Hypoxia induces glucose uptake and metabolism of adipose­derived stem cells.

It has previously been demonstrated that hypoxia has diverse stimulatory effects on adipose­derived stem cells (ASCs), however, metabolic responses under hypoxia remain to be elucidated. Thus, the present study aimed to investigate the glucose uptake and metabolism of ASCs under hypoxic conditions, and to identify the underlying molecular mechanisms. ASCs were cultured in 1% oxygen, and experiments were conducted in vitro. As determined by proteomic analysis and western blotting, GAPDH and enolase 1 (ENO1) expression were upregulated under hypoxia. In addition, lactate production was significantly increased, and mRNA levels of glycolytic enzymes, including GAPDH, ENO1, hexokinase 2 (HK2), and lactate dehydrogenase α (LDHα) were upregulated. Hypoxia­inducible factor 1­α (HIF­1α) expression was increased as demonstrated by western blotting, and a pharmacological inhibitor of HIF­1α significantly attenuated hypoxia­induced lactate production and expression of glycolytic enzymes. It was also observed that hypoxia significantly increased glucose uptake in ASCs, and glucose transporter (GLUT)1 and GLUT3 expression were upregulated under hypoxia. Pharmacological inhibition of the HIF­1α signaling pathways also attenuated hypoxia­induced GLUT1 and GLUT3 expression. These results collectively indicate that hypoxia increases glucose uptake via GLUT1 and GLUT3 upregulation, and induces lactate production of ASCs via GAPDH, ENO1, HK2, and LDHα. Furthermore, HIF­1α is involved in glucose uptake and metabolism of ASCs.

Cyclosporine Amicellar delivery system for dry eyes.

BACKGROUND: The objectives of this study were to develop stable cyclosporine A (CsA) ophthalmic micelle solutions for dry-eye syndrome and evaluate their physicochemical properties and therapeutic efficacy. MATERIALS AND METHODS: CsA-micelle solutions (MS-CsA) were created by a simple method with Cremophor EL, ethanol, and phosphate buffer. We investigated the particle size, pH, and osmolarity. In addition, long-term physical and chemical stability for MS-CsA was observed. To confirm the therapeutic efficacy, tear production in dry eye-induced rabbits was evaluated using the Schirmer tear test (STT). When compared to a commercial product, Restasis, MS-CsA demonstrated improvement in goblet-cell density and conjunctival epithelial morphology, as demonstrated in histological hematoxylin and eosin staining. RESULTS: MS-CsA had a smaller particle size (average diameter 14-18 nm) and a narrow size distribution. Physicochemical parameters, such as particle size, pH, osmolarity, and remaining CsA concentration were all within the expected range of 60 days. STT scores significantly improved in MS-CsA treated groups (P<0.05) in comparison to those of the Restasis-treated group. The number of goblet cells for rabbit conjunctivas after the administration of MS-CsA was 94.83±8.38, a significantly higher result than the 65.17±11.51 seen with Restasis. The conjunctival epithelial morphology of dry eye-induced rabbits thinned with loss of goblet cells. However, after 5 days of treatment with drug formulations, rabbit conjunctivas recovered epithelia and showed a relative increase in the number of goblet cells. CONCLUSION: The results of this study indicate the potential use of a novel MS for the ophthalmic delivery of CsA in treating dry eyes.

Sustained release of risperidone from biodegradable microspheres prepared by in-situ suspension-evaporation process.

Risperidone-loaded poly (D,L-lactide-co-glycolide) (PLGA) microspheres were prepared with a suspension-evaporation process with an aqueous suspension containing an in situ-formed aluminum hydroxide inorganic gel (SEP-AL process) and evaluated for encapsulation efficiency, particle size, surface morphology, glass transition temperature, in vitro drug release profile, and in vivo behavior. The SEP-AL microspheres were compared with conventional oil-in-water (O/W) emulsion solvent evaporation method using polyvinylalcohol (PVA) as an emulsifier (CP-PVA process). The microspheres were spherical in shape. DSC measurements showed that risperidone crystallinity was greatly reduced due to the homogeneous distribution of risperidone in PLGA microspheres. In vitro drug release profile from the microspheres showed a sigmoidal pattern of negligible initial burst up to 24h and minimal release (time-lag) for 7 days. After the lag phase, slow release took a place up to 25 days and then rapid release occurred sharply for 1 week. In vivo rat pharmacokinetic profile from the microspheres showed very low blood concentration level at the initial phase (up to 24h) followed by the latent phase up to 21 days. At the 3rd week, main phase started and the blood concentration of the drug increased up to the 5th week, and then gradually decreased. The risperidone-loaded PLGA microspheres produced by SEP-AL process showed excellent controlled release characteristics for the effective treatment of schizophrenia patients.

Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells.

Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs) or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA), significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

Preparation and evaluation of cyclosporin A-containing proliposomes: a comparison of the supercritical antisolvent process with the conventional film method.

OBJECTIVES: The objectives of this study were to prepare cyclosporin A (CsA)-containing proliposomes using the supercritical antisolvent (SAS) process and the conventional thin film method for the comparative study of proliposomal formulations and to evaluate the physicochemical properties of these proliposomes. METHODS: CsA-containing proliposomes were prepared by the SAS process and the conventional film method, composed of natural and synthetic phospholipids. We investigated particle size, polydispersity index, and zeta potential of CsA-containing proliposomes. In addition, both production yield and entrapment efficiency of CsA in different proliposomes were analyzed. Physicochemical properties of CsA-containing proliposomes were also evaluated, using differential scanning calorimetry and X-ray diffraction. The morphology and size of CsA-containing proliposomes were confirmed, using scanning electron microscopy. We checked the in vitro release of CsA from CsA-containing proliposomes prepared by different preparation methods, comparing them with Restasis(®) as a positive control and the stability of SAS-mediated proliposomes was also studied. RESULTS: CsA-containing proliposomes formed by the SAS process had a relatively smaller particle size, with a narrow size distribution and spherical particles compared with those of conventionally prepared proliposomes. The yield and entrapment efficiency of CsA in all proliposomes varied from 85% to 92% and from 86% to 89%, respectively. Differential scanning calorimetry and X-ray diffraction studies revealed that the anhydrous lactose powder used in this formulation retained its crystalline form and that CsA was present in an amorphous form. Proliposome powders were rapidly converted to liposomes on contact with water. The in vitro release study of proliposomal formulations demonstrated a similar pattern to Restasis(®). The SAS-mediated CsA-containing proliposomes were stable on storage, with no significant changes in particle size, polydispersity index, and entrapment efficiency. CONCLUSION: These results show promising features of CsA-containing proliposomal formulations, using the SAS process for the large-scale industrial application.

Supercritical fluid-mediated liposomes containing cyclosporin A for the treatment of dry eye syndrome in a rabbit model: comparative study with the conventional cyclosporin A emulsion.

BACKGROUND: The objective of this study was to compare the efficacy of cyclosporin (CsA)-encapsulated liposomes with the commercially available CsA emulsion (Restasis) for the treatment of dry eye syndrome in rabbits. METHODS: Liposomes containing CsA were prepared by the supercritical fluid (SCF) method consisted of phosphatidylcholine from soybean (SCF-S100) and egg lecithins (SCF-EPCS). An in vitro permeation study was carried out using artificial cellulose membrane in Franz diffusion cells. Dry eye syndrome was induced in male albino rabbits and further subdivided into untreated, Restasis-treated, EPCS, and S100-treated groups. Tear formation in the dry-eye-induced rabbits was evaluated using the Schirmer tear test. All formulations were also evaluated by ocular irritation tests using the Draize eye and winking methods with the determination of CsA concentration in rabbit tears. RESULTS: After the treatment, the Schirmer tear test value significantly improved in EPCS-treated (P=0.005) and S100-treated (P=0.018) groups compared to the Restasis-treated group. The AUC0₋24 h for rabbit's tear film after the administration of SCF-S100 was 32.75±9.21 µg·h/mg which was significantly higher than that of 24.59±8.69 µg·h/mg reported with Restasis. Liposomal CsA formulations used in this study showed lower irritation in rabbit eyes compared with Restasis. CONCLUSION: These results demonstrate that the novel SCF-mediated liposomal CsA promises a significant improvement in overcoming the challenges associated with the treatment of dry eyes.

Bioimaging of dexamethasone and TGF beta-1 and its biological activities of chondrogenic differentiation in hydrogel constructs.

This study examined the efficacy of poly(NiPAAm-co-AAc) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The hydrogel constructs, which consisted of embedded cells co-encapsulating dexamethasone (Dex) and TGF beta-1 or unloaded Dex, were used as controls to determine the effects of Dex and TGF beta-1 on chondrogenic differentiation. The level of Dex and TGF beta-1 released was monitored using a bioimaging method. The amount of Dex released from hydrogel was faster than that of TGF beta-1. TGF beta-1 was present in hydrogel for more than 4 weeks after the injection. The level of the cartilage associated ECM proteins was examined by immunohistochemical staining for collagen type II as well as by Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with the chondrocytes and differentiation delivery material for applications in neocartilage formation.

In vitro and in vivo test of PEG/PCL-based hydrogel scaffold for cell delivery application.

Biodegradable elastic hydrogel scaffolds based on hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(epsilon-caprolactone) (PCL) were fabricated and investigated as a delivery vehicle of rabbit chondrocytes for the formation of neocartilage. The diacrylated forms of PEG and PCL were used as building blocks to prepare a series of hydrogel scaffolds with different block compositions and, thus, different physico-chemical properties. The porous hydrogel scaffolds were prepared by using the salt leaching method that is generally used for the creation of porous scaffolds, and their in vitro cell interactions were examined using chondrocytes. The hydrogel scaffold with a relatively high PEG content showed better cell growth for chondrocytes, while the scaffold with a relatively low PEG content showed lower chondrogenic differentiation. It was observed that different kinds of scaffolds and rabbit chondrocytes were shown to have different swelling ratios in the scaffold for effective cell growth and tissue regeneration. RT-PCR results for the resultant cartilage tissue revealed that a PEG-PCL ratio of 14 to 6 scaffold was optimal for cartilage tissue formation in terms of collagen Type II, aggrecan, SOX9, and COMP gene expression. In addition, the hydrogel scaffold with a PEG-PCL ratio of 14 to 6 showed faster formation of new cartilage than those shown by other scaffolds.

Blended construct consisting of thermo-reversible hydrogels and heparinized nanoparticles for increasing the proliferation activity of the rabbit chondrocyte in vivo test.

To use nano-structured materials as a novel cell therapeutic agent, we have devised a novel method for the fabrication of nano-scaled 3D scaffolds consisting of heparin/poly(ethylenimine) (PEI) nanoparticles in a thermo-reversible hydrogel, attached via a layer-by-layer system. Bioassay results showed significant difference in DNA amount between groups. Specifically, groups with heparin/PEI nanoparticles had almost twice the glycosaminoglycan content per construct starting at day 7 as compared to controls. Gene expression of total collagen was evident in groups containing heparin/PEI nanoparticles.

Combination of ascorbate and growth factor (TGF beta-3) in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes for neocartilage formation.

This study evaluated the potential of using poly(NiPAAm-co-AAc) as an injectable drug delivery carrier and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. In particular, rabbit chondrocytes were embedded in hydrogels containing a combination of ascorbate and transforming growth factor beta-3 (TGF beta-3). Hydrogel constructs containing embedded cells either without ascorbate or a combination of ascorbate and TGF beta-3 were used as controls to determine the effects of ascorbate and TGF beta-3 on chondrogenic differentiation. The level of cartilage associated ECM proteins was examined using immunohistochemical staining for collagen type II as well as by Safranin-O and Alcian blue (GAG) staining. The results showed that ascorbate is an important factor for preparing cartilage constructs because of its action on chondrocyte phenotype modulation and proliferation.

Enhancement of cell proliferation and differentiation by combination of ascorbate and dexamethasone in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes.

The effects of inducible materials (dexamethasone and ascorbate) on chondrogenic differentiation of rabbit chondrocytes have been examined. A hydrogel construct containing dexamethasone and ascorbate up-regulated gene expression of the cartilage matrix component of collagen to give three times the collagen content per construct at day 56 as compared to controls. Alcian Blue and Safranin-O staining revealed that these constructs also had formed more hyaline cartilage than other hydrogel constructs.

Combination material delivery of dexamethasone and growth factor in hydrogel blended with hyaluronic acid constructs for neocartilage formation.

The aim of this study was to assess the efficacy of poly(NiPAAm-co-AAc) blended with hyaluronic acid (HA) as an injectable cell vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. Specially, rabbit chondrocytes were embedded in blended hydrogels co-encapsulation with dexamethasone (Dex) and growth factors for enhancing the chondrogenic differentiation. Blended hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF beta-3 or unloaded Dex and sTGF beta-3 served as controls to assess the effects of Dex on chondrogenic differentiation. Hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF beta-3 on chondrogenic differentiation. The hydrogel constructs were injected subcutaneously into the nude mice and monitored for 1, 4, and 8 weeks after the injection. The level of the cartilage-associated ECM proteins was determined by immunohistochemical (collagen type II; specific marker for chondrogenic differentiation), Safranin-O, and Alcian blue (GAG) staining. Over the same time period, the glycosamingoglycan content per cell remained constant for all formulations, indicating that the dramatic increase in cell number for samples with Dex and TGF beta-3 loaded in hydrogel constructs was accompanied by maintenance of the cell phenotypes.

Osteogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel constructs containing hydroxyapatite and bone morphogenic protein-2 (BMP-2).

The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.

Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test.

In this study, a hydrogel composite, based on the thermo-reversible hydrogel of p(NiPAAm-co-AAc) and hyaluronic acid (HA) was used as an injectable cell and growth factor carrier for cartilage tissue engineering applications. Rabbit chondrocytes were embedded in blended hydrogel composites co-encapsulated with the transforming growth factor beta-3 (TGFbeta-3). The blended hydrogel with the embedded chondrocytes and HA co-encapsulating unloaded growth factors and those with the thermo-reversible hydrogel were used as the controls to examine the effects of TGFbeta-3 on neocartilage formation. The blended hydrogel loaded with TGFbeta-3 embedded with chondrocytes were injected subcutaneously into the nude mice. The mice were monitored for 8 weeks after the injection. Both the differentiation and level of cartilage-specific ECM production were significantly higher in the presence of HA and growth factor than in the control without the growth factor. The level of cartilage associated ECM proteins was examined by immunohistochemical staining (collagen types II and X) as well as by Safranin-O and Alcian blue (GAG) staining. The results showed the potential application of blended hydrogel mixed with the growth factor to neocartilage formation.

Delivery of dexamethasone, ascorbate, and growth factor (TGF beta-3) in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes.

The aim of this study was to assess the efficacy of poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The p(NiPAAm-co-AAc) hydrogel itself without specific differentiation-inducing drugs was used as a control in order to determine the effects of these materials on chondrogenic differentiation. The level of cartilage associated extracellular matrix (ECM) proteins was examined by immunohistochemical staining for collagen type II as well as Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with chondrocytes and differentiation materials as an injectable delivery vehicle for use in neocartilage formation.

Rosiglitazone protects against cyclosporine-induced pancreatic and renal injury in rats.

Rosiglitazone (RGTZ) has protective effect against various types of injury. This study was performed to evaluate the effect of RGTZ on pancreatic and renal injury caused by cyclosporine (CsA). CsA (15 mg/kg) and RGTZ (3 mg/kg) were administered alone and together to the rats for 28 days. The effect of RGTZ on CsA-induced pancreatic injury was evaluated by intraperitoneal glucose tolerance test (IPGTT), plasma insulin concentrations and pancreatic beta-cell morphology. The effect of RGTZ on CsA-induced renal injury was evaluated by assessing renal function and pathology; mediators of inflammation and fibrosis such as angiotensin II (AngII), osteopontin (OPN) and transforming growth factor-beta1 (TGF-beta1) and apoptotic cell death. Four weeks of CsA treatment caused diabetes, renal dysfunction, typical pathologic lesions (arteriolopathy, interstitial fibrosis and inflammatory cells infiltration) and apoptotic cell death. RGTZ treatment decreased blood glucose concentration, increased plasma insulin concentration and preserved pancreatic beta islet mass. RGTZ treatment improved renal function and histopathology. Pro-inflammatory and pro-fibrotic molecules such as AngII, OPN and TGF-beta1, and apoptotic cell death also decreased with RGTZ treatment. These data suggest that RGTZ has a protective effect against CsA-induced pancreatic and renal injury.

Combined effects of losartan and pravastatin on interstitial inflammation and fibrosis in chronic cyclosporine-induced nephropathy.

BACKGROUND: Statins and angiotensin II type I receptor blockers have synergistic effects on vascular smooth-muscle-cell proliferation and the progression of renal diseases. We evaluated whether combined treatment with losartan (LSRT) and pravastatin (PRVT) affords superior protection compared with their respective monotherapies in treating chronic cyclosporine (CsA)-induced nephropathy in rats. METHODS: Rats maintained on a low salt diet were given vehicle, CsA (15 mg/kg), CsA and LSRT (10 mg/kg), CsA and PRVT (5 mg/kg), or a combination of CsA, LSRT, and PRVT for 28 days. Basic parameters (renal function, systolic blood pressure, serum high-sensitivity C-reactive protein [hs-CRP], and lipid profiles), histopathology (arteriolopathy, tubulointerstitial fibrosis, and inflammatory cell infiltration), and inflammatory and fibrotic factors (intrarenal CRP, angiotensin II, osteopontin, and transforming growth factor [TGF]-beta1) were studied. RESULTS: LSRT or PRVT treatment significantly attenuated the histopathologic changes induced by CsA, and combined treatment with LSRT and PRVT further decreased these parameters compared with giving each drug alone. Increased levels of angiotensin II, intrarenal CRP, osteopontin, and TGF-beta1 in CsA-treated rat kidney were reduced by treatment with either LSRT or PRVT and were further decreased by the combination of the two drugs. There were no significant differences in systolic blood pressure or serum lipid parameters between groups. CONCLUSIONS: Combined treatment with LSRT and PRVT provided synergistic effects in attenuating inflammatory and fibrotic processes in a rat model of chronic CsA-induced nephropathy, and this effect was independent of their hypolipidemic and hypotensive actions.

Ischemia-reperfusion injury activates innate immunity in rat kidneys.

BACKGROUND: There is growing evidence of a role of the immune system in the pathophysiology of ischemia-reperfusion (I/R) injury, but the influence of I/R injury on innate immunity is still undetermined. METHODS: Sprague-Dawley rats were used. I/R injury was induced by clamping both renal arteries for 45 min, and the rats were killed 1, 3, 5, and 7 days later. Activation of innate immunity was evaluated in terms of the expression of toll-like receptor (TLR) 2 or TLR4 mRNAs and protein, by the level of the TLR ligand (heat shock protein [HSP] 70), and maturation of dendritic cells by double-label immunohistochemistry of dendritic cells for major histocompatibility complex (MHC) class II antigen. RESULTS: I/R injury increased TLR2 and TLR4 mRNA and protein expression, and they were mainly observed on renal tubular cells. I/R injury also produced endogenous TLR ligand (HSP70) on renal tubular cells. I/R injury increased not only the numbers of dendritic cells but also the production of MHC class II antigen in dendritic cells, suggesting maturation of these cells. Activation of innate immunity was observed at day 1, peaked at days 3 to 5 after I/R injury, and thereafter gradually decreased. CONCLUSIONS: I/R injury rapidly activates the innate immune response.

Preconditioning with 1,25-dihydroxyvitamin D3 protects against subsequent ischemia-reperfusion injury in the rat kidney.

BACKGROUND/AIM: Induction of heat shock protein 70 (HSP70) is important in the tolerance of subsequent ischemia-reperfusion (I/R) injury. The aim of this study was to evaluate the effect of HSP70 induction by 1,25-dihydroxyvitamin D3 (VD3) on subsequent I/R injury in rats. METHODS: HSP70 was induced in Sprague-Dawley rats by VD3 treatment for 7 days, and the effect of VD3 pretreatment on subsequent I/R injury was evaluated in terms of renal function, tubular necrosis score, tumor necrosis factor alpha mRNA expression, mitogen-activated protein kinase expression, and proliferating cell nuclear antigen expression. RESULTS: VD3 treatment increased HSP70 expression which was localized to renal tubular cells in the outer medulla. Pretreatment with VD3 before I/R injury resulted in (1) decreased blood urea nitrogen and serum creatinine levels; (2) decreased tubular cell necrosis; (3) increased tubular cell proliferation as determined by proliferating cell nuclear antigen expression; (4) decreased tumor necrosis factor alpha mRNA expression, and (5) increased extracellular signal regulated protein kinase and decreased c-Jun N-terminal kinase expression. CONCLUSION: Our study demonstrates that VD3 is a nontoxic inducer of HSP70 and exerts a protective effect against subsequent I/R injury.

Blockade of angiotensin II with losartan attenuates transforming growth factor-beta1 inducible gene-h3 (betaig-h3) expression in a model of chronic cyclosporine nephrotoxicity.

BACKGROUND: We recently demonstrated that upregulation of the transforming growth factor (TGF)-beta1 inducible gene-h3 (betaig-h3) is associated with tubulointerstitial fibrosis (TIF) in a rat model of chronic cyclosporine A (CsA) nephrotoxicity. This study investigated the association between betaig-h3 expression and TIF during losartan treatment in this model. METHODS: Adult Sprague-Dawley rats kept on a salt-depleted diet (0.05% sodium) were treated daily for 4 weeks with vehicle (olive oil, 1 ml/kg), CsA (15 mg/kg) or both CsA and losartan (10 mg/kg in drinking water). The effect of losartan on betaig-h3 expression was evaluated using in situ hybridization, immunohistochemistry and immunoblotting. Histopathology, expressions of TGF-beta1 and intrarenal angiotensin II were compared across treatment groups. RESULTS: Concurrent administration of losartan significantly attenuated betaig-h3 mRNA and protein expression within the tubulointerstitium of CsA-treated kidneys. This was accompanied by the retardation of TIF (18 +/- 5 vs. 39 +/- 5%, p < 0.01 vs. CsA) and the expression of TGF-beta1 mRNA (336 +/- 49 vs. 685 +/- 63%, p < 0.01 vs. CsA) and the number of angiotensin II-positive glomeruli (18 +/- 5 vs. 38 +/- 6, p < 0.05 vs. CsA). CONCLUSION: Losartan is capable of abrogating the upregulation of TGF-beta1 and betaig-h3 expression, and this is associated with attenuated tubulointerstitial fibrosis in chronic CsA nephrotoxicity.