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Kidney diseases, including chronic kidney disease (CKD), diabetic nephropathy, and acute kidney injury (AKI), represent a significant global health burden. The kidneys are metabolically very active organs demanding a large amount of ATP. They are composed of highly specialized cell types in the glomerulus and subsequent tubular compartments which fine-tune metabolism to meet their numerous and diverse functions. Defective renal cell metabolism, including altered fatty acid oxidation or glycolysis, has been linked to both AKI and CKD. Mitochondria play a vital role in renal metabolism, and emerging research has identified mitochondrial sirtuins (SIRT3, SIRT4 and SIRT5) as key regulators of renal cell metabolic adaptation, especially SIRT3. Sirtuins belong to an evolutionarily conserved family of mainly NAD + -dependent deacetylases, deacylases, and ADP-ribosyl transferases. Their dependence on NAD + , used as a co-substrate, directly links their enzymatic activity to the metabolic status of the cell. In the kidney, SIRT3 has been described to play crucial roles in the regulation of mitochondrial function, and the antioxidative and antifibrotic response. SIRT3 has been found to be constantly downregulated in renal diseases. Genetic or pharmacologic upregulation of SIRT3 has also been associated with beneficial renal outcomes. Importantly, experimental pieces of evidence suggest that SIRT3 may act as an important energy sensor in renal cells by regulating the activity of key enzymes involved in metabolic adaptation. Activation of SIRT3 may thus represent an interesting strategy to ameliorate renal cell energetics. In this review, we discuss the roles of SIRT3 in lipid and glucose metabolism and in mediating a metabolic switch in a physiological and pathological context. Moreover, we highlight the emerging significance of other mitochondrial sirtuins, SIRT4 and SIRT5, in renal metabolism. Understanding the role of mitochondrial sirtuins in kidney diseases may also open new avenues for innovative and efficient therapeutic interventions and ultimately improve the management of renal injuries.

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent inherited muscle disorders and is linked to the inappropriate expression of the DUX4 transcription factor in skeletal muscles. The deregulated molecular network causing FSHD muscle dysfunction and pathology is not well understood. It has been shown that the hypoxia response factor HIF1α is critically disturbed in FSHD and has a major role in DUX4-induced cell death. In this study, we further explored the relationship between DUX4 and HIF1α. We found that the DUX4 and HIF1α link differed according to the stage of myogenic differentiation and was conserved between human and mouse muscle. Furthermore, we found that HIF1α knockdown in a mouse model of DUX4 local expression exacerbated DUX4-mediated muscle fibrosis. Our data indicate that the suggested role of HIF1α in DUX4 toxicity is complex and that targeting HIF1α might be challenging in the context of FSHD therapeutic approaches.

Introduction: COVID-19 vaccine was demonstrated to be effective in dialysis patients, but boosters are mandatory due to a rapid waning of anti-spike antibodies. A vaccination strategy based on immunologic response might be useful to maintain a favorable risk-benefit balance in this vulnerable population., Methods: CoviDial is an observational prospective study enrolling 121 dialysis patients to receive a 3-dose mRNA-1273 vaccine according to a uniform schedule. At baseline, months 1, 3, 6, 9, and 12, anti-spike antibodies against four epitopes (S1, S2, ECD-S1 + S2, RBD) were monitored with a multiplex immunodot enzymatic assay. Potential correlation between initial serologic response and subsequent COVID-19 infection was then assessed., Results: Overall, 96.2% and 96.8% of patients had anti-RBD antibodies at 3 and 12 months, respectively. All antibodies titers significantly decreased at month 6 compared to month 3. Booster vaccine induced a robust serologic response at month 9, but with a waning 3 months later, particularly for anti-S2 (37.2 ± 3.3 vs. 61.3 ± 3.0, p < 0.0001) and anti-S1 + S2 antibodies (68.4 ± 3.3 vs. 88.4 ± 2.3, p = 0.0015). Fifteen patients were later tested positive for SARS-CoV-2. At month 3, mean titers of anti-RBD, anti-S1 + S2, and anti-S2 antibodies were lower in the subsequent SARS-CoV-2 infected cohort (71.57 ± 9.01 vs. 85.79 ± 2.61, p = 0.0131; 41.07 ± 7.96 vs. 61.68 ± 3.56, p = 0.0237; 13.79 ± 5.03 vs. 39.70 ± 3.86, p = 0.0096; respectively)., Conclusion: Three doses of mRNA-1273 vaccine induce a robust but time-limited immunologic response in dialysis patients. Lower anti-spike antibodies titers after initial vaccination are associated with a higher risk to subsequently contract SARS-CoV-2, even beyond 6 months., (© 2023 The Authors. Seminars in Dialysis published by Wiley Periodicals LLC.)

Background: Hypoxia is known to modify skeletal muscle biological functions and muscle regeneration. However, the mechanisms underlying the effects of hypoxia on human myoblast differentiation remain unclear. The hypoxic response pathway is of particular interest in patients with hereditary muscular dystrophies since many present respiratory impairment and muscle regeneration defects. For example, an altered hypoxia response characterizes the muscles of patients with facioscapulohumeral dystrophy (FSHD)., Methods: We examined the impact of hypoxia on the differentiation of human immortalized myoblasts (LHCN-M2) cultured in normoxia (PO 2 : 21%) or hypoxia (PO 2 : 1%). Cells were grown in proliferation (myoblasts) or differentiation medium for 2 (myocytes) or 4 days (myotubes). We evaluated proliferation rate by EdU incorporation, used myogenin-positive nuclei as a differentiation marker for myocytes, and determined the fusion index and myosin heavy chain-positive area in myotubes. The contribution of HIF1α was studied by gain (CoCl 2 ) and loss (siRNAs) of function experiments. We further examined hypoxia in LHCN-M2-iDUX4 myoblasts with inducible expression of DUX4, the transcription factor underlying FSHD pathology., Results: We found that the hypoxic response did not impact myoblast proliferation but activated precocious myogenic differentiation and that HIF1α was critical for this process. Hypoxia also enhanced the late differentiation of human myocytes, but in an HIF1α-independent manner. Interestingly, the impact of hypoxia on muscle cell proliferation was influenced by dexamethasone. In the FSHD pathological context, DUX4 suppressed HIF1α-mediated precocious muscle differentiation., Conclusion: Hypoxia stimulates myogenic differentiation in healthy myoblasts, with HIF1α-dependent early steps. In FSHD, DUX4-HIF1α interplay indicates a novel mechanism by which DUX4 could interfere with HIF1α function in the myogenic program and therefore with FSHD muscle performance and regeneration., (© 2023. The Author(s).)

Background: Sex differences have been observed in the development of obesity-related complications in patients, as well as in animal models. Accumulating evidence suggests that sex-dependent regulation of lipid metabolism contributes to sex-specific physiopathology. Lipid accumulation in the renal tissue has been shown to play a major role in the pathogenesis of obesity-induced kidney injury. Unlike in males, the physiopathology of the disease has been poorly described in females, particularly regarding the lipid metabolism adaptation., Methods: Here, we compared the lipid profile changes in the kidneys of female and male mice fed a high-fat diet (HFD) or low-fat diet (LFD) by lipidomics and correlated them with pathophysiological changes., Results: We showed that HFD-fed female mice were protected from insulin resistance and hepatic steatosis compared to males, despite similar body weight gains. Females were particularly protected from renal dysfunction, oxidative stress, and tubular lipid accumulation. Both HFD-fed male and female mice presented dyslipidemia, but lipidomic analysis highlighted differential renal lipid profiles. While both sexes presented similar neutral lipid accumulation with obesity, only males showed increased levels of ceramides and phospholipids. Remarkably, protection against renal lipotoxicity in females was associated with enhanced renal adiponectin and AMP-activated protein kinase (AMPK) signaling. Circulating adiponectin and its renal receptor levels were significantly lower in obese males, but were maintained in females. This observation correlated with the maintained basal AMPK activity in obese female mice compared to males., Conclusions: Collectively, our findings suggest that female mice are protected from obesity-induced renal dysfunction and lipotoxicity associated with enhanced adiponectin and AMPK signaling compared to males., (© 2023. Society for Women's Health Research and BioMed Central Ltd.)

Background: We have previously demonstrated that double homeobox 4 centromeric (DUX4C) encoded for a functional DUX4c protein upregulated in dystrophic skeletal muscles. Based on gain- and loss-of-function studies we have proposed DUX4c involvement in muscle regeneration. Here, we provide further evidence for such a role in skeletal muscles from patients affected with facioscapulohumeral muscular dystrophy (FSHD)., Methods: DUX4c was studied at RNA and protein levels in FSHD muscle cell cultures and biopsies. Its protein partners were co-purified and identified by mass spectrometry. Endogenous DUX4c was detected in FSHD muscle sections with either its partners or regeneration markers using co-immunofluorescence or in situ proximity ligation assay., Results: We identified new alternatively spliced DUX4C transcripts and confirmed DUX4c immunodetection in rare FSHD muscle cells in primary culture. DUX4c was detected in nuclei, cytoplasm or at cell-cell contacts between myocytes and interacted sporadically with specific RNA-binding proteins involved, a.o., in muscle differentiation, repair, and mass maintenance. In FSHD muscle sections, DUX4c was found in fibers with unusual shape or central/delocalized nuclei (a regeneration feature) staining for developmental myosin heavy chain, MYOD or presenting intense desmin labeling. Some couples of myocytes/fibers locally exhibited peripheral DUX4c-positive areas that were very close to each other, but in distinct cells. MYOD or intense desmin staining at these locations suggested an imminent muscle cell fusion. We further demonstrated DUX4c interaction with its major protein partner, C1qBP, inside myocytes/myofibers that presented features of regeneration. On adjacent muscle sections, we could unexpectedly detect DUX4 (the FSHD causal protein) and its interaction with C1qBP in fusing myocytes/fibers., Conclusions: DUX4c upregulation in FSHD muscles suggests it contributes not only to the pathology but also, based on its protein partners and specific markers, to attempts at muscle regeneration. The presence of both DUX4 and DUX4c in regenerating FSHD muscle cells suggests DUX4 could compete with normal DUX4c functions, thus explaining why skeletal muscle is particularly sensitive to DUX4 toxicity. Caution should be exerted with therapeutic agents aiming for DUX4 suppression because they might also repress the highly similar DUX4c and interfere with its physiological role., (© 2023. The Author(s).)

Proprotein convertase substilisin/kexin 9 (PCSK9) inhibitors (PCSK9i) revolutionised the lipid-lowering therapy. However, a risk of type 2 diabetes mellitus (T2DM) is evoked under PCSK9i therapy. In this review, we summarise the current knowledge on the link of PCSK9 with T2DM. A significant correlation was found between PCSK9 and insulin, homeostasis model assessment (HOMA) of insulin resistance and glycated haemoglobin. PCSK9 is also involved in inflammation. PCSK9 loss-of-function variants increased T2DM risk by altering insulin secretion. Local pancreatic low PCSK9 regulates β-cell LDLR expression which in turn promotes intracellular cholesterol accumulation and hampers insulin secretion. Nevertheless, the association of PCSK9 loss-of-function variants and T2DM is inconsistent. InsLeu and R46L polymorphisms were associated with T2DM, low HOMA for β-cell function and impaired fasting glucose, while the C679X polymorphism was associated with low fasting glucose in Black South African people. Hence, we assume that the impact of these variants on glucose homeostasis may vary depending on the genetic background of the studied populations and the type of effect caused by those genetic variants on the PCSK9 protein. Accordingly, these factors should be considered when choosing a genetic variant of PCSK9 to assess the impact of long-term use of PCSK9i on glucose homeostasis.

Aristolochic acid nephropathy (AAN) is a progressive tubulointerstitial nephritis caused by the intake of aristolochic acids (AA) contained in Chinese herbal remedies or contaminated food. AAN is characterized by tubular atrophy and interstitial fibrosis, characterizing advanced kidney disease. It is established that sustained or recurrent acute kidney injury (AKI) episodes contribute to the progression of CKD. Therefore, the study of underlying mechanisms of AA-induced nephrotoxicity could be useful in understanding the complex AKI-to-CKD transition. We developed a translational approach of AKI-to-CKD transition by reproducing human AAN in rodent models. Indeed, in such models, an early phase of acute tubular necrosis was rapidly followed by a massive interstitial recruitment of activated monocytes/macrophages followed by cytotoxic T lymphocytes, resulting in a transient AKI episode. A later chronic phase was then observed with progressive tubular atrophy related to dedifferentiation and necrosis of tubular epithelial cells. The accumulation of vimentin and αSMA-positive cells expressing TGFβ in interstitial areas suggested an increase in resident fibroblasts and their activation into myofibroblasts resulting in collagen deposition and CKD. In addition, we identified 4 major actors in the AKI-to-CKD transition: (1) the tubular epithelial cells, (2) the endothelial cells of the interstitial capillary network, (3) the inflammatory infiltrate, and (4) the myofibroblasts. This review provides the most comprehensive and informative data we were able to collect and examines the pending questions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Baudoux, Jadot, Declèves, Antoine, Colet, Botton, De Prez, Pozdzik, Husson, Caron and Nortier.)

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial ROS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated mitochondrial ROS (mitoROS) levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitoROS, and affects mitochondrial health through lipid peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial ROS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O 2 tension is required., (Copyright © 2022. Published by Elsevier B.V.)

Muscular dystrophies (MDs) are a group of inherited degenerative muscle disorders characterized by a progressive skeletal muscle wasting. Respiratory impairments and subsequent hypoxemia are encountered in a significant subgroup of patients in almost all MD forms. In response to hypoxic stress, compensatory mechanisms are activated especially through Hypoxia-Inducible Factor 1 α (HIF-1α). In healthy muscle, hypoxia and HIF-1α activation are known to affect oxidative stress balance and metabolism. Recent evidence has also highlighted HIF-1α as a regulator of myogenesis and satellite cell function. However, the impact of HIF-1α pathway modifications in MDs remains to be investigated. Multifactorial pathological mechanisms could lead to HIF-1α activation in patient skeletal muscles. In addition to the genetic defect per se , respiratory failure or blood vessel alterations could modify hypoxia response pathways. Here, we will discuss the current knowledge about the hypoxia response pathway alterations in MDs and address whether such changes could influence MD pathophysiology.

(1) Background: White adipose tissue (WAT) is a dynamic and plastic tissue showing high sensitivity to carbohydrate supply. In such a context, the WAT may accordingly modulate its mitochondrial metabolic activity. We previously demonstrated that a partial replacement of glucose by galactose in a culture medium of 3T3-L1 cells leads to a poorer adipogenic yield and improved global mitochondrial health. In the present study, we investigate key mitochondrial metabolic actors reflecting mitochondrial adaptation in response to different carbohydrate supplies. (2) Methods: The metabolome of 3T3-L1 cells was investigated during the differentiation process using different glucose/galactose ratios and by a targeted approach using 1 H-NMR (Proton nuclear magnetic resonance) spectroscopy; (3) Results: Our findings indicate a reduction of adipogenic and metabolic overload markers under the low glucose/galactose condition. In addition, a remodeling of the mitochondrial function triggers the secretion of metabolites with signaling and systemic energetical homeostasis functions. Finally, this study also sheds light on a new way to consider the mitochondrial metabolic function by considering noncarbohydrates related pathways reflecting both healthier cellular and mitochondrial adaptation mechanisms; (4) Conclusions: Different carbohydrates supplies induce deep mitochondrial metabolic and function adaptations leading to overall adipocytes function and profile remodeling during the adipogenesis.

Exercise training is now recognized as an interesting therapeutic strategy in managing obesity and its related disorders. However, there is still a lack of knowledge about its impact on obesity-induced chronic kidney disease (CKD). Here, we investigated the effects of a delayed protocol of endurance exercise training (EET) as well as the underlying mechanism in obese mice presenting CKD. Mice fed a high-fat diet (HFD) or a low-fat diet (LFD) for 12 weeks were subsequently submitted to an 8-weeks EET protocol. Delayed treatment with EET in obese mice prevented body weight gain associated with a reduced calorie intake. EET intervention counteracted obesity-related disorders including glucose intolerance, insulin resistance, dyslipidaemia and hepatic steatosis. Moreover, our data demonstrated for the first time the beneficial effects of EET on obesity-induced CKD as evidenced by an improvement of obesity-related glomerulopathy, tubulo-interstitial fibrosis, inflammation and oxidative stress. EET also prevented renal lipid depositions in the proximal tubule. These results were associated with an improvement of the AMPK pathway by EET in renal tissue. AMPK-mediated phosphorylation of ACC and ULK-1 were particularly enhanced leading to increased fatty acid oxidation and autophagy improvement with EET in obese mice.

Chronic kidney disease (CKD) is prevalent in 9.1% of the global population and is a significant public health problem associated with increased morbidity and mortality. CKD is associated with highly prevalent physiological and metabolic disturbances such as hypertension, obesity, insulin resistance, cardiovascular disease, and aging, which are also risk factors for CKD pathogenesis and progression. Podocytes and proximal tubular cells of the kidney strongly express AMP-activated protein kinase (AMPK). AMPK plays essential roles in glucose and lipid metabolism, cell survival, growth, and inflammation. Thus, metabolic disease-induced renal diseases like obesity-related and diabetic chronic kidney disease demonstrate dysregulated AMPK in the kidney. Activating AMPK ameliorates the pathological and phenotypical features of both diseases. As a metabolic sensor, AMPK regulates active tubular transport and helps renal cells to survive low energy states. AMPK also exerts a key role in mitochondrial homeostasis and is known to regulate autophagy in mammalian cells. While the nutrient-sensing role of AMPK is critical in determining the fate of renal cells, the role of AMPK in kidney autophagy and mitochondrial quality control leading to pathology in metabolic disease-related CKD is not very clear and needs further investigation. This review highlights the crucial role of AMPK in renal cell dysfunction associated with metabolic diseases and aims to expand therapeutic strategies by understanding the molecular and cellular processes underlying CKD.

Obesity is an alarming yet increasing phenomenon worldwide, and more effective obesity management strategies have become essential. In addition to the numerous anti-adipogenic treatments promising a restauration of a healthy white adipose tissue (WAT) function, numerous studies reported on the critical role of nutritional parameters in obesity development. In a metabolic disorder context, a better control of nutrient intake is a key step in slowing down adipogenesis and therefore obesity. Of interest, the effect on WAT remodeling deserves deeper investigations. Among the different actors of WAT plasticity, the mitochondrial network plays a central role due to its dynamics and essential cellular functions. Hence, the present in vitro study, conducted on the 3T3-L1 cell line, aimed at evaluating the incidence of modulating the carbohydrates intake on adipogenesis through an integrated assessment of mitochondrial structure, dynamics, and functions-correlated changes. For this purpose, our experimental strategy was to compare the occurrence of adipogenesis in 3T3-L1 cells cultured either in a high-glucose (HG) medium (25 mM) or in a low-glucose (LG) medium (5 mM) supplemented with equivalent galactose (GAL) levels (20 mM). The present LG-GAL condition was associated, in differentiating adipocytes, to a reduced lipid droplet network, lower expressions of early and late adipogenic genes and proteins, an increased mitochondrial network with higher biogenesis marker expression, an equilibrium in the mitochondrial fusion/fission pattern, and a decreased expression of mitochondrial metabolic overload protein markers. Therefore, those main findings show a clear effect of modulating glucose accessibility on 3T3-L1 adipogenesis through a combined effect of adipogenesis modulation and overall improvement of the mitochondrial health status. This nutritional approach offers promising opportunities in the control and prevention of obesity.

Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose-response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is being intensively studied, particularly its evolution, in the increasingly available sequences between countries/continents with classical phylogenetic tree representation. More recently, certain protein mutations have been correlated with specific functional impacts. Our clinical data from patients suggest that clinical symptoms differ between European countries. Among other factors, SARS-CoV-2 mutations could explain these disparities. Our analyses point to an association of diverse mutations, including co-evolving ones, in a few SARS-CoV-2 proteins within specific countries. We therefore suggest combining clinical information from patients and the determination of the associated SARS-CoV-2 genome to better understand the specific symptoms., (© 2020 The Authors.)

Aristolochic acid nephropathy (AAN) is characterized by interstitial fibrosis, proximal tubular atrophy, and hypoxia. A correlation between a reduced peritubular capillary density and the severity of fibrosis has been demonstrated. As calcium, redox and energetic homeostasis are crucial in maintaining endothelial cell function and survival, we aimed to investigate AA-induced disturbances involved in endothelial cell injury. Our results showed a cytotoxic effect of AA on EAhy926 endothelial cells. Exposure of aortic rings to AA impaired vascular relaxation to Acetylcholine (ACh). Increased levels of intracellular reactive oxygen species (ROS) were observed in cells exposed to AA. Pre-treatment with antioxidant N-acetyl cysteine inhibited AA-induced cell death. Superoxide dismutase resulted in restoring ACh-induced relaxation. An increase in intracellular calcium level ([Ca 2+ ] i ) was observed on endothelial cells. Calcium chelators BAPTA-AM or APB, a specific inhibitor of IP 3 R, improved cell viability. Moreover, AA exposure led to reduced AMP-activated protein kinase (AMPK) expression. AICAR, an activator of AMPK, improved the viability of AA-intoxicated cells and inhibited the rise of cytosolic [Ca 2+ ] i levels. This study provides evidence that AA exposure increases ROS generation, disrupts calcium homeostasis and decreases AMPK activity. It also suggests that significant damage observed in endothelial cells may enhance microcirculation defects, worsening hypoxia and tubulointerstitial lesions., Competing Interests: Declaration of Competing Interest The authors declare that they have no knownfinancial or nonfinancial conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

High-fat diet (HFD) causes renal lipotoxicity that is ameliorated with AMP-activated protein kinase (AMPK) activation. Although bioactive eicosanoids increase with HFD and are essential in regulation of renal disease, their role in the inflammatory response to HFD-induced kidney disease and their modulation by AMPK activation remain unexplored. In a mouse model, we explored the effects of HFD on eicosanoid synthesis and the role of AMPK activation in ameliorating these changes. We used targeted lipidomic profiling with quantitative MS to determine PUFA and eicosanoid content in kidneys, urine, and renal arterial and venous circulation. HFD increased phospholipase expression as well as the total and free pro-inflammatory arachidonic acid (AA) and anti-inflammatory DHA in kidneys. Consistent with the parent PUFA levels, the AA- and DHA-derived lipoxygenase (LOX), cytochrome P450, and nonenzymatic degradation (NE) metabolites increased in kidneys with HFD, while EPA-derived LOX and NE metabolites decreased. Conversely, treatment with 5-aminoimidazole-4-carboxamide-1-β-D-furanosyl 5'-monophosphate (AICAR), an AMPK activator, reduced the free AA and DHA content and the DHA-derived metabolites in kidney. Interestingly, kidney and circulating AA, AA metabolites, EPA-derived LOX, and NE metabolites are increased with HFD; whereas, DHA metabolites are increased in kidney in contrast to their decreased circulating levels with HFD. Together, these changes showcase HFD-induced pro- and anti-inflammatory eicosanoid dysregulation and highlight the role of AMPK in correcting HFD-induced dysregulated eicosanoid pathways., (Copyright © 2019 Declèves et al.)

Aristolochic Acid (AA) nephropathy (AAN) is a progressive tubulointerstitial nephritis characterized by an early phase of acute kidney injury (AKI) leading to chronic kidney disease (CKD). The reduced nitric oxide (NO) bioavailability reported in AAN might contribute to renal function impairment and progression of the disease. We previously demonstrated that L-arginine (L-Arg) supplementation is protective in AA-induced AKI. Since the severity of AKI may be considered a strong predictor of progression to CKD, the present study aims to assess the potential benefit of L-Arg supplementation during the transition from the acute phase to the chronic phase of AAN. C57BL/6J male mice were randomly subjected to daily i.p. injections of vehicle or AA for 4 days. To determine whether renal AA-induced injuries were linked to reduced NO production, L-Arg was added to drinking water from 7 days before starting i.p. injections, until the end of the protocol. Mice were euthanized 5, 10 and 20 days after vehicle or AA administration. AA-treated mice displayed marked renal injury and reduced NO bioavailability, while histopathological features of AAN were reproduced, including interstitial cell infiltration and tubulointerstitial fibrosis. L-Arg treatment restored renal NO bioavailability and reduced the severity of AA-induced injury, inflammation and fibrosis. We concluded that reduced renal NO bioavailability contributes to the processes underlying AAN. Furthermore, L-Arg shows nephroprotective effects by decreasing the severity of acute-to-chronic transition in experimental AAN and might represent a potential therapeutic tool in the future.

The term "aristolochic acid nephropathy" (AAN) is used to include any form of toxic interstitial nephropathy that is caused either by ingestion of plants containing aristolochic acids (AA) as part of traditional phytotherapies (formerly known as "Chinese herbs nephropathy"), or by the environmental contaminants in food (Balkan endemic nephropathy). It is frequently associated with urothelial malignancies. Although products containing AA have been banned in most of countries, AAN cases remain regularly reported all over the world. Moreover, AAN incidence is probably highly underestimated given the presence of AA in traditional herbal remedies worldwide and the weak awareness of the disease. During these two past decades, animal models for AAN have been developed to investigate underlying molecular and cellular mechanisms involved in AAN pathogenesis. Indeed, a more-in-depth understanding of these processes is essential to develop therapeutic strategies aimed to reduce the global and underestimated burden of this disease. In this regard, our purpose was to build a broad overview of what is currently known about AAN. To achieve this goal, we aimed to summarize the latest data available about underlying pathophysiological mechanisms leading to AAN development with a particular emphasis on the imbalance between vasoactive factors as well as a focus on the vascular events often not considered in AAN.

Background: The platelet-derived growth factor receptor β (PDGFRβ)+ perivascular cell activation becomes increasingly recognized as a main source of scar-associated kidney myofibroblasts and recently emerged as a new cellular therapeutic target., Aims: In this regard, we first confirmed the presence of PDGFRβ+ perivascular cells in a human case of end-stage aristolochic acid nephropathy (AAN) and thereafter we focused on the early fibrosis events of transforming growth factor β (TGFβ) inhibition in a rat model of AAN., Materials and Methods: Neutralizing anti-TGFβ antibody (1D11) and its control isotype (13C4) were administered (5 mg/kg, i.p.) at Days -1, 0, 2 and 4; AA (15 mg/kg, sc) was injected daily., Results: At Day 5, 1D11 significantly suppressed p-Smad2/3 signaling pathway improving renal function impairment, reduced the score of acute tubular necrosis, peritubular capillaritis, interstitial inflammation and neoangiogenesis. 1D11 markedly decreased interstitial edema, disruption of tubular basement membrane loss of brush border, cytoplasmic edema and organelle ultrastructure alterations (mitochondrial disruption and endoplasmic reticulum edema) in proximal tubular epithelial cells. Moreover, 1D11 significantly inhibited p-PERK activation and attenuated dysregulation of unfolded protein response (UPR) pathways, endoplasmic reticulum and mitochondrial proteostasis in vivo and in vitro., Conclusions: The early inhibition of p-Smad2/3 signaling pathway improved acute renal function impairment, partially prevented epithelial-endothelial axis activation by maintaining PTEC proteostasis and reduced early PDGFRβ+ pericytes-derived myofibroblasts accumulation.

AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Aristolochic acids (AA) are nephrotoxic and profibrotic agents, leading to chronic kidney disease. As some controversial studies have reported a nephroprotective effect of exogenous recombinant human bone morphogenetic protein (rhBMP)-7 in several models of renal fibrosis, we investigated the putative effect of rhBMP-7 to prevent progressive tubulointerstitial damage after AA intoxication in vitro and in vivo. In vitro, the toxicity of AA on renal tubular cells was demonstrated by an increase in vimentin as well as a decrease in β-catenin expressions, reflecting a dedifferentiation process. Increased fibronectin and interleukin-6 levels were measured in the supernatants. Enhanced α-SMA mRNA levels associated to decreased E-cadherin mRNA levels were also measured. Incubation with rhBMP-7 only prevented the increase in vimentin and the decrease in β-catenin expressions. In vivo, in a rat model of AA nephropathy, severe tubulointerstitial lesions induced by AA after 10 and 35 days (collagen IV deposition and tubular atrophy), were not prevented by the rhBMP-7 treatment. Similarly, rhBMP-7 did not ameliorate the significant increase in urinary concentrations of transforming growth factor-β. In summary, our in vitro data demonstrated a poor beneficial effect of rhBMP-7 to reverse cell toxicity while, in vivo, there was no beneficial effect of rhBMP-7. Therefore, further investigations are needed to confirm the exact role of BMP-7 in progressive chronic kidney disease., (Copyright © 2015 John Wiley & Sons, Ltd.)

Renal ischemia-reperfusion injury (IRI) is a pathological process that may lead to acute renal failure and chronic dysfunction in renal allografts. During IRI, hyaluronan (HA) accumulates in the kidney, but suppression of HA accumulation during IRI protects the kidney from ischemic insults. Here we tested whether Hyal1-/- and Hyal2-/- mice display exacerbated renal damage following unilateral IRI due to a higher HA accumulation in the post-ischemic kidney compared with that in the kidney of wild-type mice. Two days after IRI in male mice there was accumulation of HA and CD44 in the kidney, marked tubular damage, infiltration, and increase creatininemia in wild-type mice. Knockout mice exhibited higher amounts of HA and higher creatininemia. Seven days after injury, wild-type mice had a significant decrease in renal damage, but knockout mice still displayed exacerbated inflammation. HA and CD44 together with α-smooth muscle actin and collagen types I and III expression were increased in knockout compared with wild-type mice 30 days after IRI. Thus, both HA-degrading enzymes seem to be protective against IRI most likely by reducing HA accumulation in the post-ischemic kidney and decreasing the inflammatory processes. Deficiency in either HYAL1 or HYAL2 leads to enhanced HA accumulation in the post-ischemic kidney and consequently worsened inflammatory response, increased tubular damage, and fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease with a poor prognosis and is characterized by the accumulation of fibrotic tissue in lungs resulting from a dysfunction in the healing process. In humans, the pathological process is patchy and temporally heterogeneous and the exact mechanisms remain poorly understood. Different animal models were thus developed. Among these, intratracheal administration of bleomycin (BML) is one of the most frequently used methods to induce lung fibrosis in rodents. In the present study, we first characterized histologically the time-course of lung alteration in rats submitted to BLM instillation. Heterogeneous damages were observed among lungs, consisting in an inflammatory phase at early time-points. It was followed by a transition to a fibrotic state characterized by an increased myofibroblast number and collagen accumulation. We then compared instillation and aerosolization routes of BLM administration. The fibrotic process was studied in each pulmonary lobe using a modified Ashcroft scale. The two quantification methods were confronted and the interobserver variability evaluated. Both methods induced fibrosis development as demonstrated by a similar progression of the highest modified Ashcroft score. However, we highlighted that aerosolization allows a more homogeneous distribution of lesions among lungs, with a persistence of higher grade damages upon time.

Purpose of Review: To provide a perspective by investigating the potential cross-talk between the adipose tissue and the kidney during obesity., Recent Findings: It is well established that excessive caloric intake contributes to organ injury. The associated increased adiposity initiates a cascade of cellular events that leads to progressive obesity-associated diseases such as kidney disease. Recent evidence has indicated that adipose tissue produces bioactive substances that contribute to obesity-related kidney disease, altering the renal function and structure. In parallel, proinflammatory processes within the adipose tissue can also lead to pathophysiological changes in the kidney during the obese state., Summary: Despite considerable efforts to better characterize the pathophysiology of obesity-related metabolic disease, there are still a lack of efficient therapeutic strategies. New strategies focused on regulating adipose function with respect to AMP-activated protein kinase activation, NADPH oxidase function, and TGF-β may contribute to reducing adipose inflammation that may also provide renoprotection.

Hyaluronidase 1 (HYAL1) and hyaluronidase 2 (HYAL2) are the major hyaluronidases acting synergistically to degrade hyaluronan (HA). In the kidney, HA is distributed heterogeneously. Our goal was to determine the consequences of a lack of either HYAL1 or HYAL2 (using specific knockout mice) on renal function and on renal HA accumulation. Experiments were performed in Hyal1(-/-) and Hyal2(-/-) mice and in their wild-type controls. HA concentration was measured in the plasma and kidney tissue and its distribution through the different kidney zones was examined by immunohistochemistry. Relative mRNA expressions of HYAL1, HYAL2 and the 3 main HA synthases were evaluated by quantitative RT-PCR. Results: Kidney function was not impaired in the knockout mice but they displayed elevated HA concentrations in the plasma and in the kidney. Hyal1(-/-) mice presented an accumulation of HA inside the proximal tubular cells whereas Hyal2(-/-) mice showed HA accumulation in the interstitial space. In the cortex and in the outer medulla, HYAL1 mRNA expression was up-regulated in Hyal2(-/-) mice. From our study we conclude that somatic hyaluronidases are not required for renal function. However, HYAL1 is necessary for the breakdown of intracellular HA in the cortex, whereas HYAL2 is essential for the degradation of extracellular HA in all kidney regions., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: Kidney ischemia-reperfusion is a form of acute kidney injury resulting in a cascade of cellular events prompting rapid cellular damage and suppression of kidney function. A cellular response to ischemic stress is the activation of AMP-activated protein kinase (AMPK), where AMPK induces a number of homeostatic and renoprotective mechanisms, including autophagy. However, whether autophagy is beneficial or detrimental in ischemia-reperfusion remains controversial. We investigated the effects of agonist induction of AMPK activity on autophagy and cell stress proteins in the model of kidney ischemia-reperfusion. Methods: AMPK agonists, AICAR (0.1 g/kg) and metformin (0.3 g/kg), were administered 24 h prior to ischemia, with kidneys harvested at 24 h of reperfusion. Results: We observed a paradoxical decrease in AMPK activity accompanied by increases in mammalian target of rapamycin (mTOR) C1 activity and p62/SQSTM1 expression. These results led us to propose that AMPK and autophagy are insufficient to properly counter the cellular insults in ischemia-reperfusion. Agonist induction of AMPK activity with AICAR or metformin increased macroautophagy protein LC3 and normalized p62/SQSTM1 expression and mTOR activity. Ischemia-reperfusion increases in Beclin-1 and PINK1 expressions, consistent with increased mitophagy, were also mitigated with AMPK agonists. Stress-responsive and apoptotic marker expressions increase in ischemia-reperfusion and are significantly attenuated with agonist administration, as are early indicators of fibrosis. Conclusions: Our data suggest that levels of renoprotective AMPK activity and canonical autophagy are insufficient to maintain cellular homeostasis, contributing to the progression of ischemia-reperfusion injury. We further demonstrate that induction of AMPK activity can provide beneficial cellular effects in containing injury in ischemia-reperfusion. © 2014 S. Karger AG, Basel.

The Wistar-Furth (WF) rat strain is usually used in models of full major histocompatibility complex-mismatched kidney transplantation. Because these rats have been demonstrated to be resistant to several models of chronic kidney disease, the aim of the present study was to investigate their potential resistance to renal ischaemia-reperfusion (I/R) injury compared with another strain, namely Wistar-Hanover (WH) rats. Anaesthetized male WH and WF rats were submitted to I/R by occlusion of the left renal artery and contralateral nephrectomy. Urine, blood and tissue samples were collected at different time points after I/R to evaluate renal function, inflammation and tubular injury, along with determination of nitric oxide synthase (NOS) expression and thromboxane A2 (TxA2 ) production. Post-ischaemic renal function was better preserved in WF than WH rats, as evidenced by reduced levels of creatininaemia, urinary neutrophil gelatinase-associated lipocalin excretion and proteinuria. In addition, WF rats had less intrarenal inflammation than WH rats after I/R injury. These observations were associated with maintenance of neuronal NOS expression, along with lower induction of inducible NOS expression in WF versus WH rats. Moreover, WF rats excreted a significantly lower amount of TxB2 . The results indicate that WF rats are more resistant to an I/R injury than WH rats in terms of renal function and inflammation. These observations are associated with differential regulation of intrarenal NOS expression, as well as a reduction in thromboxane production, which could contribute to a better outcome for the postischaemic kidney in WF rats., (© 2014 Wiley Publishing Asia Pty Ltd.)

The endoplasmic reticulum (ER) stress response is activated in the diabetic kidney and functions to reduce ER protein accumulation and improve cellular function. We previously showed that tribbles homolog 3 (TRB3), an ER stress-associated protein, is upregulated in the diabetic kidney. Here, we investigated whether absence of TRB3 alters outcomes in diabetic nephropathy. Type 1 diabetes was induced in TRB3 wild-type and knockout ((-/-)) mice by low-dose streptozotocin, and the mice were followed for 12 weeks. Diabetic TRB3(-/-) mice developed higher levels of albuminuria and increased expression of inflammatory cytokine and chemokine mRNA in renal cortices relative to wild-type littermates, despite similar hyperglycemia. Diabetic TRB3(-/-) mice also expressed higher levels of ER stress-associated molecules in both the renal cortices and glomeruli. This change was associated with higher renal cortical phosphorylation of AKT at serine 473 (Ser(473)), which is the AKT site phosphorylated by mammalian target of rapamycin complex-2 (mTORC2). We show in renal tubular cells that TRB3 binds to mTOR and the rapamycin-insensitive companion of mTOR (Rictor), a protein specific to mTORC2. Finally, we demonstrate in murine tubular cells that TRB3 can inhibit secretion of IL-6. Thus, TRB3 reduces albuminuria and inflammatory gene expression in diabetic kidney disease by a mechanism that may involve inhibition of the mTORC2/AKT pathway and may prove to be a novel therapeutic target., (Copyright © 2014 by the American Society of Nephrology.)

Chronic kidney disease (CKD) is becoming a worldwide epidemic, driven largely by the dramatic rise in the prevalence of diabetes and obesity. Novel targets and treatments for CKD are, therefore, desperately needed-to both mitigate the burden of this disease in the general population and reduce the necessity for renal replacement therapy in individual patients. This Review highlights new insights into the mechanisms that contribute to CKD, and approaches that might facilitate the development of disease-arresting therapies for CKD. Particular focus is given to therapeutic approaches using antifibrotic agents that target the transforming growth factor β superfamily. In addition, we discuss new insights regarding the roles of vascular calcification, the NADPH oxidase family, and inflammation in the pathogenesis of CKD. We also highlight a new understanding regarding kidney energy sensing pathways (AMPK, sirtuins, and mTOR) in a variety of kidney diseases and how they are linked to inflammation and fibrosis. Finally, exciting new insights have been made into the role of mitochondrial function and mitochondrial biogenesis in relation to progressive kidney disease. Prospective therapeutics based on these findings will hopefully renew hope for clinicians and patients in the near future.

AMP-activated protein kinase (AMPK) is an important energy sensor that may be critical in regulating renal lipid accumulation. To evaluate the role of AMPK in mediating renal lipid accumulation, C57BL/6J mice were randomized to a standard diet, a high-fat diet, or a high-fat diet plus AICAR (an AMPK activator) for 14 weeks. Renal functional and structural studies along with electron microscopy were performed. Mice given the high-fat diet had proximal tubule injury with the presence of enlarged clear vacuoles, and multilaminar inclusions concurrent with an increase of tissue lipid and overloading of the lysosomal system. The margins of the clear vacuoles were positive for the endolysosomal marker, LAMP1, suggesting lysosome accumulation. Characterization of vesicles by special stains (Oil Red O, Nile Red, Luxol Fast Blue) and by electron microscopy showed they contained onion skin-like accumulations consistent with phospholipids. Moreover, cholesteryl esters and phosphatidylcholine-containing phospholipids were significantly increased in the kidneys of mice on a high-fat diet. AMPK activation with chronic AICAR treatment prevented the clinical and structural effects of high-fat diet. Thus, high-fat diet contributes to a dysfunction of the lysosomal system and altered lipid metabolism characterized by cholesterol and phospholipid accumulation in the kidney. AMPK activation normalizes the changes in renal lipid content despite chronic exposure to lipid challenge.

Obesity-related kidney disease occurs as a result of complex interactions between metabolic and hemodynamic effects. Changes in microvascular perfusion may play a major role in kidney disease; however, these changes are difficult to assess in vivo. Here, we used perfusion ultrasound imaging to evaluate cortical blood flow in a mouse model of high-fat diet-induced kidney disease. C57BL/6J mice were randomized to a standard diet (STD) or a high-fat diet (HFD) for 30 wk and then treated either with losartan or a placebo for an additional 6 wk. Noninvasive ultrasound perfusion imaging of the kidney was performed during infusion of a microbubble contrast agent. Blood flow within the microvasculature of the renal cortex and medulla was derived from imaging data. An increase in the time required to achieve full cortical perfusion was observed for HFD mice relative to STD. This was reversed following treatment with losartan. These data were concurrent with an increased glomerular filtration rate in HFD mice compared with STD- or HFD-losartan-treated mice. Losartan treatment also abrogated fibro-inflammatory disease, assessed by markers at the protein and messenger level. Finally, a reduction in capillary density was found in HFD mice, and this was reversed upon losartan treatment. This suggests that alterations in vascular density may be responsible for the elevated perfusion time observed by imaging. These data demonstrate that ultrasound contrast imaging is a robust and sensitive method for evaluating changes in renal microvascular perfusion and that cortical perfusion time may be a useful parameter for evaluating obesity-related renal disease.

Background: Ischaemia-reperfusion injury (IRI) to the kidney is a complex pathophysiological process that leads to acute renal failure and chronic dysfunction in renal allografts. It was previously demonstrated that during IRI, hyaluronan (HA) accumulates in the cortical and external medullary interstitium along with an increased expression of its main receptor, CD44, on inflammatory and tubular cells. The HA-CD44 pair may be involved in persistent post-ischaemic inflammation. Thus, we sought to determine the role of HA in the pathophysiology of ischaemia-reperfusion (IR) by preventing its accumulation in post-ischaemic kidney., Methods: C57BL/6 mice received a diet containing 4-methylumbelliferone (4-MU), a potent HA synthesis inhibitor. At the end of the treatment, unilateral renal IR was induced and mice were euthanized 48 h or 30 days post-IR., Results: 4-MU treatment for 14 weeks reduced the plasma HA level and intra-renal HA content at 48 h post-IR, as well as CD44 expression, creatininemia and histopathological lesions. Moreover, inflammation was significantly attenuated and proliferation was reduced in animals treated with 4-MU. In addition, 4-MU-treated mice had a significantly reduced expression of α-SMA and collagen types I and III, i.e. less renal fibrosis, 30 days after IR compared with untreated mice., Conclusion: Our results demonstrate that HA plays a significant role in the pathogenesis of IRI, perhaps in part through reduced expression of CD44. The suppression of HA accumulation during IR may protect renal function against ischaemic insults.

Ultrasound can selectively and specifically visualize upregulated vascular receptors through the detection of bound microbubbles. However, most current ultrasound molecular imaging methods incur delays that result in longer acquisition times and reduced frame rates. These delays occur for two main reasons: 1) multi-pulse imaging techniques are used to differentiate microbubbles from tissue and 2) acquisition occurs after free bubble clearance (>6 minutes) in order to differentiate bound from freely circulating microbubbles. In this paper, we validate tumor imaging with a broadband single pulse molecular imaging method that is faster than the multi-pulse methods typically implemented on commercial scanners. We also combine the single pulse method with interframe filtering to selectively image targeted microbubbles without waiting for unbound bubble clearance, thereby reducing acquisition time from 10 to 2 minutes. The single pulse imaging method leverages non-linear bubble behavior by transmitting at low and receiving at high frequencies (TLRH). We implemented TLRH imaging and visualized the accumulation of intravenously administrated integrin-targeted microbubbles in a phantom and a Met-1 mouse tumor model. We found that the TLRH contrast imaging has a ~2-fold resolution improvement over standard contrast pulse sequencing (CPS) imaging. By using interframe filtering, the tumor contrast was 24.8±1.6 dB higher after the injection of integrin-targeted microbubbles than non-targeted control MBs, while echoes from regions lacking the target integrin were suppressed by 26.2±2.1 dB as compared with tumor echoes. Since real-time three-dimensional (3D) molecular imaging provides a more comprehensive view of receptor distribution, we generated 3D images of tumors to estimate their volume, and these measurements correlated well with expected tumor sizes. We conclude that TLRH combined with interframe filtering is a feasible method for 3D targeted ultrasound imaging that is faster than current multi-pulse strategies.

Background: The turnover of hyaluronan (HA), especially the production of low-molecular-weight fragments of HA, was examined in a model of unilateral renal ischaemia-reperfusion (IR) in rats., Methods: HA was extracted from the outer and inner stripe of the outer medulla (OSOM and ISOM) at different times following IR. Its fragmentation was measured using membrane filtration and size-exclusion chromatography. Quantitative reverse transcription-polymerase chain reaction, zymography and immunohistochemistry were used to assess the expression and localization of various forms of HA synthase (HAS) and hyaluronidase (HYAL). Macrophage infiltration was evaluated using immunohistochemistry., Results: HA accumulated at Day 1 mostly as high-molecular-weight (HMW) species with an elution profile similar to a reference 2500 kDa HA and at Day 14 mostly as medium- to low-size fragments. Within 1 day, HAS1 messenger RNA was up-regulated > 50- and 35-fold in OSOM and ISOM, respectively. Thereafter, HAS1 tended to normalize, while HAS2 increased steadily. Both synthetic enzymes were localized around tubules and in the interstitium. Conversely, HYAL1, HYAL2 and global hyaluronidase activity were repressed during the first 24 h. The patterns were identical in the OSOM and ISOM despite markedly different amounts of HA at baseline. There was no obvious correlation between HA deposits and macrophage infiltration., Conclusions: In the post-ischaemic kidney, HA starts to accumulate at Day 1 mostly as HMW species. Later on, a large proportion becomes degraded into smaller fragments. This pattern is explained by coordinated changes in the expression of HA synthases and hyaluronidases, especially an early induction of HAS1. The current data open the door to timed pharmacological interventions blocking the production of HA fragments.

Background/aims: Acute kidney injury (AKI) contributes to significant morbidity and mortality in the intensive care unit (ICU). Plasma levels of interleukin (IL)-6 predict the development of AKI and are associated with higher mortality in ICU patients with AKI. Most studies in AKI have focused on the tubulo-interstitium, despite evidence of glomerular involvement. In the following study, our goals were to investigate the expression of IL-6 and its downstream mediators in septic-induced AKI., Methods: Podocytes were treated in vitro with lipopolysaccharide (LPS) and mice were treated with LPS, and we evaluated IL-6 expression by real-time PCR, ELISA and in situ RNA hybridization., Results: Following LPS stimulation, IL-6 is rapidly and highly induced in cultured podocytes and in vivo in glomeruli and infiltrating leukocytes. Surprisingly, in direct response to exogenous IL-6, podocytes produce lipocalin-2/neutrophil gelatinase-associated lipocalin (Lcn2/Ngal). LPS also potently induces Lcn2/Ngal expression in podocytes in culture and in glomeruli in vivo. Intense Lcn2/Ngal expression is also observed in IL-6 knockout mice, suggesting that while IL-6 may be sufficient to induce glomerular Lcn2/Ngal expression, it is not essential., Conclusions: The glomerulus is involved in septic AKI, and we demonstrate that podocytes secrete key mediators of AKI including IL-6 and Lcn2/Ngal., (Copyright © 2012 S. Karger AG, Basel.)

The mechanisms underlying the association between obesity and progressive renal disease are not well understood. Exposure to a high-fat diet decreases levels of the cellular energy sensor AMPK in many organs, including the kidney, but whether AMPK contributes to the pathophysiology of kidney disease induced by a high-fat diet is unknown. In this study, we randomly assigned C57BL/6J mice to a standard or high-fat diet. After 1 week, mice fed a high-fat diet exhibited an increase in body weight, renal hypertrophy, an increase in urine H(2)O(2) and urine MCP-1, and a decrease in circulating adiponectin levels and renal AMPK activity. Urine ACR progressively increased after 4 weeks of a high-fat diet. After 12 weeks, kidneys of mice fed a high-fat diet demonstrated a marked increase in markers of fibrosis and inflammation, and AMPK activity remained significantly suppressed. To determine whether inhibition of AMPK activity explained these renal effects, we administered an AMPK activator along with a high-fat diet for 1 week. Although AMPK activation did not abrogate the weight gain, it reduced the renal hypertrophy, urine H(2)O(2), and urine and renal MCP-1. In vitro, AMPK activation completely inhibited the induction of MCP-1 by palmitic acid in mesangial cells. In conclusion, these data suggest that the energy sensor AMPK mediates the early renal effects of a high-fat diet.

The predominant transcription factors regulating key genes in diabetic kidney disease have not been established. The transcription factor upstream stimulatory factor 1 (USF1) is an important regulator of glucose-mediated transforming growth factor (TGF)-β1 expression in mesangial cells; however, its role in the development of diabetic kidney disease has not been evaluated. In the present study, wild-type (WT; USF1 +/+), heterozygous (USF1 +/-), and homozygous (USF1 -/-) knockout mice were intercrossed with Akita mice (Ins2/Akita) to induce type 1 diabetes. Mice were studied up to 36 wk of age. The degree of hyperglycemia and kidney hypertrophy were similar in all groups of diabetic mice; however, the USF1 -/- diabetic mice had significantly less albuminuria and mesangial matrix expansion than the WT diabetic mice. TGF-β1 and renin gene expression and protein were substantially increased in the WT diabetic mice but not in USF1 -/- diabetic mice. The underlying pathway by which USF1 is regulated by high glucose was investigated in mesangial cell culture. High glucose inhibited AMP-activated protein kinase (AMPK) activity and increased USF1 nuclear translocation. Activation of AMPK with AICAR stimulated AMPK activity and reduced nuclear accumulation of USF1. We thus conclude that USF1 is a critical transcription factor regulating diabetic kidney disease and plays a critical role in albuminuria, mesangial matrix accumulation, and TGF-β1 and renin stimulation in diabetic kidney disease. AMPK activity may play a key role in high glucose-induced regulation of USF1.

Rationale and Objectives: Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of antiangiogenic therapies. Our objectives are to develop a robust ultrasound contrast agent platform using microbubbles (MB) to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index (MI) imaging processing method and corresponding quantification method. The MB and the imaging methods were evaluated in a mouse model of breast cancer in vivo., Materials and Methods: We used a cyclic arginine-glycine-aspartic acid (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting αvβ3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine αvβ3 integrin protein and αvβ3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for αvβ3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted MB were visualized using a low MI contrast imaging pulse sequence, and quantified by intensity normalization and 2-dimensional Fourier transform analysis., Results: The cRGD ligand concentration on the MB surface was ∼8.2 × 10(6) molecules per MB. At a wall shear stress of 1.0 dynes/cm, cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant αvβ3 integrin relative to nontargeted MB and cRAD-MB controls. Similarly, cRGD-MB showed significantly greater adhesion to bEnd.3 EC compared with nontargeted MB and cRAD-MB. In addition, cRGD-MB, but not nontargeted MB or cRAD-MB, showed significantly enhanced contrast signals with a high tumor-to-background ratio. The adhesion of cRGD-MB to bEnd.3 was reduced by 80% after using anti-αv monoclonal antibody to treat bEnd.3. The normalized image intensity amplitude was ∼0.8, 7 minutes after the administration of cRGD-MB relative to the intensity amplitude at the time of injection, while the spatial variance in image intensity improved the detection of bound agents. The accumulation of cRGD-MB was blocked by preadministration with an anti-αv blocking antibody., Conclusions: The results demonstrate the functionality of a novel MB contrast agent covalently coupled to an RGD peptide for ultrasound molecular imaging of αvβ3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low MI.

Microbubbles and ultrasound enhance the cellular uptake of drugs (including gene constructs) into the kidney. Microbubble induced modifications to the size selectivity of the filtration capacity of the kidney may enable drugs to enter previously inaccessible compartments of the kidney. So far, negative renal side-effects such as capillary bleeding have been reported only in rats, with no apparent damage in larger models such as pigs and rabbits. Although local delivery is accomplished by applying ultrasound only to the target area, efficient delivery using conventional microbubbles has depended on the combined injection of both drugs and microbubbles directly into the renal artery. Conjugation of antibodies to the shell of microbubbles allows for the specific accumulation of microbubbles in the target tissue after intravenous injection. This exciting approach opens new possibilities for both drug delivery and diagnostic ultrasound imaging in the kidney., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Diabetic nephropathy is the most common and most rapidly growing cause of end-stage renal failure in developed countries. Diabetic nephropathy results from complex interactions between genetic, metabolic and hemodynamic factors. Improvements in our understanding of the pathogenesis of fibrosis associated with diabetic kidney disease have led to the identification of several novel targets for the treatment of diabetic nephropathy. Albuminuria is a useful clinical marker of diabetic nephropathy, as it can be used to predict a decline in renal function. A reduction in albuminuria might not, however, be reflective of a protective effect of therapies focused on ameliorating renal fibrosis. Although new strategies for slowing down the progression of several types of renal disease have emerged, the challenge of arresting the relentless progression of diabetic nephropathy remains. In this Review, we discuss novel pharmacological approaches that aim to improve the renal outcomes of diabetic nephropathy, including the use of direct renin inhibitors and statins. We also discuss the promise of using antifibrotic agents to treat diabetic nephropathy. The need for novel biomarkers of diabetic nephropathy is also highlighted.

This work explores the distribution of various markers expressed by interstitial cells in rat kidneys after ischemic injury (35 minutes) during regeneration of S3 tubules of outer stripe of outer medulla (OSOM). Groups of experimental animals (n = 4) were sacrificed every two hours during the first 24 hours post-ischemia as well as 2, 3, 7, 14 days post-ischemia. The occurrence of lineage markers was analyzed on kidney sections by immunohistochemistry and morphometry during the process of tubular regeneration. In postischemic kidneys, interstitial cell proliferation, assessed by 5-bromo-2'-deoxyuridine (BrdU) and Proliferating Cell Nuclear Antigen (PCNA) labeling, was prominent in outer medulla and reach a maximum between 24 and 72 hours after reperfusion. This population was characterized by the coexpression of vimentin and nestin. The density of -Neural Cell Adhesion Molecule (NCAM) positive interstitial cells increased transiently (18-72 hours) in the vicinity of altered tubules. We have also localized a small population of alpha-Smooth Muscle Actin (SMA)-positive cells confined to chronically altered areas and characterized by a small proliferative index. In conclusion, we observed in the postischemic kidney a marked proliferation of interstitial cells that underwent transient phenotypical modifications. These interstitial cells could be implicated in processes leading to renal fibrosis.

Background: In this study, we have examined rat kidneys after ischaemic injury (35 min) with regard to the dynamics of S3 tubule regeneration., Methods: One day before ischaemia, each rat received four successive i.p. injections of BrdU (5-bromo-2'-deoxyuridine: 80 mg/kg) at 2 h intervals. Groups of experimental animals (n = 4) were killed every 2 h during the first 24 h post-ischaemia as well as 2, 3, 7 and 14 days post-ischaemia. Renal sections were processed to characterize by immunohistochemistry the distribution and phenotype of BrdU-positive cells., Results: Renal regeneration after ischaemia was associated with a typical sequence of transient events: (1) absence of immunostaining during the first 8 h after reperfusion; (2) between 8 and 16 h, detection of a small population of BrdU-positive cells (CD44(+), vimentin(+), CD45(-)) restricted to the lumen of blood vessels characterized by the endothelial expression of selectin E; (3) between 16 and 24 h, progressive decrease of labelled cells in renal capillaries and a concomitant increase in the interstitial compartment; (4) after 1 day, labelled cells disappeared progressively from peritubular interstitium and were mainly observed in regenerating S3 tubules, and (5) after 3 days numerous positive cells were only present in regenerated tubules., Conclusions: Our data suggest that positive cells (BrdU(+), CD44(+), vimentin(+) and CD45(-)) observed in kidney tubules after ischaemia could originate from an extrarenal source and reach the renal parenchyma via blood vessels. We postulate that these immature cells migrate to injured tubules, proliferate and finally differentiate into mature epithelial cells leading to the replacement of a majority (>80%) of altered S3 cells.

Ischemia/reperfusion (I/R) injury in the kidney involves hemodynamic and cellular dysfunctions as well as leukocyte infiltration. Functional recovery occurs via cell proliferation and/or migration. To determine the roles of hyaluronan (HA) and its main receptor CD44 in renal postischemic processes, we compared their localization and expression with that of neutrophils, macrophages, and PCNA-positive (regenerative) cells as characterized by immunohistochemistry, up to 28 days after I/R in uninephrectomized rats. Observations covered all kidney zones, i.e. cortex (C), outer and inner stripes of outer medulla (OSOM, ISOM), and inner medulla (IM). In controls, HA was localized to the interstitium of IM and ISOM, and CD44 was mostly present on the basolateral membranes of collecting ducts in ISOM, the thin descending limb of Henle's loop and macula densa cells. After I/R, HA and CD44 staining appeared in C and OSOM at 12 h and persisted throughout the regenerative period, i.e. until day 7. Thereafter, they regressed but remained associated with remodeling areas. CD44 expression was found de novo on the apical pole of regenerating, not fully differentiated tubular cells and on some interstitial cells. It was prominent on all infiltrating neutrophils, as soon as 2 h post-I/R, and on 30% of the macrophages, including those in late HA-rich inflammatory granulomas. CD44 is probably involved in early leukocyte infiltration, in tubular regeneration, and in macrophage activity, while HA modifies the physico-chemical environment of interstitial and migrating cells. Based on its presence in remodeling areas, the HA-CD44 pair may be implicated in persistent postichemic inflammation as observed in chronic allograft nephropathy.

A significant renal vasodilation was observed previously after an acute cyclo-oxygenase (COX) inhibition induced with indomethacin. Because this effect could be due to COX-dependent intrarenal metabolization of arachidonic acid through cytochrome P450 (CYP450) pathways, the aim of the present study was to investigate, in vivo, possible interactions between COX and CYP450 mono-oxygenases. Mean arterial pressure (MAP) and renal blood flow (RBF), using an electromagnetic flow transducer for RBF evaluation, were measured continuously in 71 anaesthetized euvolaemic rats. Appropriate solvents (vehicle), 3 mg/kg indomethacin, 17-octadecynoic acid (17-ODYA; 2 mmol/L), either miconazole (MI; 1.5 mmol/L) or N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 5 mg/kg) and N'-hydroxyphenylformamidine (HET0016; 5 or 10 mg/kg) were administered to inhibit either COX, CYP450 mono-oxygenases, epoxygenases or hydroxylase, respectively. The CYP450 and COX inhibitors were also combined as follows: 17-ODYA/indomethacin, MI (or MS-PPOH)/indomethacin, HET0016/indomethacin and indomethacin/HET0016. Mean arterial pressure and RBF were not modified by vehicle, 17-ODYA or MI (or MS-PPOH). However, MAP decreased slightly (P < 0.05; paired t-test, 5 d.f.) and RBF increased transiently (P < 0.05; anova, 5 d.f.) after HET0016. In contrast, MAP decreased by 10 mmHg (P < 0.05) and RBF increased by 10% (P < 0.05) after indomethacin. This enhancement was prevented by 17-ODYA or MI (or MS-PPOH), but not by HET0016. Moreover, RBF increased step-wise to 21% in the indomethacin/HET0016 experiment (P < 0.05). Consequently, changes from baseline in renal vascular resistance differed among treatments, averaging -2 +/- 3 (vehicle), -13 +/- 3 (indomethacin; P < 0.05 vs vehicle), -4 +/- 3 (17-ODYA/indomethacin), -3 +/- 4 (MI or MS-PPOH/indomethacin), -15 +/- 3 (HET0016/indomethacin; P < 0.05) and -22 +/- 4% (indomethacin/HET0016; P < 0.05). In conclusion, these results demonstrate that the renal vasodilation induced by indomethacin can be prevented by prior inhibition of CYP450 mono-oxygenases and further suggest that the CYP450 epoxygenases pathway may prevail.