target in patients at risk of or with ischemia-induced acute kidney injury., (© 2022 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Fibroblast growth factor (FGF) 21, a hormone that increases insulin sensitivity, has shown promise as a therapeutic agent to improve metabolic dysregulation. Here we report that FGF21 directly targets cardiac myocytes by binding β-klotho and FGF receptor (FGFR) 4. In combination with high glucose, FGF21 induces cardiac myocyte growth in width mediated by extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. While short-term FGF21 elevation can be cardio-protective, we find that in type 2 diabetes (T2D) in mice, where serum FGF21 levels are elevated, FGFR4 activation induces concentric cardiac hypertrophy. As T2D patients are at risk for heart failure with preserved ejection fraction (HFpEF), we propose that induction of concentric hypertrophy by elevated FGF21-FGFR4 signaling may constitute a novel mechanism promoting T2D-associated HFpEF such that FGFR4 blockade might serve as a cardio-protective therapy in T2D. In addition, potential adverse cardiac effects of FGF21 mimetics currently in clinical trials should be investigated., (© 2022. The Author(s).)

Elevations in plasma phosphate concentrations (hyperphosphatemia) occur in chronic kidney disease (CKD), in certain genetic disorders, and following the intake of a phosphate-rich diet. Whether hyperphosphatemia and/or associated changes in metabolic regulators, including elevations of fibroblast growth factor 23 (FGF23) directly contribute to specific complications of CKD is uncertain. Here, we report that similar to patients with CKD, mice with adenine-induced CKD develop inflammation, anemia, and skeletal muscle wasting. These complications are also observed in mice fed high phosphate diet even without CKD. Ablation of pathologic FGF23-FGFR4 signaling did not protect mice on an increased phosphate diet or mice with adenine-induced CKD from these sequelae. However, low phosphate diet ameliorated anemia and skeletal muscle wasting in a genetic mouse model of CKD. Our mechanistic in vitro studies indicate that phosphate elevations induce inflammatory signaling and increase hepcidin expression in hepatocytes, a potential causative link between hyperphosphatemia, anemia, and skeletal muscle dysfunction. Our study suggests that high phosphate intake, as caused by the consumption of processed food, may have harmful effects irrespective of pre-existing kidney injury, supporting not only the clinical utility of treating hyperphosphatemia in CKD patients but also arguing for limiting phosphate intake in healthy individuals., Competing Interests: BC, KH, IC, CY, DK, DW, GJ, IS, MH No competing interests declared, OG has received honoraria and grant support from Akebia and Amgen, grant support from GSK, honoraria from Ardelyx, Reata, and AstraZeneca, and serves on the Data Monitoring Committee for QED, JB has ownership interest in Ferrumax Pharmaceuticals and has been a consultant for Incyte Corporation, and Alnylam Pharmaceuticals, CF has served as a consultant for Bayer and Calico Labs, and he is the founder and currently the CSO of a startup biotech company (Alpha Young LLC), (© 2022, Czaya et al.)

In the context of transplantation, complement activation is associated with poor prognosis and outcome. While complement activation in antibody-mediated rejection is well-known, less is known about complement activation in acute T cell-mediated rejection (TCMR). There is increasing evidence that complement contributes to the clearance of apoptotic debris and tissue repair. In this regard, we have analysed published human kidney biopsy transcriptome data clearly showing upregulated expression of complement factors in TCMR. To clarify whether and how the complement system is activated early during acute TCMR, experimental syngeneic and allogeneic renal transplantations were performed. Using an allogeneic rat renal transplant model, we also observed upregulation of complement factors in TCMR in contrast to healthy kidneys and isograft controls. While staining for C4d was positive, staining with a C3d antibody showed no C3d deposition. FACS analysis of blood showed the absence of alloantibodies that could have explained the C4d deposition. Gene expression pathway analysis showed upregulation of pro-apoptotic factors in TCMR, and apoptotic endothelial cells were detected by ultrastructural analysis. Monocytes/macrophages were found to bind to and phagocytise these apoptotic cells. Therefore, we conclude that early C4d deposition in TCMR may be relevant to the clearance of apoptotic cells.

Purpose of Review: Chronic kidney disease-mineral and bone disorder (CKD-MBD) has become a global health crisis with very limited therapeutic options. Dentin matrix protein 1 (DMP1) is a matrix extracellular protein secreted by osteocytes that has generated recent interest for its possible involvement in CKD-MBD pathogenesis. This is a review of DMP1 established regulation and function, and early studies implicating DMP1 in CKD-MBD., Recent Findings: Patients and mice with CKD show perturbations of DMP1 expression in bone, associated with impaired osteocyte maturation, mineralization, and increased fibroblast growth factor 23 (FGF23) production. In humans with CKD, low circulating DMP1 levels are independently associated with increased cardiovascular events. We recently showed that DMP1 supplementation lowers circulating FGF23 levels and improves bone mineralization and cardiac outcomes in mice with CKD. Mortality rates are extremely high among patients with CKD and have only marginally improved over decades. Bone disease and FGF23 excess contribute to mortality in CKD by increasing the risk of bone fractures and cardiovascular disease, respectively. Previous studies focused on DMP1 loss-of-function mutations have established its role in the regulation of FGF23 and bone mineralization. Recent studies show that DMP1 supplementation may fill a crucial therapeutic gap by improving bone and cardiac health in CKD., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive muscle weakness. Skeletal muscle is a prime source for biomarker discovery since it is one of the earliest sites to manifest disease pathology. From a prior RNA sequencing project, we identified FGF23 as a potential muscle biomarker in ALS. Here, we validate this finding with a large collection of ALS muscle samples and found a 13-fold increase over normal controls. FGF23 was also increased in the SOD1 G93A mouse, beginning at a very early stage and well before the onset of clinical symptoms. FGF23 levels progressively increased through end-stage in the mouse. Immunohistochemistry of ALS muscle showed prominent FGF23 immunoreactivity in the endomysial connective tissue and along the muscle membrane and was significantly higher around grouped atrophic fibers compared to non-atrophic fibers. ELISA of plasma samples from the SOD1 G93A mouse showed an increase in FGF23 at end-stage whereas no increase was detected in a large cohort of ALS patients. In conclusion, FGF23 is a novel muscle biomarker in ALS and joins a molecular signature that emerges in very early preclinical stages. The early appearance of FGF23 and its progressive increase with disease progression offers a new direction for exploring the molecular basis and response to the underlying pathology of ALS.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia that mainly affects the elderly. Several reports have demonstrated that aging is involved in the underlying pathogenic mechanisms of IPF. α-Klotho (KL) has been well characterized as an "age-suppressing" hormone and can provide protection against cellular senescence and oxidative stress. In this study, KL levels were assessed in human plasma and primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF-FB) and in lung tissue from mice exposed to bleomycin, which showed significant downregulation when compared with controls. Conversely, transgenic mice overexpressing KL were protected against bleomycin-induced lung fibrosis. Treatment of human lung fibroblasts with recombinant KL alone was not sufficient to inhibit transforming growth factor-β (TGF-β)-induced collagen deposition and inflammatory marker expression. Interestingly, fibroblast growth factor 23 (FGF23), a proinflammatory circulating protein for which KL is a coreceptor, was upregulated in IPF and bleomycin lungs. To our surprise, FGF23 and KL coadministration led to a significant reduction in fibrosis and inflammation in IPF-FB; FGF23 administration alone or in combination with KL stimulated KL upregulation. We conclude that in IPF downregulation of KL may contribute to fibrosis and inflammation and FGF23 may act as a compensatory antifibrotic and anti-inflammatory mediator via inhibition of TGF-β signaling. Upon restoration of KL levels, the combination of FGF23 and KL leads to resolution of inflammation and fibrosis. Altogether, these data provide novel insight into the FGF23/KL axis and its antifibrotic/anti-inflammatory properties, which opens new avenues for potential therapies in aging-related diseases like IPF.

Kidney transplantation is the preferred treatment for patients with end-stage renal disease. Despite effective immunosuppressants, acute allograft rejections pose a major threat to graft survival. In early stages, acute rejections are still potentially reversible, and early detection is crucial to initiate the necessary treatment options and to prevent further graft dysfunction or even loss of the complete graft. Currently, invasive core needle biopsy is the reference standard to diagnose acute rejection. However, biopsies carry the risk of graft injuries and cannot be immediately performed on patients receiving anticoagulation drugs. Therefore, non-invasive assessment of the whole organ for specific and rapid detection of acute allograft rejection is desirable. We herein provide a review summarizing current imaging-based approaches for non-invasive diagnosis of acute renal allograft rejection., Competing Interests: None.

To date, allogeneic kidney transplantation remains the best available therapeutic option for patients with end-stage renal disease regarding overall survival and quality of life. Despite the advancements in immunosuppressive drugs and protocols, episodes of acute allograft rejection, a sterile inflammatory process, continue to endanger allograft survival. Since effective treatment for acute rejection episodes is available, instant diagnosis of this potentially reversible graft injury is imperative. Although histological examination by invasive core needle biopsy of the graft remains the gold standard for the diagnosis of ongoing rejection, it is always associated with the risk of causing substantial graft injury as a result of the biopsy procedure itself. At the same time, biopsies are not immediately feasible for a considerable number of patients taking anticoagulants due to the high risk of complications such as bleeding and uneven distribution of pathological changes within the graft. This can result in the wrong diagnosis due to the small size of the tissue sample taken. Therefore, there is a need for a tool that overcomes these problems by being noninvasive and capable of assessing the whole organ at the same time for specific and fast detection of acute allograft rejection. In this article, we review current state-of-the-art approaches for noninvasive diagnostics of acute renal transplant inflammation, i.e., rejection. We especially focus on nonradiation-based methods using magnetic resonance imaging (MRI) and ultrasound.

Acute cellular renal allograft rejection (AR) frequently occurs after kidney transplantations. It is a sterile T-cell mediated inflammation leading to increased local glucose metabolism. Here we demonstrate in an allogeneic model of Brown Norway rat kidneys transplanted into uninephrectomized Lewis rats the successful implementation of the recently developed glucose chemical exchange saturation transfer (glucoCEST) magnetic resonance imaging. This technique is a novel method to assess and differentiate AR. Renal allografts undergoing AR showed significantly increased glucoCEST contrast ratios of cortex to medulla of 1.61 compared to healthy controls (1.02), syngeneic Lewis kidney to Lewis rat transplants without rejection (0.92), kidneys with ischemia reperfusion injury (0.99) and kidneys affected by cyclosporine A toxicity (1.10). Receiver operating characteristic curve analysis showed an area under the curve value of 0.92, and the glucoCEST contrast ratio predicted AR with a sensitivity of 100% and a specificity of 69% at a threshold level over 1.08. In defined animal models of kidney injuries, the glucoCEST contrast ratios of cortex to medulla correlated positively with mRNA expression levels of T-cell markers (CD3, CD4, CD8a/b), but did not correlate to impaired renal perfusion. Thus, the glucoCEST parameter may be valuable for the assessment and follow up treatment of AR., (Copyright © 2017 International Society of Nephrology. All rights reserved.)

Kidney transplantation is the best available treatment for patients with end stage renal disease. Despite the introduction of effective immunosuppressant drugs, episodes of acute allograft rejection still endanger graft survival. Since efficient treatment of acute rejection is available, rapid diagnosis of this reversible graft injury is essential. For diagnosis of rejection, invasive core needle biopsy of the graft is the "gold-standard". However, biopsy carries the risk of significant graft injury and is not immediately feasible in patients taking anticoagulants. Therefore, a non-invasive tool assessing the whole organ for specific and fast detection of acute allograft rejection is desirable. We herein review current imaging-based state of the art approaches for non-invasive diagnostics of acute renal transplant rejection. We especially focus on new positron emission tomography-based as well as targeted ultrasound-based methods.

Purpose: We propose CD3-antibody-mediated contrast-enhanced ultrasonography using human T-lymphocytes for image-based diagnosis of acute allograft rejection (AR) established in a rat renal transplantation model., Materials and Methods: 15 minutes after tail vein injection of 30 × 10(6) human T-lymphocytes, contrast media/microbubbles conjugated with an anti-human CD3 antibody was applied to uni-nephrectomized 10-week-old allogeneically transplanted male rats (Lewis-Brown Norway (LBN) to Lewis, aTX) and ultrasound was performed to investigate the transplanted kidney as well as the native kidney. In vivo results were confirmed via immunohistochemical stainings of CD3 after post mortem dissection. Syngeneically transplanted rats (LBN to LBN, sTX), rats with ischemia/reperfusion injury (IRI, 45 min. warm ischemia), and rats subjected to acute cyclosporin A toxicity (CSA) (cyclosporine 50 mg/kg BW for 2 days i. p.) served as controls., Results: Accumulation of human T-lymphocytes was clearly detected by antibody-mediated sonography und was significantly increased in allografts undergoing AR (5.41 ± 1.32 A. U.) when compared to native control kidneys (0.70 ± 0.08 A. U.). CD3 signal intensity was low in native kidneys, sTX (0.99 ± 0.30 A. U.), CSA (0.10 ± 0.02 A. U.) and kidneys with IRI (0.46 ± 0.29 A. U.). Quantification of the ultrasound signal correlated significantly with the T-cell numbers obtained by immunohistochemical analysis (R2 = 0.57)., Conclusion: Contrast-enhanced sonography using CD3-antibodies is an option for quick and highly specific assessment of AR in a rat model of renal transplantation., (© Georg Thieme Verlag KG Stuttgart · New York.)

Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Chronic kidney disease (CKD) is associated with an increased risk of heart failure (HF). Elevated plasma concentrations of soluble Flt-1 (sFlt-1) have been linked to cardiovascular disease in CKD patients, but whether sFlt-1 contributes to HF in CKD is still unknown. To provide evidence that concludes a pathophysiological role of sFlt-1 in CKD-associated HF, we measured plasma sFlt-1 concentrations in 586 patients with angiographically documented coronary artery disease and renal function classified according to estimated glomerular filtration rate (eGFR). sFlt-1 concentrations correlated negatively with eGFR and were associated with signs of heart failure, based on New York Heart Association functional class and reduced left ventricular ejection fraction (LVEF), and early mortality. Additionally, rats treated with recombinant sFlt-1 showed a 15 % reduction in LVEF and a 29 % reduction in cardiac output compared with control rats. High sFlt-1 concentrations were associated with a 15 % reduction in heart capillary density (number of vessels/cardiomyocyte) and a 24 % reduction in myocardial blood volume. Electron microscopy and histological analysis revealed mitochondrial damage and interstitial fibrosis in the hearts of sFlt-1-treated, but not control rats. In 5/6-nephrectomised rats, an animal model of CKD, sFlt-1 antagonism with recombinant VEGF121 preserved heart microvasculature and significantly improved heart function. Overall, these findings suggest that a component of cardiovascular risk in CKD patients could be directly attributed to sFlt-1. Assessment of patients with CKD confirmed that sFlt-1 concentrations were inversely correlated with renal function, while studies in rats suggested that sFlt-1 may link microvascular disease with HF in CKD.

Background and Objectives: The endothelial glycocalyx (eGC), a mesh of anionic biopolymers covering the luminal surface of endothelial cells, is considered as an intravascular compartment that protects the vessel wall against pathogenic insults in cardiovascular disease. We hypothesized that chronic kidney disease (CKD) is associated with reduced eGC integrity and subsequent endothelial dysfunction., Methods & Results: Shedding of two major components of the eGC, namely syndecan-1 (Syn-1) and hyaluronan (HA), was measured by ELISA in 95 patients with CKD (stages 3-5) and 31 apparently healthy controls. Plasma levels of Syn-1 and HA increased steadily across CKD stages (5- and 5.5-fold, respectively P < 0.001) and were independently associated with impaired renal function after multivariate adjustment. Furthermore, Syn-1 and HA correlated tightly with plasma markers of endothelial dysfunction such as soluble fms-like tyrosine kinase-1 (sFlt-1), soluble vascular adhesion molecule-1 (sVCAM-1), von-Willebrand-Factor (vWF) and angiopoietin-2 (P < 0.001). Experimentally, excessive shedding of the eGC, evidenced by 11-fold increased Syn-1 plasma levels, was also observed in an established rat model of CKD, the 5/6-nephrectomized rats. Consistently, an atomic force microscopy-based approach evidenced a significant decrease in eGC thickness (360 ± 79 vs. 157 ± 29 nm, P = 0.001) and stiffness (0.33 ± 0.02 vs. 0.22 ± 0.01 pN/nm, P < 0.001) of aorta endothelial cell explants isolated from CKD rats., Conclusion: Our findings provide evidence for damage of the atheroprotective eGC as a consequence of CKD and potentially open a new avenue to pathophysiology and treatment of cardiovascular disease in renal patients., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Molecular imaging techniques such as single photon emission computed tomography (SPECT) or positron emission tomography are promising tools for noninvasive diagnosis of acute allograft rejection (AR). Given the importance of renal transplantation and the limitation of available donors, detailed analysis of factors that affect transplant survival is important. Episodes of acute allograft rejection are a negative prognostic factor for long-term graft survival. Invasive core needle biopsies are still the "goldstandard" in rejection diagnostics. Nevertheless, they are cumbersome to the patient and carry the risk of significant graft injury. Notably, they cannot be performed on patients taking anticoagulant drugs. Therefore, a noninvasive tool assessing the whole organ for specific and fast detection of acute allograft rejection is desirable. We herein review SPECT- and PET-based approaches for noninvasive molecular imaging-based diagnostics of acute transplant rejection.

Unlabelled: We proposed small-animal PET with (18)F-FDG-labeled T lymphocytes as a new method for image-based diagnosis of acute allogeneic renal transplant rejection (AR) established in a rat model., Methods: One and 2 h after tail vein injection of 30 × 10(6) ex vivo (18)F-FDG-labeled human T cells into male 10-wk-old uninephrectomized, allogeneically transplanted rats (aTX; Lewis-brown Norway [LBN] to Lewis), whole-body radioactivity distribution was assessed in vivo by small-animal PET (postoperative day 4), and percentage injected dose (%ID) as a parameter of T-cell infiltration was assessed and compared between graft and native kidney. In vivo results were confirmed by autoradiography and staining of human CD3 after postmortem dissection. Syngeneically transplanted rats (sTX) (LBN to LBN), rats with ischemia-reperfusion injury (IRI) (45-min warm ischemia), and rats subjected to acute cyclosporine A (CSA) toxicity (50 mg/kg for 2 d intraperitoneally) served as controls., Results: The accumulation of labeled cells was significantly elevated in allografts with AR (1.07 ± 0.28 %ID), compared with native control kidneys (0.49 ± 0.18 %ID) (P < 0.0001). No differences were found among native controls, sTX, CSA toxicity, and kidneys with IRI. In vivo uptake of (18)F-FDG cells measured in the PET scanner correlated with results obtained by autoradiography, histologic evaluation, and polymerase chain reaction., Conclusion: We proposed graft PET imaging using (18)F-FDG-labeled T cells as a new option to detect rat renal AR with a low dose of (18)F-FDG in a noninvasive, fast, and specific manner in rats.

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function (1). At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models. Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR (2). We herein present in addition small animal positron emission tomography (PET) using (18)F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation (3). Further, this method can be applied for follow-up to improve monitoring of transplant rejection (4).

Chronic kidney disease (CKD) is a public health epidemic that increases risk of death due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiovascular disease in individuals with CKD. Elevated levels of FGF23 have been linked to greater risks of LVH and mortality in patients with CKD, but whether these risks represent causal effects of FGF23 is unknown. Here, we report that elevated FGF23 levels are independently associated with LVH in a large, racially diverse CKD cohort. FGF23 caused pathological hypertrophy of isolated rat cardiomyocytes via FGF receptor-dependent activation of the calcineurin-NFAT signaling pathway, but this effect was independent of klotho, the coreceptor for FGF23 in the kidney and parathyroid glands. Intramyocardial or intravenous injection of FGF23 in wild-type mice resulted in LVH, and klotho-deficient mice demonstrated elevated FGF23 levels and LVH. In an established animal model of CKD, treatment with an FGF-receptor blocker attenuated LVH, although no change in blood pressure was observed. These results unveil a klotho-independent, causal role for FGF23 in the pathogenesis of LVH and suggest that chronically elevated FGF23 levels contribute directly to high rates of LVH and mortality in individuals with CKD.

Aims: Chronic kidney disease is directly associated with cardiovascular complications. Heart remodelling, including fibrosis, hypertrophy, and decreased vascularization, is frequently present in renal diseases. Our objective was to investigate the impact of calcineurin inhibitors (CNI) on cardiac remodelling and function in a rat model of renal disease., Methods and Results: Male Sprague Dawley rats were divided into six groups: sham-operated rats, 5/6 nephrectomized rats (Nx) treated with vehicle, CNI (cyclosporine A 5.0 or 7.5, or tacrolimus 0.5 mg/kg/day) or hydralazine (20 mg/kg twice a day) for 14 days, starting on the day of surgery. Creatinine clearance was significantly lower and blood pressure significantly higher in Nx rats when compared with controls. Morphological and echocardiographic analyses revealed increased left ventricular hypertrophy and decreased number of capillaries in Nx rats. Treatment with CNI affected neither the renal function nor the blood pressure, but prevented the development of cardiac hypertrophy and improved vascularization. In addition, regional blood volume improved as confirmed by contrast agent-based echocardiography. Hydralazine treatment did not avoid heart remodelling in this model. Gene expression analysis verified a decrease in hypertrophic genes in the heart of CNI-treated rats, while pro-angiogenic and stem cell-related genes were upregulated. Moreover, mobilization of stem/progenitor cells was increased through manipulation of the CD26/SDF-1 system., Conclusion: We conclude from our studies that CNI-treatment significantly prevented cardiac remodelling and improved heart function in Nx rats without affecting renal function and blood pressure. This sheds new light on possible therapeutic strategies for renal patients at high cardiovascular risk.

Renal ischemia-reperfusion (IR) injury (IRI) is a common and important trigger of acute renal injury (AKI). It is inevitably linked to transplantation. Involving both, the innate and the adaptive immune response, IRI causes subsequent sterile inflammation. Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity. Considering the important role of cytoskeletal reorganization, mainly regulated by RhoGTPases, in the development of IRI we hypothesized that a preventive, selective inhibition of the Rho effector Rho-associated coiled coil containing protein kinase (ROCK) by hydroxyfasudil may improve renal IRI outcome. Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals. In addition, renal perfusion (as assessed by (18)F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, (18)F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis. We conclude from our study that ROCK-inhibition by hydroxyfasudil significantly improves kidney function in a rat model of acute renal IRI and is therefore a potential new therapeutic option in humans.

The number of patients necessitating treatment for end-stage organ failure, and therefore, the number of allograft recipients, increases. Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection (AR) is still a major risk for graft survival. Hence, early detection and treatment of AR is crucial to limit the inflammatory process and preserve the function of the transplant. Nowadays, AR can only be definitively diagnosed by biopsy. As an invasive procedure, biopsy carries the risk of significant graft injury and is not feasible in patients taking anticoagulant medication. Moreover, limited sampling site (randomly taken exceedingly small portions of tissue) may lead to false negative results, i.e., when rejection is focal or patchy. Thus, in diagnostics, entirely image-based methods would be superior. As AR is characterized by recruitment of activated leukocytes into the transplant several diagnostic strategies exist. We herein review the current approaches (experimental and clinical scenarios with a special focus on renal AR) in noninvasive molecular imaging-based diagnostics of acute AR using either single photon (gamma) imaging or positron emission tomography.

Background: Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs., Methods: We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry., Results: Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1--a cytokine responsible for EPC mobilization--is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1., Conclusions: We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.

Unlabelled: We propose (18)F-FDG PET as a method to monitor acute rejection of allogeneic renal transplants in a rat model., Methods: Allogeneically transplanted (aTX) rats (binephrectomized Lewis-brown Norway to Lewis) served as the renal transplant model. aTX rats treated with cyclosporine A (CSA) served as a therapy monitoring group. Healthy control rats, rats with acute CSA nephrotoxicity, rats with acute tubular necrosis, syngeneically transplanted (sTX) rats, and aTX rats treated with CSA since postoperative day 0 served as controls. After surgery, renal glucose metabolism was assessed in vivo serially up to postoperative day 7 by performing small-animal PET 3 h after intravenous injection of 30 MBq of (18)F-FDG. Mean radioactivity (cps/mm(3) of tissue) was measured and the percentage injected dose calculated. Results were confirmed by histologic, functional, and autoradiographic analysis., Results: Renal (18)F-FDG uptake was significantly elevated at postoperative day 4 in aTX rats, when compared with control, sTX, acute tubular necrosis, or CSA-treated rats (P < 0.05). In vivo (18)F-FDG uptake correlated with the results of autoradiography and with inflammatory infiltrates observed on histologic examination. Notably, (18)F-FDG PET assessed the response to therapy 48 h earlier than the time at which serum creatinine decreased and when histologic examination still showed signs of allograft rejection. In aTX rats, the CSA-susceptible graft infiltrate was dominated by activated cytotoxic T cells and monocytes/macrophages., Conclusion: (18)F-FDG PET is an option to noninvasively assess early response to therapy in rat renal allograft rejection.

The epithelial sodium channel (ENaC) mediates the first step in Na+ reabsorption in epithelial cells such as kidney, colon, and airways and may consist of four homologous subunits (alpha, beta, gamma, delta). Predominantly, the alpha-subunit is expressed in these epithelia, and it usually forms functional channels with the beta- and gamma-subunits. The delta-subunit was first found in human brain and kidney, but the expression was also detected in human cell lines of lung, pancreatic, and colonic origin. When co-expressed with beta and gamma accessory subunits in heterologous systems, the two known isoforms of the delta-ENaC subunit (delta1 and delta2) can build amiloride-sensitive Na+ channels. In the present study we demonstrate the expression and function of the delta-subunit in human nasal epithelium (HNE). We cloned and sequenced the full-length cDNA of the delta-ENaC subunit and were able to show that in nasal tissue at least isoform 1 is expressed. Furthermore, we performed Western blot analyses and compared the cell surface expression of the delta-subunit with the classically expressed alpha-subunit by using immunofluorescence experiments. Thereby, we could show that the quantity of both subunits is almost similar. In addition, we show the functional expression of the delta-ENaC subunit with measurements in modified Ussing chambers, and demonstrate that in HNE a large portion of the Na+ transport is mediated by the delta-ENaC subunit. Therefore, we suppose that the delta-subunit may possess an important regulatory function and might interact with other ENaC subunits or members of the DEG/ENaC family in the human respiratory epithelium.