TRANSPLANTATION OF SUBRETINAL STEM CELL-DERIVED RETINAL PIGMENT EPITHELIUM FOR STARGARDT DISEASE: A Phase I Clinical Trial.

PURPOSE: To assess the safety of injecting human embryonic stem cell retinal pigment epithelial cell dose to treat Stargardt disease. METHODS: In this prospective, Phase I clinical trial, human embryonic stem cell retinal pigment epithelial cells in suspension were injected into the subretinal space in eyes with the worse best-corrected visual acuity (BCVA). After vitrectomy/posterior hyaloid removal, a partial retinal detachment was created and the human embryonic stem cell retinal pigment epithelial cells were administered. Phacoemulsification with intraocular lens implantation was performed in eyes with lens opacity. All procedures were optical coherence tomography-guided. The 12-month follow-up included retinal imaging, optical coherence tomography, visual field/electrophysiologic testing, and systemic evaluation. The main outcome was the absence of ocular/systemic inflammation or rejection, tumor formation, or toxicity during follow-up. RESULTS: The mean baseline BCVAs in the phacoemulsification and no phacoemulsification groups were similar (1.950 ± 0.446 and 1.575 ± 0.303, respectively). One year postoperatively, treated eyes showed a nonsignificant increase in BCVA. No adverse effects occurred during follow-up. Intraoperative optical coherence tomography was important for guiding all procedures. CONCLUSION: This surgical procedure was feasible and safe without cellular migration, rejection, inflammation, or development of ocular or systemic tumors during follow-up.

Diabetic Foot Wounds Treated With Human Amniotic Membrane and Low-level Laser Therapy: A Pilot Clinical Study.

BACKGROUND: Low-level laser therapy (LLLT) and human amniotic membrane (HAM) application have been shown to be viable options for use in wound healing. PURPOSE: This study sought to compare LLLT and HAM to a control treatment (hydrogel, saline, and gauze) in persons with diabetes mellitus (DM) and foot ulcers. METHODS: Using a prospective pilot clinical study design, patients receiving care at a health center that specializes in the treatment of diabetic foot wounds between November 2016 and August 2017 were recruited. Eligible patients had to be 30 to 59 years of age; diagnosed with type 2 DM (postprandial capillary glucose levels between 140 and 350 mg/dL); and have uninfected, granulating stage 2 or 3 foot ulcers measuring less than 7 cm by 3 cm. Immunosuppressed and malnourished patients or those with neoplasms or in critical condition were not eligible to participate. Patients received the control treatment (2 mg hydrogel, saline, and gauze), HAM (patches of thawed HAM, applied with overlapping edges), or LLLT (phototherapy session, 2 mg hydrogel, saline, and gauze) for 28 days. Variables, wound area measurements, Pressure Ulcer Scale for Healing (PUSH) scores, and Visual Analog Scale (VAS) scores were used to assess wound improvement progress and pain on days 7, 14, 21, and 28. Descriptive statistics were used to analyze the participant anthropometric and clinical profiles. The Kolmogorov-Smirnov test was used to analyze the sample distribution. The Kruskal-Wallis test with Dunn's post-test was used to evaluate differences in PUSH and VAS scores and wound size for intergroup analysis, and the Mann-Whitney U test was used for the same outcomes in intragroup analysis. The level of significance was 5% (P < .05). RESULTS: Twenty-seven (27) patients participated (mean age, 51.4 years; mean body mass index, 26.5 kg/m2), with 9 patients in each treatment group. No statistically significant differences were noted in clinical or anthropometric variables among the groups, but mean baseline wound areas were different (2.6 cm² for the control, 1.9 cm² for the LLLT, and 5.5 cm² for the HAM groups). Intragroup comparisons showed a significant reduction in PUSH score in the LLT group between days 0 and 21 (8.2 vs 4.9; P < .01) and days 21 to 28 (4.9 vs 3.2; P < .001). In all treatment groups the percent reduction was significantly different between days 7 and 28. No outcomes were significantly different between groups. CONCLUSION: Diabetic foot ulcer wound area as well as PUSH and VAS scores showed more improvement for patients with DM receiving LLLT or HAM than for the control group, but the differences were not significant. Larger studies are needed to compare these treatment modalities.

Analysis of potential contamination factors in musculoskeletal tissues.

Tissue Banks have become the main source for bone grafts, due to preference for homologous tissues. Notwithstanding the use of aseptic techniques for procurement of tissues and judicious selection of donors, microorganisms are frequently found in procured bones. Purpose of this study is to evaluate the factors that increase safety of procurement and minimize discard of procured tissues. Microbiological contamination was analyzed in 1271 musculoskeletal tissues removed from 138 multi-organ donors over a period extending from 2006 to 2016. Effects of various risk factors related with contamination were estimated using a logistic regression model. Microbiological contamination rate in the tissues was 17.1%; low pathogenic microorganisms were cultivated in 12.9% of the tissues, while highly pathogenic ones were cultivated in 4.2% of the tissues. Evolution of one single team was monitored during that period, verifying a fall in the general contamination level from 22.5 to 9.2%. Absence of antibiotics increased low pathogenic contamination risk. Every additional day in intensive care unit (ICU) increased the risk of highly pathogenic contamination. Time elapsed between death and the beginning of removal procedures was found to be relevant for both low pathogenic and highly pathogenic microorganisms. Among the studied factors, the following contributed for a significant increase in contamination by microorganisms in removed tissues: lack of use of prophylactic antibiotic therapy in donors, quantity of removed tissues, length of admission in ICU and the time elapsed between aortic clamping and beginning of the removal procedure.

Supra-organization and optical anisotropies of the extracellular matrix in the amniotic membrane and limbal stroma before and after explant culture.

In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure-induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties.

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro.

PURPOSE: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. METHODS: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. RESULTS: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. CONCLUSION: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro / Avaliação do efeito da riboflavina e luz ultravioleta sobre os ceratócitos in vitro

ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.

RESUMO Objetivo: Avaliar o efeito da aplicação da luz ultravioleta e riboflavina sobre ceratócitos da córnea humana in vitro. Métodos: Os ceratócitos foram obtidos a partir das rimas corneoesclerais remanescentes da trepanação de córneas previamente utilizadas em cirurgias de transplante de córnea e cultivadas em meio DMEM/F12 com 2% de FBS até atingir confluência. As culturas de células foram caracterizadas por imunofluorescência com os anticorpos K3 (marcador de células epiteliais), Thy-1 (marcador de fibroblasto) SMA (marcador de miofibroblasto) e Lumican (marcador de ceratócitos). Imunofluorescência também foi feita após o tratamento. As células do estroma da córnea foram cobertas com colágeno (200 µL e 500 µL) e 0,1% de riboflavina e exposta a luz UVA a 370 nm por 30 minutos. Após 24 horas, citotoxicidade foi determinada por ensaio de MTT e a viabilidade celular foi feita por Hoechst 33342/Iodeto de propideo. Resultados: As culturas de células atingiram confluência em aproximadamente 20 dias. Imunofluorescência apontou alta expressão para o marcador de ceratócitos (Lumican) e expressão negativa par os marcadores de células epiteliais (K3), fibroblasto (Thy-1) e miofibroblasto (α-SMA). Após o cross linking a análise de MTT mostrou baixa taxa de toxicidade e com a coloração de Hoechst 33342/Iodeto de propideo baixa taxa de apoptose e necrose respectivamente em todos os grupos que continham colágeno. Conclusão: As culturas de ceratócitos foram obtidas e caracterizadas por imunofluorescência através do marcador Lumican com sucesso. O ensaio de MTT e a coloração por Hoechst 33342 e iodeto de propídio, apresentaram maior índice de morte celular nos grupos que não continham colágeno, provando que protege as células contra os efeitos da luz UVA.

Toxoplasma Gondii Infection of Chicken Embryos Causes Retinal Changes and Modulates HSP90B1 Gene Expression: A Promising Ocular Toxoplasmosis Model.

HSP90B1 is a gene that codifies heat shock protein 108 (HSP108) that belongs to a group of proteins induced under stress situation, and it has close relation with the nervous system, especially in the retina. Toxoplasma gondii causes ocular toxoplasmosis that has been associated with a late manifestation of the congenital toxoplasmosis although experimental models show that morphological alterations are already present during embryological development. Here, we used 18 eyes of Gallus domesticus embryos in 7th and 20th embryonic days to establish a model of congenital ocular toxoplasmosis, experimentally infected in its fifth day correlating with HSP90B1 gene expression. Embryos' eyes were histologically evaluated, and gene expression was performed by real-time polymerase chain reaction (PCR). Our data showed parasite present in the choroid, unusual migration of retinal pigment epithelium, and chorioretinal scars, and a tendency to a lower expression of the HSP90B1 gene upon experimental infection. This is a promising model to better understand T. gondii etiopathogeny.

Conjunctival epithelial cells cultivated ex vivo from patients with total limbal stem cell deficiency.

PURPOSE: Reconstruction of the ocular surface is challenging. As an alternative to mucosal and limbal epithelial, we study the feasibility of cultivated human conjunctival epithelial (HCjE) cells of patients with total limbal stem cell deficiency (LSCD). METHODS: We studied superior forniceal conjunctival biopsies harvested from 9 living donors with total LSCD of several etiologies who underwent surgery for ocular surface reconstruction. The conjunctival explants were cultivated on serum and growth factor supplemented DMEM/F12 under submerged conditions on denuded human amniotic membrane and tissue culture dishes. The area of cell growth was assessed. Cell morphology was analyzed by light microscopy, impression cytology, and transmission electron microscopy. Cultures were evaluated for epithelial cytokeratins (CK3, CK19), proliferation marker (Ki-67), and putative stem cells markers (ABCG2 and p63). Confocal immunofluorescence was also performed to assess CK3, CK19, Ki-67, ABCG2, and p63. RESULTS: The HCjE cells cultivated ex vivo were successfully expanded on denuded amniotic membrane but with a slower growth than in the tissue culture dish. Transmission electron microscopy showed stratified epithelium with microvilli, desmosomes, and hemidesmosomes. Impression cytology showed PAS+ cells that resembled goblet cells. Immunocytochemical analysis showed positivity for CK3, CK19, Ki-67, ABCG2, and p63. Confocal immunofluorescence was positive for CK3, CK19, Ki-67, ABCG2, and p63. CONCLUSIONS: Our results showed that it is possible to cultivate HCjE cells ex vivo of patients with ocular surface diseases. This method is important for ocular surface reconstruction in patients with bilateral total LSCD.

Transplantation of conjunctival epithelial cells cultivated ex vivo in patients with total limbal stem cell deficiency.

PURPOSE: To report the outcomes of transplantation of autologous conjunctival epithelial cells cultivated ex vivo (EVCAU) in patients with total limbal stem cell deficiency (LSCD). METHODS: EVCAU were cultivated on denuded human amniotic membrane and transplanted in 12 eyes of 10 patients with total LSCD. We evaluated the improvement in the defined clinical parameters of LSCD (loss of corneal epithelial transparency, superficial corneal neovascularization and epithelial irregularity/recurrent epithelial breakdown), vision acuity, impression cytology, immunocytochemical analysis (CK3/CK19), and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. Histologic and immunohistochemical features were studied in 3 corneal buttons of patients submitted to penetrating keratoplasty after EVCAU. RESULTS: Cultivated conjunctival epithelium formed 4 to 5 layers with the formation of basement membrane-like structures. Immunocytochemical analysis showed positivity for CK3, CK19, MUC5AC, Ki-67, P63, and ABCG2. The improvement of the clinical parameters for this treatment in our cohort was 10 of 12 (83.3%) in a mean follow-up time of 18.5 months (range, 15-26 months), and these eyes showed an improvement in impression cytology, immunocytochemistry, and in vivo confocal analysis. Corneal buttons showed a well-formed epithelium with 5 to 6 layers, with rare cells periodic acid-Schiff+, and positivity for CK3, CK19, P63, connexin 43, and MUC5AC. CONCLUSION: We demonstrated the preliminary results of transplantation of EVCAU for corneal surface reconstruction in cases with total LSCD. Future studies are needed to further assess the long-term efficacy of this procedure.

Mesangial cells cultured from pregnant rats display reduced reactivity to angiotensin II: the role of relaxin, nitric oxide and AT2 receptor.

BACKGROUND/AIMS: Pregnancy is characterized by vasodilatation and increased glomerular filtration rate (GFR), despite overstimulation of the renin angiotensin system (RAS). The mesangial cells (MCs) influences GFR and when cultured from pregnant rats displays refractoriness to Ang II. We evaluated the role of relaxin (RLX) and its receptor (RXFP1), nitric oxide (NO) and the AT2 receptor in this response. METHODS: MCs cultured from kidneys of virgin (V) and pregnant (P) Wistar rats were treated with RLX or AT2 receptor blocker PD123319 or NO synthase inhibitor L-NAME. After 24 hr, intracellular calcium concentration ([Ca]i) was recorded before and after the addition of Ang II. RESULTS: MCs from V group expressed AT2, RLX and RXFP1, whose levels were increased in P cells. Ang II induced a 150% increase in [Ca] i in the V cells and 85% (p<0.05) in the P cells. V cells treated with RLX displayed a similar response to that observed in P cells, suggesting that RLX can modulate the reactivity of the MCs to Ang II. L-NAME and PD123319 did not interfere in this response. CONCLUSION: Results suggest that RLX is a mediator of the refractoriness of the MCs to Ang II during pregnancy.

Role of chymase in diabetic nephropathy.

Chymase is an alternative pathway for angiotensin-converting enzyme in angiotensin II (Ang II) formation, and its expression is increased in human diabetic kidneys and in human mesangial cells (MCs) stimulated with high glucose. In addition, chymase activates transforming growth factor (TGF-ß1) via an Ang II-independent pathway. The aim of this study was to evaluate the role of chymase on TGF-ß1 activation in diabetic rats and in rat MCs (RMCs) stimulated with high glucose (HG). Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, intravenous). After 30 (D30) or 60 (D60) days, chymase activity and the expression of profibrotic markers were evaluated. RMCs were stimulated with HG in the presence or absence of 50 µmol/L chymostatin, a chymase inhibitor, or 100 nmol/L of losartan, an Ang II antagonist. Chymase activity and expression increased in D60 kidneys, with increased expression of fibronectin, type I and III collagen, TGF-ß1 and Smad 3 and with no change in Smad 7 expression. RMCs exposed to HG presented increases in chymase activity and expression, together with upregulation in fibrosis markers and in the TGF-ß1 signaling pathway. All these effects were reversed by chymostatin and by losartan, but type 1 angiotensin II receptor blockade did not interfere with the Smad 3 and 7 pathway. Similar to HG-stimulated RMCs, control RMCs treated with chymase responded with increased expression of TGF-ß1, Smad 3 and fibrosis markers. These effects were reversed by chymostatin but not by losartan. The results indicate an important role for chymase in inducing fibrosis through TGF-ß1 activation, parallel with Ang II effects.

(Pro)renin receptor: another member of the system controlled by angiotensin II?

The prorenin receptor [(P)RR] is upregulated in the diabetic kidney and has been implicated in the high glucose (HG)-induced overproduction of profibrotic molecules by mesangial cells (MCs), which is mediated by ERK1/2 phosphorylation. The regulation of (P)RR gene transcription and the mechanisms by which HG increases (P)RR gene expression are not fully understood. Because intracellular levels of angiotensin II (AngII) are increased in MCs stimulated with HG, we used this in vitro system to evaluate the possible role of AngII in (P)RR gene expression and function by comparing the effects of AT1 receptor blockers (losartan or candesartan) and (P)RR mRNA silencing (siRNA) in human MCs (HMCs) stimulated with HG. HG induced an increase in (P)RR and fibronectin expression and in ERK1/2 phosphorylation. These effects were completely reversed by (P)RR siRNA and losartan but not by candesartan (an angiotensin receptor blocker that, in contrast to losartan, blocks AT1 receptor internalization). These results suggest that (P)RR gene activity may be controlled by intracellular AngII and that HG-induced ERK1/2 phosphorylation and fibronectin overproduction are primarily induced by (P)RR activation. This relationship between AngII and (P)RR may constitute an additional pathway of MC dysfunction in response to HG stimulation.

Escherichia coli lipopolysaccharide impairs the calcium signaling pathway in mesangial cells: role of angiotensin II receptors.

Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. The ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca(2+)]i) mobilization was analyzed using angiotensin II (AngII). The effect of LPS on the levels of the renin-angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 microg/mL). AngII-induced [Ca(2+)]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). The mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca(2+)]i and an impaired response to AngII-induced rise in [Ca(2+)]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca(2+)]i peak, suggesting an involvement of AT2R in these responses. The expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO.

Regulation of glucose uptake in mesangial cells stimulated by high glucose: role of angiotensin II and insulin.

Mesangial cells (MCs) play a central role in the pathogenesis of diabetic nephropathy (DN). MC dysfunction arises from excessive glucose uptake through insulin-independent glucose transporter (GLUT1). The role of the insulin-dependent transporter (GLUT4) remains unknown. This study evaluated the effect of high glucose on GLUT1, GLUT4, and fibronectin expression levels. Glucose uptake was determined in the absence and presence of insulin. Angiotensin II has been implicated as a mediator of MC abnormalities in DN, and its effects on the GLUTs expression were evaluated in the presence of losartan. MCs were exposed to normal (NG, 10 mM) or high (HG, 30 mM) glucose for 1, 4, 12, 24, and 72 hrs. Glucose uptake was elevated from 1 hr up to 24 hrs of HG, but returned to NG levels after 72 hrs. HG induced an early (1-, 4-, and 12-hrs) rise in GLUT1 expression, returning to NG levels after 72 hrs, whereas GLUT4 was overexpressed at later timepoints (24 and 72 hrs). HG during 4 hrs induced a 40% rise in glucose uptake, which was unaffected by insulin. In contrast, after 72 hrs, glucose uptake was increased by 50%, only under insulin stimulus. Losartan blunted the effects of HG on GLUT1, GLUT4, and fibronectin expression and on glucose uptake. Results suggest that MCs can be highly susceptible to the HG environment since they uptake glucose in both an insulin-independent and insulin-dependent manner. The beneficial effects of angiotensin II inhibition in DN may also involve a decrease in the rate of glucose uptake by MCs.

ACE-dependent and chymase-dependent angiotensin II generation in normal and glucose-stimulated human mesangial cells.

High glucose (HG) increases angiotensin II (AngII) generation in mesangial cells (MC). Chymase, an alternative AngII-generating enzyme, is upregulated in the glomeruli of diabetic kidneys. In this study, we examined AngII synthesis by human MC via angiotensin-converting enzyme (ACE)-dependent and chymase-dependent pathways under normal glucose (NG, 5 mM) and HG (30 mM) conditions. NG cells expressed ACE and chymase mRNA. Under NG conditions the chymase inhibitor chymostatin reduced AngII levels in cell lysates and in the culture medium, and the ACE inhibitor captopril had no effect. HG induced a 3-fold increase in chymase mRNA and protein but not in ACE mRNA; however, HG induced a 10-fold increase in intracellular ACE activity. The increase in AngII generation induced by HG was found in the cell lysate but not in the culture medium. The rise in intracellular AngII was not prevented by captopril or by chymostatin. Moreover, captopril inhibited extracellular ACE activity but failed to block intracellular ACE activity; these results suggested that captopril was unable to reach intra-cellular ACE. Losartan did not change the intracellular AngII content in either NG or HG conditions, and this lack of change suggested that the increase in AngII was due to intracellular generation. Together these results suggest that chymase may be active in human MC and that both ACE and chymase are involved in increased AngII generation during the HG stimulus by different mechanisms, including an upregulation of chymase mRNA and a rise in intracellular ACE activity, favoring the generation and accumulation of intracellular AngII.

Renin similar to the submaxillary gland form is expressed in mouse mesangial cells: subcellular localization and all generation under control and glucose-stimulated conditions.

It was analyzed the forms of renin produced by a mouse immortalized mesangial cell line (MIC) and their ability to generate angiotensin II (AII). The synthesis, localization and secretion of renin and AII by MIC were evaluated under conditions of normal (10 mM) or high (30 mM) glucose concentration. Two major bands of 35 kDa and 70 kDa were observed in SDS-PAGE. The amino-terminal sequencing revealed the presence of prorenin and renin in these bands with higher homology with the submaxillary gland form of renin. Renin and AII were detected in cell lysate and in culture medium, indicating that MIC synthesize and secrete these peptides. Renin was localized in the cytoplasm while AII was seen predominantly inside the nucleus. High glucose induced an increase in the synthesis and secretion of renin and AII. Results suggest that MIC produce AII and a renin form similar to the submandibular. Intracellular AII may be directed at the nucleus and/or be secreted, indicating that AII may directly influences gene expression in these cells. The mechanisms of synthesis and secretion of renin and AII are potentially modified by high glucose concentration, suggesting a possible role of AII produced by mesangial cells in diabetic nephropathy.