Sex differences in obesity-induced renal lipid accumulation revealed by lipidomics: a role of adiponectin/AMPK axis.

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.

Obesity-related complications can differ between men and women due to sex-specific differences in how fats are handled. Here, we studied the effects of high-fat diet on the kidneys of male and female mice. We found that despite gaining similar weight, obese female mice were better protected against insulin resistance, liver fat accumulation, and kidney damage caused by obesity than males. In particular, female mice were protected against lipid accumulation in the kidneys. We further analyzed the lipid profile in the kidneys of both male and female mice and observed differences in the amount and nature of the accumulated lipids. Male mice had increased levels of specific lipids, which may contribute to their higher risk of kidney damage. In contrast, female mice showed better lipid metabolism adaptation, which helped to protect their kidneys. This study also revealed an association between higher levels of the protein hormone adiponectin and higher activity of the cellular energy master regulator protein AMPK in obese females. These proteins may help prevent obesity-induced kidney damage. In obese males, these protective proteins are reduced and are associated with kidney damage. In conclusion, this study suggests that female mice are naturally shielded from obesity-induced kidney damage and lipid accumulation in the kidneys. Obesity in males is associated with the presence of potentially toxic lipids and dysregulated renal metabolism. Understanding these sex-related differences in obesity-related complications could lead to better management and treatment of kidney problems in both men and women.

Experimental Aristolochic Acid Nephropathy: A Relevant Model to Study AKI-to-CKD Transition.

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 TGFß 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.

Adipose tissue hyaluronan production improves systemic glucose homeostasis and primes adipocytes for CL 316,243-stimulated lipolysis.

Plasma hyaluronan (HA) increases systemically in type 2 diabetes (T2D) and the HA synthesis inhibitor, 4-Methylumbelliferone, has been proposed to treat the disease. However, HA is also implicated in normal physiology. Therefore, we generated a Hyaluronan Synthase 2 transgenic mouse line, driven by a tet-response element promoter to understand the role of HA in systemic metabolism. To our surprise, adipocyte-specific overproduction of HA leads to smaller adipocytes and protects mice from high-fat-high-sucrose-diet-induced obesity and glucose intolerance. Adipocytes also have more free glycerol that can be released upon beta3 adrenergic stimulation. Improvements in glucose tolerance were not linked to increased plasma HA. Instead, an HA-driven systemic substrate redistribution and adipose tissue-liver crosstalk contributes to the systemic glucose improvements. In summary, we demonstrate an unexpected improvement in glucose metabolism as a consequence of HA overproduction in adipose tissue, which argues against the use of systemic HA synthesis inhibitors to treat obesity and T2D.

Alginate@TiO2 hybrid microcapsules with high in vivo biocompatibility and stability for cell therapy.

Designing new materials to encapsulate living therapeutic cells for the treatment of the diseases caused by protein or hormone deficiencies is a great challenge. The desired materials need to be biocompatible towards both entrapped cells and host organisms, have long-term in vivo stability after implantation, allow the diffusion of nutrients and metabolites, and ensure perfect immune-isolation. The current work investigates the in vivo biocompatibility and stability of alginate@TiO2 hybrid microcapsules and the immune-isolation of entrapped HepG2 cells, to assess their potential for cell therapy. A comparison was made with alginate-silica hybrid microcapsules (ASA). These two hybrid microcapsules are implanted subcutaneously in female Wistar rats. The inflammatory responses of the rats are monitored by the histological examination of the implants and the surrounding tissues, to indicate their in vivo biocompatibility towards the hosts. The in vivo stability of the microcapsules is evaluated by the recovery rate of the intact microcapsules after implantation. The immune-isolation of the entrapped cells is assessed by their morphology, membrane integrity and intracellular enzymatic activity. The results show high viability of the entrapped cells and insignificant inflammation of the hosts, suggesting the excellent biocompatibility of alginate@TiO2 and ASA microcapsules towards both host organisms and entrapped cells. Compared to the ASA microcapsules, more intact alginate@TiO2 hybrid microcapsules are recovered 2-day and 2-month post-implantation and more cells remain alive, proving their better in vivo biocompability, stability, and immune-isolation. The present study demonstrates that the alginate@TiO2 hybrid microcapsule is a highly promising implantation material for cell therapy.

Critical Role for AMPK in Metabolic Disease-Induced Chronic Kidney Disease.

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.

Delayed Exercise Training Improves Obesity-Induced Chronic Kidney Disease by Activating AMPK Pathway in High-Fat Diet-Fed Mice.

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.

Endothelial Glycocalyx as a Shield Against Diabetic Vascular Complications: Involvement of Hyaluronan and Hyaluronidases.

The endothelial glycocalyx (EG), which covers the apical surface of the endothelial cells and floats into the lumen of the vessels, is a key player in vascular integrity and cardiovascular homeostasis. The EG is composed of PGs (proteoglycans), glycoproteins, glycolipids, and glycosaminoglycans, in particular hyaluronan (HA). HA seems to be implicated in most of the functions described for EG such as creating a space between blood and the endothelium, controlling vessel permeability, restricting leukocyte and platelet adhesion, and allowing an appropriate endothelial response to flow variation through mechanosensing. The amount of HA in the EG may be regulated by HYAL (hyaluronidase) 1, the most active somatic hyaluronidase. HYAL1 seems enriched in endothelial cells through endocytosis from the bloodstream. The role of the other main somatic hyaluronidase, HYAL2, in the EG is uncertain. Damage to the EG, accompanied by shedding of one or more of its components, is an early sign of various pathologies including diabetes mellitus. Shedding increases the blood or plasma concentration of several EG components, such as HA, heparan sulfate, and syndecan. The plasma levels of these molecules can then be used as sensitive markers of EG degradation. This has been shown in type 1 and type 2 diabetic patients. Recent experimental studies suggest that preserving the size and amount of EG HA in the face of diabetic insults could be a useful novel therapeutic strategy to slow diabetic complications. One way to achieve this goal, as suggested by a murine model of HYAL1 deficiency, may be to inhibit the function of HYAL1. The same approach may succeed in other pathological situations involving endothelial dysfunction and EG damage.

Evaluation of inducible nitric oxide synthase inhibition on kidney function and structure in high-fat diet-induced kidney disease.

NEW FINDINGS: What is the central question of this study? The metabolic pathways regulating the effects of obesity on the kidney remain unknown. We sought to determine whether inducible nitric oxide synthase (iNOS) is involved in the underlying mechanisms of high-fat diet-induced kidney disease using a specific iNOS inhibitor, N6-(1-iminoethyl)-l-lysine hydrochloride (L-NIL). What is the main finding and its importance? We did not demonstrate an upregulation of iNOS renal expression after high caloric intake, suggesting that iNOS might not be a crucial player in the development of obesity-induced kidney disease. Although L-NIL treatment clearly ameliorated systemic metabolic parameters, the effect on loss of renal function, impairment of tubular integrity, oxidative stress and inflammation appeared to be more moderate. Central obesity is related to caloric excess, promoting deleterious cellular responses in targeted organs. Nitric oxide (NO) has been determined as a key player in the pathogenesis of metabolic diseases. Here, we investigated the implication of inducible NO synthase (iNOS) in the development of obesity-induced kidney disease. C57Bl/6 male mice were randomized to a low-fat diet (LFD) or a high-fat diet (HFD) and treated with N6-(1-iminoethyl)-l-lysine hydrochloride (L-NIL), a specific iNOS inhibitor, for 16 weeks. Mice fed an HFD exhibited a significant increase in body weight, fasting blood glucose and plasma concentrations of non-esterified fatty acids, triglyceride and insulin. Inhibition of iNOS prevented these changes in mice fed an HFD. Interestingly, the significant increase in albuminuria and mesangial matrix expansion were not ameliorated with L-NIL, whereas a significant decrease in proteinuria, N-acetyl-ß-d-glucosaminidase excretion and renal triglyceride content were found, suggesting that iNOS inhibition is more suitable for tubular function than glomerular function. The urinary concentration of hydrogen peroxide, a stable product of reactive oxygen species production, that was found to be increased in mice fed an HFD, was significantly reduced with L-NIL. Finally, despite a moderate effect of L-NIL on inflammatory processes in the kidney, we demonstrated a positive impact of this treatment on adipocyte hypertrophy and on adipose tissue inflammation. These results suggest that inhibition of iNOS leads to a moderate beneficial effect on kidney function in mice fed an HFD. Further studies are needed for better understanding of the role of iNOS in obesity-induced kidney disease.

Genetically increased angiotensin I-converting enzyme alters peripheral and renal vascular reactivity to angiotensin II and bradykinin in mice.

Angiotensin I-converting enzyme (ACE) levels in humans are under strong genetic influence. Genetic variation in ACE has been linked to risk for and progression of cardiovascular and renal diseases. Causality has been documented in genetically modified mice, but the mechanisms underlying causality are not completely elucidated. To further document the vascular and renal consequences of a moderate genetic increase in ACE synthesis, we studied genetically modified mice carrying three copies of the ACE gene (three-copy mice) and littermate wild-type animals (two-copy mice). We investigated peripheral and renal vascular reactivity to angiotensin II and bradykinin in vivo by measuring blood pressure and renal blood flow after intravenous administration and also reactivity of isolated glomerular arterioles by following intracellular Ca2+ mobilization. Carrying three copies of the ACE gene potentiated the systemic and renal vascular responses to angiotensin II over the whole range of peptide concentration tested. Consistently, the response of isolated glomerular afferent arterioles to angiotensin II was enhanced in three-copy mice. In these mice, signaling pathways triggered by endothelial activation by bradykinin or carbachol in glomerular arterioles were also altered. Although the nitric oxide (NO) synthase (NOS)/NO pathway was not functional in arterioles of two-copy mice, in muscular efferent arterioles of three-copy mice NOS3 gene expression was induced and NO mediated the effect of bradykinin or carbachol. These data document new and unexpected vascular consequences of a genetic increase in ACE synthesis. Enhanced vasoconstrictor effect of angiotensin II may contribute to the risk for cardiovascular and renal diseases linked to genetically high ACE levels. NEW & NOTEWORTHY A moderate genetic increase in angiotensin I-converting enzyme (ACE) in mice similar to the effect of the ACE gene D allele in humans unexpectedly potentiates the systemic and renal vasoconstrictor responses to angiotensin II. It also alters the endothelial signaling pathways triggered by bradykinin or carbachol in glomerular efferent arterioles.

Restored nitric oxide bioavailability reduces the severity of acute-to-chronic transition in a mouse model of aristolochic acid nephropathy.

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.

An Integrated View of Aristolochic Acid Nephropathy: Update of the Literature.

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.

Blocking TGF-ß Signaling Pathway Preserves Mitochondrial Proteostasis and Reduces Early Activation of PDGFRß+ Pericytes in Aristolochic Acid Induced Acute Kidney Injury in Wistar Male Rats.

BACKGROUND: The platelet-derived growth factor receptor ß (PDGFRß)+ 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 PDGFRß+ 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 ß (TGFß) inhibition in a rat model of AAN. MATERIALS AND METHODS: Neutralizing anti-TGFß 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 PDGFRß+ pericytes-derived myofibroblasts accumulation.

Hyaluronidase 1 Deficiency Preserves Endothelial Function and Glycocalyx Integrity in Early Streptozotocin-Induced Diabetes.

Hyaluronic acid (HA) is a major component of the glycocalyx involved in the vascular wall and endothelial glomerular permeability barrier. Endocytosed hyaluronidase HYAL1 is known to degrade HA into small fragments in different cell types, including endothelial cells. In diabetes, the size and permeability of the glycocalyx are altered. In addition, patients with type 1 diabetes present increased plasma levels of both HA and HYAL1. To investigate the potential implication of HYAL1 in the development of diabetes-induced endothelium dysfunction, we measured endothelial markers, endothelium-dependent vasodilation, arteriolar glycocalyx size, and glomerular barrier properties in wild-type and HYAL1 knockout (KO) mice with or without streptozotocin (STZ)-induced diabetes. We observed that 4 weeks after STZ injections, the lack of HYAL1 1) prevents diabetes-induced increases in soluble P-selectin concentrations and limits the impact of the disease on endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation; 2) increases glycocalyx thickness and maintains glycocalyx structure and HA content during diabetes; and 3) prevents diabetes-induced glomerular barrier dysfunction assessed using the urinary albumin-to-creatinine ratio and urinary ratio of 70- to 40-kDa dextran. Our findings suggest that HYAL1 contributes to endothelial and glycocalyx dysfunction induced by diabetes. HYAL1 inhibitors could be explored as a new therapeutic approach to prevent vascular complications in diabetes.

Protective effect of nitric oxide in aristolochic acid-induced toxic acute kidney injury: an old friend with new assets.

Aristolochic acid (AA) nephropathy (AAN), a progressive tubulointerstitial injury of toxic origin, is characterized by early and transient acute tubular necrosis. This process has been demonstrated to be associated with reduced nitric oxide (NO) production, which can disrupt the regulation of renal function. In this study, we tested the hypothesis that L-arginine (L-Arg) supplementation could restore renal function and reduce renal injury after AA intoxication. C57BL/6 J male mice were randomly subjected to daily i.p. injection of either sterile saline solution or AA (2.5 mg kg(-1)) for 4 days. To determine whether AA-induced renal injuries were linked to reduced NO production, L-Arg, a substrate for NO synthase, was supplemented (5%) in drinking water. Mice intoxicated with AA exhibited features of rapid-onset acute kidney injury, including polyuria, significantly increased plasma creatinine concentrations, proteinuria and fractional excretion of sodium (P < 0.05), along with severe proximal tubular cell injury and increased NADPH oxidase 2 (Nox2)-derived oxidative stress (P < 0.05). This was associated with a significant reduction in NO bioavailability. L-Arg supplementation in AA-treated mice significantly increased NO bioavailability, which in turn improved renal function (creatininaemia, polyuria, proteinuria, fractional excreted sodium and N-acetyl-ß-D-glucosaminidase enzymuria) and renal structure (tubular necrosis and tubular cell apoptosis). These changes were associated with significant reductions in Nox2 expression and in production of reactive oxygen species and with an increase in antioxidant concentrations. Our results demonstrate that preservation of NO bioavailability leads to renal protection in AA-induced acute kidney injury by reducing oxidative stress and maintaining renal function.

Lack of hyaluronidases exacerbates renal post-ischemic injury, inflammation, and fibrosis.

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.

Hyaluronidase 1 and hyaluronidase 2 are required for renal hyaluronan turnover.

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.

Protection of Wistar-Furth rats against postischaemic acute renal injury: role for nitric oxide and thromboxane?

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.

Inhibition of hyaluronan is protective against renal ischaemia-reperfusion injury.

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.

A hybrid assembly by encapsulation of human cells within mineralised beads for cell therapy.

BACKGROUND: The design of new technologies for treatment of human disorders such as protein deficiencies is a complex and difficult task. Particularly, the construction of artificial organs, based on the immunoisolation of protein-secreting cells, requires the use of suitable materials which have to be biocompatible with the immunoisolated cells and avoid any inappropriate host response. METHODOLOGY/PRINCIPAL FINDINGS: This work investigates the in vivo behavior of mechanically resistant hybrid beads which can be considered as a model for artificial organ for cell therapy. This hybrid system was designed and fabricated via the encapsulation of living cells (HepG2) within alginate-silica composites. Two types of beads (alginate-silica hybrid (AS) or alginate/silica hybrid subsequently covered by an external layer of pure alginate (ASA)), with or without HepG2 cells, were implanted into several female Wistar rats. After four weeks, the potential inflammatory local response that might be due to the presence of materials was studied by histochemistry. The results showed that the performance of ASA beads was quite promising compared to AS beads, where less abnormal rat behaviour and less inflammatory cells in histological sections were observed in the case of ASA beads. CONCLUSIONS/SIGNIFICANCE: The current study highlights that alginate-silica composite materials coated with an extra-alginate shell offer much promise in the development of robust implantation devices and artificial organs.

Do segmental reflexes play a role in the distribution of external intercostal EMG activity in the rabbit?

External intercostal muscle is considered as an inspiratory muscle. Its electromyographic (EMG) activity is exquisitely sensitive to proprioceptive afferents and is predominant in zones with the greatest respiratory effect. In the current series of studies, we assessed the contribution of segmental reflexes to this particular distribution of activity in anesthetized rabbits. We first established a parallel between gradients of activity and mechanical advantage. The sensitivity of external intercostal EMG activity to rib displacement was then assessed in the 3rd and 5th interspaces and in the dorsal, middle and ventral muscle bundles of the 3rd interspace. Finally, persistence of the dorso-ventral gradient was tested after a dorsal rhizotomy (interspaces 3-5). There were no differences in the sensitivity to rib motion in the studied positions. After rhizotomy, the dorso-ventral gradient of activity persisted at rest and with inspiratory resistive loading. We, therefore, conclude that the neuromechanical matching of respiratory drive in the external intercostal muscles is not the result of segmental reflexes.