RESUMO
BACKGROUND: Peritoneal dialysis (PD) is limited by reduced efficacy over time. We previously showed that a Janus kinase 1/2 inhibitor (JAK1/2i) reduced inflammation, hypervascularity and fibrosis induced by 4.25% dextrose dialysate (4.25%D) intraperitoneally (IP) infused for 10 days in rats with normal kidney function. JAK/STAT signalling mediates inflammatory pathways, including angiotensin signalling. We now tested the effect of long-term JAK1/2i and/or an angiotensin receptor blocker (ARB) on peritoneal membrane (PM) in polycystic kidneys (PCK) rats infused with 4.25%D. METHODS: Except for controls, all PCK rats had a tunnelled PD catheter: (1) no infusions; (2) 4.25%D; (3) 4.25%D + JAK1/2i (5 mg/kg); (4) 4.25%D +losartan (5 mg/kg); and (5) 4.25%D + losartan +JAK1/2i (5 mg/kg each) IP BID × 16 weeks (N = 5/group). PM VEGFR2 staining areas and submesothelial compact zone (SMCZ) width were morphometrically measured. Peritoneal equilibration testing measured peritoneal ultrafiltration (UF) by calculating dialysate glucose at time 0 and 90 min (D/D0 glucose). RESULTS: 4.25%D caused hypervascularity, SMCZ widening, fibrosis and UF functional decline in PCK rats. Angiogenesis was significantly attenuated by JAK1/2i ± ARB but not by ARB monotherapy. Both treatments reduced SMCZ area. UF was preserved consistently by dual therapy (p < 0.05) but with inconsistent responses by monotherapies. CONCLUSION: Long-term JAK1/2i ± ARB reduced angiogenesis and fibrosis, and the combination consistently maintained UF. In clinical practice, angiotensin inhibition has been advocated to maintain residual kidney function. Our study suggests that adding JAK1/2i to angiotensin inhibition may preserve PM structure and UF.
Assuntos
Diálise Peritoneal , Insuficiência Renal Crônica , Ratos , Animais , Soluções para Diálise/metabolismo , Diálise Peritoneal/efeitos adversos , Losartan/metabolismo , Losartan/farmacologia , Antagonistas de Receptores de Angiotensina/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Peritônio/metabolismo , Fibrose , Glucose/metabolismo , Angiotensinas/metabolismo , Angiotensinas/farmacologia , Insuficiência Renal Crônica/metabolismoRESUMO
BACKGROUND: Matrix Gla Protein (MGP) is a potent inhibitor of ectopic calcification and modulates bone morphogenesis. Little is known about MGP expression or function in kidney. METHODS: We investigated renal MGP expression in Sprague-Dawley rats after 5/6 nephrectomy (5/6 Nx) and in human kidney biopsies in the Nephrotic Syndrome Study Network (NEPTUNE) cohort. We analyzed associations between glomerular (nâ¯=â¯182) and tubulointerstitial (TI) (nâ¯=â¯219) MGP mRNA levels and the disease activity/histologic features in NEPTUNE patients. Additionally, uncarboxylated and carboxylated MGP (ucMGP and cMGP, respectively) were localized by immunohistochemistry and quantitated in kidney tissues of patients at different stages of CKD (nâ¯=â¯18). RESULTS: Renal MGP expression was increased in rats after 5/6 Nx. In NEPTUNE data, baseline estimated glomerular filtration rate (eGFR) negatively correlated with glomerular and TI MGP expression (pâ¯<0.001). TI MGP expression strongly correlated with interstitial fibrosis, tubular atrophy, acute tubular injury, and interstitial inflammation, independent of eGFR. Kaplan-Meier analysis and multivariable Cox regression showed that higher levels of TI MGP expression were associated with an increased risk for the composite of 40% decline in eGFR and end-stage renal disease (ESRD) (HR, 3.31; 95% CI, 1.31 to 6.32; pâ¯=0.02). Glomerular and tubulointerstitial cells demonstrated nuclear and cytoplasmic cMGP and ucMGP staining, and eGFR inversely correlated with quantified glomerular cMGP staining (pâ¯<0.05). CONCLUSIONS: Our data demonstrate that renal MGP expression is increased in human and experimental CKD, and is associated with renal outcome. Additional studies are needed to determine its mechanism of action.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas da Matriz Extracelular/genética , Humanos , Rim/metabolismo , Rim/patologia , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/patologia , Proteína de Matriz GlaRESUMO
BACKGROUND: We tested minocycline as an anti-proteinuric adjunct to renin-angiotensin-aldosterone system inhibitors (RAASi) in diabetic nephropathy (DN) and measured urinary biomarkers to evaluate minocycline's biological effects. DESIGN: Prospective, single center, randomized, placebo-controlled, intention-to-treat pilot trial. Inclusion. Type 2 diabetes/DN; Baseline creatinine clearance >30 mL/min; proteinuria ≥1.0 g/day; Age ≥30 years; BP <150/95 mm Hg; intolerant of/at maximum RAASi dose. Protocol. 3-wk screening; Baseline randomization; Urine and blood measures at months 1, 2, 4, and Month 6 study completion. Urine interleukin-6 (IL-6) and osteoprotegerin were measured in a subset. Primary outcome. Natural log of urine protein/creatinine (ln U P:Cr) ratio at Month 6 vs Baseline. RESULTS: 30 patients completed the study. The 15% decline in U P: Cr in minocycline patients (6 month P:Cr ÷ Baseline P:Cr, 0.85 vs. 0.92) was not significant (p = 0.27). Creatinine clearance did not differ in the 2 groups. Urine IL-6:Cr (p = 0.03) and osteoprotegerin/Cr (p = 0.046) decrements were significant. Minocycline modified the relationship between urine IL-6 and proteinuria, suggesting a protective biological effect. CONCLUSIONS: Although the decline in U P:Cr in minocycline patients was not statistically significant, the significant differences in urine IL-6 and osteoprotegerin suggest that minocycline may confer cytoprotection in patients with DN, providing a rationale for further study. TRIAL REGISTRATION: Clinicaltrials.gov NCT01779089.
Assuntos
Albuminas/análise , Nefropatias Diabéticas/tratamento farmacológico , Interleucina-6/análise , Minociclina/uso terapêutico , Osteoprotegerina/análise , Adulto , Creatinina/sangue , Creatinina/urina , Nefropatias Diabéticas/urina , Feminino , Humanos , Interleucina-6/sangue , Interleucina-6/urina , Masculino , Pessoa de Meia-Idade , Osteoprotegerina/sangue , Osteoprotegerina/urina , Projetos Piloto , Efeito Placebo , Estudos Prospectivos , Proteínas/análise , Resultado do TratamentoRESUMO
BACKGROUND: Tumoral calcinosis is an autosomal recessive disorder characterized by ectopic calcification and hyperphosphatemia. METHODS: We describe a family with tumoral calcinosis requiring amputations. The predominant metabolic anomaly identified in three affected family members was hyperphosphatemia. Biochemical and phenotypic analysis of 13 kindred members, together with exome analysis of 6 members, was performed. RESULTS: We identified a novel Q67K mutation in fibroblast growth factor 23 (FGF23), segregating with a null (deletion) allele on the other FGF23 homologue in three affected members. Affected siblings had high circulating plasma C-terminal FGF23 levels, but undetectable intact FGF23 or N-terminal FGF23, leading to loss of FGF23 function. CONCLUSIONS: This suggests that in human, as in experimental models, severe prolonged hyperphosphatemia may be sufficient to produce bone differentiation proteins in vascular cells, and vascular calcification severe enough to require amputation. Genetic modifiers may contribute to the phenotypic variation within and between families.
Assuntos
Calcinose/genética , DNA/genética , Fatores de Crescimento de Fibroblastos/genética , Hiperostose Cortical Congênita/genética , Hiperfosfatemia/genética , Mutação , Fosfatos/sangue , Calcificação Vascular/genética , Adulto , Alelos , Calcinose/sangue , Calcinose/complicações , Análise Mutacional de DNA , Ensaio de Imunoadsorção Enzimática , Exoma , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Genótipo , Humanos , Hiperostose Cortical Congênita/sangue , Hiperostose Cortical Congênita/complicações , Hiperfosfatemia/sangue , Hiperfosfatemia/complicações , Imuno-Histoquímica , Masculino , Calcificação Vascular/sangue , Calcificação Vascular/etiologiaRESUMO
Peritoneal membrane pathology limits long-term peritoneal dialysis (PD). Here, we tested whether JAK/STAT signaling is implicated and if its attenuation might be salutary. In cultured mesothelial cells, PD fluid activated, and the pan-JAK inhibitor P6 reduced, phospho-STAT1 and phospho-STAT3, periostin secretion, and cleaved caspase-3. Ex vivo, JAK was phosphorylated in PD effluent cells from long-term but not new PD patients. MCP-1 and periostin were increased in PD effluent in long term compared with new patients. In rats, twice daily, PD fluid infusion induced phospho-JAK, mesothelial cell hyperplasia, inflammation, fibrosis, and hypervascularity after 10 days of exposure to PD fluid. Concomitant instillation of a JAK1/2 inhibitor virtually completely attenuated these changes. Thus, our studies directly implicate JAK/STAT signaling in the mediation of peritoneal membrane pathology as a consequence of PD.
Assuntos
Soluções para Diálise/efeitos adversos , Janus Quinases/metabolismo , Peritônio/patologia , Peritonite/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Animais , Caspase 3/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Quimiocina CCL2/metabolismo , Células Epiteliais , Feminino , Humanos , Hiperplasia/induzido quimicamente , Janus Quinases/antagonistas & inibidores , Masculino , Neovascularização Patológica/induzido quimicamente , Nitrilas , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/induzido quimicamente , Peritônio/irrigação sanguínea , Peritonite/induzido quimicamente , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND/AIMS: Chronic kidney disease involves inflammation/oxidative stress, which contributes to progressive kidney injury. METHODS: Male Sprague-Dawley rats underwent 5/6 nephrectomy (Nx) or sham Nx and were sacrificed after 2 days, 2 weeks and 4 weeks. Microarray analysis expression sets over time suggested the evolution of renal lymphocyte infiltration and antigen-presenting cell (APC) activation after 5/6Nx. RT-PCR analysis also confirmed the migration and activation of lymphocytes and APCs through the upregulation of CD3, CXCR3/CXCL10 and CCR7/CCL19 mRNA in remnant kidney (RK). Purified T lymphocytes from spleen and unilateral ureteral obstruction (UUO) kidney were incubated with oxidized low-density lipoprotein (Ox-LDL)-treated major histocompatibility complex class II (MHC II)-expressing APCs. Culture supernatant was collected for mouse IFN-γ ELISA and cell proliferation was measured. RESULTS: Ox-LDL deposited predominantly in renal tubulointerstitial areas of RK, increased over time, and co-stained with lectin-like Ox-LDL receptor in affected renal tubular cells. Both Ox-LDL and renal-specific glycoprotein Tamm-Horsfall protein were identified in renal lymph nodes. Cells co-staining for major MHC II and Ox-LDL were observed in RK and draining renal lymph nodes after 5/6Nx. Similarly, Ox-LDL was also present in tubules after UUO, CD3-positive T cells were present in the interstitium, and Ox-LDL-treated MHC II-expressing APCs induced proliferation and IFN-γ production in renal tubulointerstitial T lymphocytes isolated from kidneys after UUO. CONCLUSIONS: These data demonstrate that the tubulointerstitial inflammatory infiltrate that accompanies chronic kidney disease reflects, at least in part, the development of autoimmunity to novel antigens generated during renal injury.
Assuntos
Autoimunidade , Nefropatias/imunologia , Lipoproteínas LDL/imunologia , Linfócitos T/metabolismo , Animais , Complexo CD3/metabolismo , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL19/metabolismo , Quimiocina CXCL10/metabolismo , Doença Crônica , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/efeitos dos fármacos , Interferon gama/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais/imunologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Lipoproteínas LDL/farmacologia , Linfonodos/metabolismo , Masculino , Análise em Microsséries , Nefrectomia , Nefrite Intersticial/imunologia , Nefrite Intersticial/metabolismo , Nefrite Intersticial/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores CCR7/metabolismo , Receptores CXCR3/metabolismo , Receptores Depuradores Classe E/metabolismo , Linfócitos T/fisiologia , Obstrução Ureteral/imunologia , Uromodulina/metabolismoRESUMO
BACKGROUND: Periostin acts as an adhesion molecule during bone formation. Knowledge of its expression in kidney injury is scant. METHODS: We investigated periostin function and expression in vivo in Sprague-Dawley rats after 5/6 nephrectomy (Nx), in DBA2J mice with streptozotocin-induced diabetic nephropathy (SZ-DN) and unilateral ureteral obstruction (UUO) and in vitro in mouse distal collecting tubular cells (MDCT) and in tissue and urine from chronic kidney disease (CKD) patients. RESULTS: Periostin messenger RNA was increased after 5/6Nx and SZ-DN demonstrating generalizability of the increment in renal injury. Periostin was expressed predominantly in distal tubule (DT) epithelial cell cytoplasm in situ, in cells shed into the lumen, and, in lesser abundance, in glomeruli undergoing obsolescence, arterioles and in the tubulointerstitium in extracellular and intracellular locations. In affected DT after 5/6Nx, periostin expression appeared de novo, E-cadherin became undetectable and tubule cells displayed the mesenchymal marker proteins fibroblast-specific protein-1 (FSP1) and matrix metalloproteinase-9 (MMP9). Periostin overexpression in cultured MDCT cells dramatically induced MMP9 and FSP1 protein and suppressed E-cadherin. Periostin short interfering RNA blocked these changes. Urine periostin excretion increased over time after 5/6Nx, and it was also excreted in the urine of CKD patients. Urine periostin enzyme-linked immunosorbent assay at a cutoff of 32.66 pg/mg creatinine demonstrated sensitivity and specificity for distinguishing patients with CKD from healthy people (92.3 and 95.0%, respectively) comparing favorably with urine neutrophil gelatinase-associated lipocalin. CONCLUSION: These data demonstrate that periostin is a mediator and marker of tubular dedifferentiation and a promising tissue and urine biomarker for kidney injury in experimental models and in clinical renal disease.
Assuntos
Biomarcadores/metabolismo , Moléculas de Adesão Celular/metabolismo , Túbulos Renais/patologia , Células-Tronco Mesenquimais/patologia , Insuficiência Renal Crônica/metabolismo , Envelhecimento , Animais , Western Blotting , Estudos de Casos e Controles , Moléculas de Adesão Celular/genética , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Túbulos Renais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Nefrectomia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , UrináliseRESUMO
We sought to find a urinary biomarker for chronic kidney disease and tested hematopoietic growth factor inducible neurokinin-1 (HGFIN, also known as Gpnmb/Osteoactivin) as it was found to be a kidney injury biomarker in microarray studies. Here, we studied whether HGFIN is a marker of kidney disease progression. Its increase in kidney disease was confirmed by real-time PCR after 5/6 nephrectomy, in streptozotocin-induced diabetes, and in patients with chronic kidney disease. In the remnant kidney, HGFIN mRNA increased over time reflecting lesion chronicity. HGFIN was identified in the infarct portion of the remnant kidney in infiltrating hematopoietic interstitial cells, and in distal nephron tubules of the viable remnant kidney expressed de novo with increasing time. In vitro, it localized to cytoplasmic vesicles and cell membranes. Epithelial cells lining distal tubules and sloughed luminal tubule cells of patients expressed HGFIN protein. The urine HGFIN-to-creatinine ratio increased over time after 5/6 nephrectomy; increased in patients with proteinuric and polycystic kidney disease; and remained detectable in urine after prolonged freezer storage. The urine HGFIN-to-creatinine ratio compared favorably with the urine neutrophil gelatinase-associated lipocalin (NGAL)-to-creatinine ratio (both measured by commercial enzyme-linked immunosorbent assays (ELISAs)), and correlated strongly with proteinuria, but weakly with estimated glomerular filtration rate and serum creatinine. Thus, HGFIN may be a biomarker of progressive kidney disease.
Assuntos
Nefropatias/diagnóstico , Glicoproteínas de Membrana/urina , Adulto , Idoso , Animais , Autofagia , Biomarcadores/urina , Creatinina/urina , Diabetes Mellitus Experimental/metabolismo , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Nefropatias/urina , Masculino , Pessoa de Meia-Idade , Nefrectomia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , EstreptozocinaRESUMO
BACKGROUND/AIMS: Altered extracellular matrix (ECM) remodeling and podocyte apoptosis are characteristic features of diabetic nephropathy (DN). Aliskiren (ALI) inhibits the renin-catalyzed conversion of angiotensinogen to angiotensin I. This study tested ALI's effect on podocyte ECM accretion and survival in a high-glucose environment in vitro. METHODS: Conditionally immortalized mouse podocytes were incubated in normal glucose (NG; 5.5 mM) or high glucose (HG; 40 mM) for 24-48 h with and without ALI (20 nM). Real-time RT-PCR was performed for fibronectin (FN), collagen α5(type IV) (Cola5IV), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), and tissue inhibitor of metalloproteinases 1 and 2 (TIMP1 and TIMP2). Western blots were performed for FN, Cola5IV, MMP2, MMP9, TIMP1 and cleaved (activated) caspase-3. RESULTS: ALI significantly reduced the mRNA and protein levels of FN, Cola5IV and TIMP1, and the mRNA of TIMP2 and cleaved caspase-3. ALI had no effect on MMP2 mRNA or protein or MMP9 mRNA tested under HG conditions. Under NG conditions, ALI had no effect on FN, Cola5IV, MMP2, MMP9 and activated caspase-3 proteins. ALI decreased the activated caspase-3 protein and evidence of apoptosis by TUNEL staining observed in podocytes cultured under HG conditions. CONCLUSION: These results show for the first time that renin inhibition with ALI mitigates the profibrotic and apoptotic effects of HG in cultured podocytes. These data strengthen the therapeutic rationale for renin inhibition with ALI beyond its hemodynamic effects.
Assuntos
Amidas/farmacologia , Apoptose/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fumaratos/farmacologia , Glucose/farmacologia , Podócitos/efeitos dos fármacos , Renina/antagonistas & inibidores , Animais , Células Cultivadas , Colágeno Tipo IV/efeitos dos fármacos , Nefropatias Diabéticas/fisiopatologia , Fibronectinas/efeitos dos fármacos , Metaloproteinases da Matriz/biossíntese , Podócitos/metabolismo , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Curcumin has anti-inflammatory, anti-oxidant, and anti-proliferative properties, and depending upon the experimental circumstances, may be pro- or anti-apoptotic. Many of these biological actions could ameliorate diabetic nephropathy. METHODS/DESIGN: Mouse podocytes, cultured in basal or high glucose conditions, underwent acute exposure to curcumin. Western blots for p38-MAPK, COX-2 and cleaved caspase-3; isoelectric focusing for HSP25 phosphorylation; and DNase I assays for F- to G- actin cleavage were performed for in vitro analyses. In vivo studies examined the effects of dietary curcumin on the development of diabetic nephropathy in streptozotocin (Stz)-induced diabetes in DBA2J mice. Urinary albumin to creatinine ratios were obtained, high performance liquid chromatography was performed for urinary curcuminoid measurements, and Western blots for p38-MAPK and total HSP25 were performed. RESULTS: Curcumin enhanced the phosphorylation of both p38MAPK and downstream HSP25; inhibited COX-2; induced a trend towards attenuation of F- to G-actin cleavage; and dramatically inhibited the activation of caspase-3 in vitro. In curcumin-treated DBA2J mice with Stz-diabetes, HPLC measurements confirmed the presence of urinary curcuminoid. Nevertheless, dietary provision of curcumin either before or after the induction of diabetes failed to attenuate albuminuria. CONCLUSIONS: Apart from species, strain, early differences in glycemic control, and/or dosing effects, the failure to modulate albuminuria may have been due to a decrement in renal HSP25 or stimulation of the 12/15 lipoxygenase pathway in DBA2J mice fed curcumin. In addition, these studies suggest that timed urine collections may be useful for monitoring curcumin dosing and renal pharmacodynamic effects.
Assuntos
Albuminúria/tratamento farmacológico , Curcumina/farmacologia , Nefropatias Diabéticas/tratamento farmacológico , Proteínas de Choque Térmico HSP27/metabolismo , Extratos Vegetais/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Actinas/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Inibidores de Caspase , Cromatografia Líquida de Alta Pressão , Curcumina/análogos & derivados , Curcumina/farmacocinética , Curcumina/uso terapêutico , Ciclo-Oxigenase 2/metabolismo , Nefropatias Diabéticas/metabolismo , Dieta , Rim/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Fosforilação , Podócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Beta-cell apoptosis occurs in diabetes mellitus (DM). Heat shock protein (HSP) 27 (human homolog of rodent HSP25) mitigates stress-induced apoptosis but has not been studied in beta-cells. We tested whether HSP27 overexpression attenuates streptozotocin (SZ)-induced DM in vivo and cytokine-induced islet apoptosis in vitro. DM was ascertained by ip glucose tolerance testing, and fasting serum insulin/glucose was measured. Pancreas was stained for insulin, HSP27, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and insulin content was measured. HSP25/27 was measured by immunoblotting, isoelectric focusing, and RT-PCR. Islet HSP25/27 oligomerization and inhibitory kappaB protein kinase gamma (nuclear factor kappaB essential modulator) binding were assessed by coimmunoprecipitation. HSP27 transgene (TG) in pancreas localized predominantly in beta-cells. Baseline pancreatic insulin levels in wild-type (WT) and HSP27TG mice were similar, but lower in WT than HSP27TG after SZ (P < 0.01). Intraperitoneal glucose tolerance testing confirmed protection from SZ-DM in HSP27TG. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and inducible nitric oxide synthase staining were increased in WT vs. HSP27TG islets (P < 0.05) after SZ. Caspase-3 activity was lower in islets from HSP27TG vs. WT mice after cytokine stress in vitro (P < 0.05). There was more HSP25 plus 27 protein from HSP27TG islets than HSP25 from WT (P < 0.01). HSP25 protein but not mRNA was increased in HSP27TG mice. Isoelectric focusing showed similar relative HSP phosphorylation in HSP27TG and WT (P > 0.05). HSP27 bound native HSP25 in TG islets; both bound to inhibitory kappaB protein kinase gamma (nuclear factor kappaB essential modulator). These data show islet protection by HSP27 by mitigation of apoptosis, possibly through nuclear factor kappaB regulation.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/prevenção & controle , Proteínas de Choque Térmico HSP27/biossíntese , Ilhotas Pancreáticas/citologia , Animais , Caspase 3/metabolismo , Proteínas de Choque Térmico/biossíntese , Humanos , Quinase I-kappa B/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares , Proteínas de Neoplasias/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Multimerização ProteicaRESUMO
Longstanding diabetes causes renal injury with early dropout of podocytes, albuminuria, glomerular and tubulointerstitial fibrosis, and progressive renal failure. The renal pathology seems to be driven, in part, by TGF-beta and is associated with a loss of renal bone morphogenic protein-7 (BMP-7) expression. Here, the hypothesis that maintenance of renal (especially podocyte) BMP-7 by transgenic expression reduces diabetic renal injury was tested. Diabetic mice that expressed the phosphoenolpyruvate carboxykinase promoter-driven BMP-7 transgene and nondiabetic, transgenic mice as well as diabetic and nondiabetic wild-type controls were studied for up to 1 yr. Transgenic expression of BMP-7 in glomerular podocytes and proximal tubules prevents podocyte dropout and reductions in nephrin levels in diabetic mice. Maintenance of BMP-7 also reduces glomerular fibrosis and interstitial collagen accumulation as well as collagen I and fibronectin expression. Diabetic wild-type mice develop progressive albuminuria, which is substantially reduced in transgenic mice. These effects of the BMP-7 transgene occur without changing renal TGF-beta levels. It is concluded that maintenance of renal BMP-7 during the evolution of diabetic nephropathy reduces diabetic renal injury, especially podocyte dropout. The findings also establish a role for endogenous glomerular BMP-7 as an autocrine regulator of podocyte integrity in vivo.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Nefropatias Diabéticas/prevenção & controle , Animais , Proteína Morfogenética Óssea 7 , Diabetes Mellitus Experimental/fisiopatologia , Fibrose , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Regiões Promotoras Genéticas , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad5/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The 12/15-lipoxygenase (12/15-LO) pathway is activated in diabetes mellitus (DM), increasing 12(S)-hydroxyeicosatetraenoic acid (12-HETE). We showed that a 12-LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC) inhibited 12/15-LO activity in vivo and assessed the efficacy of another 12/15-LO inhibitor, N-benzyl-N-hydroxy-5-phenylpentamidine (BHPP), to diminish urinary 12-HETE and ameliorate diabetic nephropathy (DN) over 4 months. Rats studied were control (C, n=8), DM (n=6), and rats injected with BHPP (C+BHPP, n=4) and (DM+BHPP, n=5). BHPP 3 mg/kg/day decreased urinary (U) 12-HETE/creatinine (cr) by 30-50% after one injection and after 1 week of daily injections in DM rats. U 12-HETE/cr excretion increased paradoxically in controls given BHPP. There was a highly significant relationship between U 12-HETE/cr excretion and U alb/cr (r=0.79, P<10(-5)), demonstrating that renal 12/15-LO pathway activation is associated with albuminuria. BHPP did not inhibit glomerular collagen synthesis or improve histology. More sustained 12-LO inhibition may improve albuminuria in DN.
Assuntos
Amidas/farmacologia , Benzilaminas/farmacologia , Ácidos Cafeicos/farmacologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/enzimologia , Inibidores de Lipoxigenase , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/urina , Albuminúria , Animais , Colágeno Tipo IV/metabolismo , Creatinina/urina , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Inibidores de Lipoxigenase/farmacologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism is implicated in extracellular matrix (ECM) synthesis, but its role in podocytes has not been studied. This study tested whether 12-LO induction by diabetes or by high glucose (HG) in cultured podocytes alters glomerular basement membrane by activating signal transduction pathways culminating in ECM synthesis. Sprague-Dawley rats received an injection of diluent (control [C]) or streptozotocin 65 mg/kg (DM) and were killed at 1 or 4 mo. Glomerular 12-LO mRNA and protein levels were higher in DM than in C glomeruli at 1 and 4 mo, and 12-LO localized predominantly in podocytes. Glomerular p38 mRNA and protein were higher in DM at months 1 and 4, but phospho-p38 mitogen-activated protein (MAPK) was increased only at month 1. Glomerular collagen alpha5(IV)/glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio was increased in DM at month 1 but not at month 4, whereas collagen alpha5(IV) protein was higher at both 1 and 4 mo. Mouse podocytes were cultured in media with 25 mM glucose (HG) with or without the 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) or with 5.5 mM glucose + 19.5 mM mannitol (low glucose [LG+M]) for 10 d at 37 degrees C. 12-LO mRNA and protein levels were higher in HG than in LG+M as was the p38 MAPK/GAPDH mRNA ratio. Phospho-p38 MAPK protein but not total p38 MAPK was higher in HG compared with LG+M. Collagen alpha5(IV)/GAPDH mRNA ratio and protein were higher in HG than in LG+M. 12-LO inhibition by CDC decreased HG-induced phospho-p38 MAPK and the phospho-p38/total p38 MAPK ratio, collagen alpha5(IV)/GAPDH mRNA ratio, and collagen alpha5(IV) protein expression. In summary, diabetes in vivo and exposure of podocytes to HG in vitro stimulated 12-LO, p38 MAPK, and collagen alpha5(IV) mRNA and (activated) protein. 12-LO inhibition by CDC diminished the expression of podocyte phospho-p38 MAPK and collagen alpha5(IV) mRNA and protein. These findings implicate 12-LO and the p38 MAPK signaling pathway in the mediation of ECM synthesis by podocytes in diabetes.
Assuntos
Araquidonato 12-Lipoxigenase/fisiologia , Colágeno Tipo V/fisiologia , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Células Epiteliais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Animais , Araquidonato 12-Lipoxigenase/genética , Colágeno Tipo V/genética , Glucose/farmacologia , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Masculino , Proteínas Quinases Ativadas por Mitógeno/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: mRNAs of pathogenetic importance in the development of diabetic nephropathy were measured in subjects with type 1 diabetes to determine whether these might be used to predict progression from normoalbuminuria to microalbuminuria. We proposed that conversion from normoalbuminuria to microalbuminuria would be most likely in subjects whose connective tissue growth factor (CTGF) and collagen mRNAs were above the 95% confidence interval (CI) for live renal donors and within the 95% CI for subjects with abnormal albuminuria. METHODS: Glomerular CTGF, collagen alpha2(IV), and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were measured in microdissected glomeruli from living renal donors (n = 10), and subjects with normoalbuminuria (n = 12), microalbuminuria (n = 5), and overt proteinuria (n = 6). RESULTS: After 44 +/- 2 months of follow-up, one subject converted from normoalbuminuria to microalbuminuria. Although the data are limited, progression from normoalbuminuria to microalbuminuria occurred in the only normoalbuminuric subject whose mRNA levels were above the live renal donors' 95% CI for CTGF and collagen alpha2(IV) and within the 95% CI of subjects with abnormal albuminuria. No clinical or histopathologic finding distinguished the progressor from the nonprogressors at the time of biopsy. CONCLUSION: This case report provides proof-of-principle that a panel of glomerular mRNA markers chosen because of their pathogenetic relevance may be useful adjuncts to albuminuria and histology in predicting clinical stability or clinical progression in diabetic nephropathy.
Assuntos
Albuminúria/genética , Diabetes Mellitus Tipo 1/genética , Nefropatias Diabéticas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Glomérulos Renais/metabolismo , RNA Mensageiro/análise , Adulto , Albuminúria/etiologia , Albuminúria/fisiopatologia , Colágeno Tipo IV/análise , Fator de Crescimento do Tecido Conjuntivo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Substâncias de Crescimento/análise , Humanos , Proteínas Imediatamente Precoces/análise , Glomérulos Renais/fisiologia , Glomérulos Renais/fisiopatologia , Doadores Vivos , Masculino , Técnicas de Diagnóstico Molecular/métodos , Valor Preditivo dos TestesRESUMO
Bone morphogenetic protein-7 (BMP7), a member of the transforming growth factor-beta (TGF-beta) superfamily of cytokines, is highly expressed in renal tubules and generally promotes maintenance of epithelial phenotype. It was examined whether, during the evolution of experimental diabetic nephropathy, the renal expression of BMP7 and BMP7 receptors declines, and the hypothesis that loss of BMP7 activity is profibrogenic in proximal tubular cells was tested. Moreover, in vitro studies in cultured proximal tubular cells were performed to examine putative mechanisms that cause these changes. At 15 wk of streptozotocin-induced diabetes, renal expression of BMP7 is declined by about half, and it decreased further by 30 wk to <10% of timed controls. Renal expression of the high-affinity BMP type II receptor and the type I receptor Alk2 (activin receptor-like kinase-2) decreased. Alk3 tended to decrease, but Alk6 remained unchanged. During the evolution of diabetic nephropathy, the secreted BMP antagonist gremlin increased substantially. In cultured tubular cells, TGF-beta reduced BMP7 and Alk3 expression and increased gremlin but did not interrupt BMP7-induced activation of smad5 or Erk1 and -2. In contrast, BMP7 did not alter TGF-beta expression. Neutralization of endogenous BMP7 in cultured proximal tubular cells raised the expression of fibronectin and tended to increase collagen alpha(1) III mRNA levels. In conclusion, in experimental diabetic nephropathy, renal tubular BMP7 and some of its receptors decreased and gremlin, a secreted BMP antagonist, increased. Some, but not all, of these changes are explained by increased TGF-beta. The loss of BMP7 activity per se is profibrogenic in tubular cells.