RESUMEN
OBJECTIVE: To study the effects of radiotherapy upon progression of crescentic glomerulonephritis in rats. METHODS: Male SD rats were divided into three groups: (1) control (n=12), sham-operation; (2) crescentic glomerulonephritis (n=23), intravenously inject with nephrotoxic serum (NTS); (3) radiotherapy (n=55), a single low-dose irradiation of 0.5 Gy X-ray to both kidneys at Days 6, 13, 20 and 27 after NTS injection, and sacrificed at different time points among control and crescentic glomerulonephritis rats. Radiotherapy rats have received local kidney irradiation at Days 6, 13, 20 and 27 after bolus NTS injection and would be referred to as NTS7dRa1d, NTS14dRa1d, NTS21dRa1d and NTS28dRa1d, respectively. RESULTS: For NTS7dRa1d and NTS14dRa1d rats of radiotherapy, the levels of serum creatinine, glomerular hypercellularity, crescents and global sclerosis were significantly lower at Days 8 (P < 0.05), 15, and 22 post-irradiation as compared with group of crescentic glomerulonephritis of similar time intervals (P < 0.01). The extent of tubulointerstitial damage was also reduced, and radiotherapy associated histological improvements were accompanied by reduced macrophage infiltration in glomeruli and interstitium. The numbers of PCNA- and ED1-positive cells were reduced in the kidneys at Day 1 postirradiation in NTS7dRa1d and NTS14dRa1d rats as compared with group of crescentic glomerulonephritis at similar time intervals (P < 0.05). A larger number of TUNEL-positive cells were noted at Day 1 postirradiation in rats irradiated at Days 6 & 13 after NTS injection as compared with group of crescentic glomerulonephritis at similar time intervals (P < 0.05). With regards to immunostaining for macrophages ED1 and TUNEL, serial sections of irradiated nephritic kidney showed that fewer ED1-positive macrophages were stained for TUNEL. As evaluated expression of active caspases 3 & 7 was noted in irradiated kidneys as compared with the corresponding group of crescentic glomerulonephritis at similar time intervals. Western blot analysis showed marked increase in the expression of active caspase 3 & 7 in irradiated kidneys as compared with NTS injection only the expression of a marked increase in the expression of p53 protein, closely related to radiation-induced apoptosis, was also observed in irradiated kidneys as compared with NTS injection only. CONCLUSION: Radiotherapy inhibits the progression of experimental crescentic glomerulonephritis through inducing apoptosis by a p53-dependent pathway.
Asunto(s)
Glomerulonefritis/metabolismo , Glomerulonefritis/radioterapia , Animales , Apoptosis , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Progresión de la Enfermedad , Genes p53 , Glomerulonefritis/patología , Riñón/metabolismo , Riñón/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Connective tissue growth factor (CTGF) is a potent fibrogenic cytokine which has been associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. We investigated the role of CTGF on the progression of a rat model of radiation nephropathy. METHODS: The model of radiation nephropathy in rats was established as follows: control group (n = 12), underwent only laparotomy; irradiated group (n = 20), underwent a laparotomy, then the rats were subjected to a single dose 25 Gy X-ray to the kidneys. The rats were followed up one, three, six and nine months after renal exposure to radiation. RESULTS: Renal dysfunction was noted early in irradiated rats. Histological analysis showed focal glomerular sclerotic lesions at an early stage after irradiation. Radiation-induced glomerular and tubulointerstitial injuries were particularly severe the sixth month after irradiation as compared to the control group (P < 0.01). By immunohistochemistry, increased expression of CTGF was noted in the irradiated kidneys, which began to increase from the first month after irradiation, and remained significantly higher at the sixth and ninth month after irradiation (P < 0.01). Upon Western blot analysis CTGF protein expression showed an increase in the radiation treated kidneys compared with the control rats. The expression of CTGF closely correlated with the progression of radiation nephropathy. The expression of alpha-smooth muscle actin, vimentin, type III collagen and type IV collagen was also high in the irradiated kidney as compared to control rat kidneys (P < 0.05), and was most severe at the sixth and ninth month after irradiation (P < 0.01). By double immunostaining, CTGF expressing cells were found to be alpha-SMA-positive myofibroblasts and vimentin-positive tubular epithelial cells. Glomerular expression of CTGF closely correlated with the glomerular expression of alpha-SMA (r = 0.628, P < 0.01), vimentin (r = 0.462, P < 0.05) and accumulation of type IV collagen (r = 0.584, P < 0.01) in the irradiated kidney. Similarly, the expression of CTGF was positively correlated with the expression of alpha-SMA (r = 0.613, P < 0.01), vimentin (r = 0.629, P < 0.01), deposition of type III collagen (r = 0.741, P < 0.001) and type IV collagen (r = 0.799, P < 0.0001) in the tubulointerstitium of the irradiated kidney. Finally the expression of CTGF after the irradiation of the kidney positively correlated with the levels of blood urea nitrogen and serum creatinine. CONCLUSION: Overexpression of CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in radiation nephropathy.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/fisiología , Enfermedades Renales/etiología , Riñón/efectos de la radiación , Traumatismos Experimentales por Radiación/etiología , Actinas/análisis , Animales , Colágeno Tipo III/análisis , Colágeno Tipo IV/análisis , Factor de Crecimiento del Tejido Conjuntivo/análisis , Masculino , Ratas , Ratas Wistar , Vimentina/análisisAsunto(s)
Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Actinas/metabolismo , Animales , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Desmina/metabolismo , Nefropatías Diabéticas/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Vimentina/metabolismoRESUMEN
AIM: Irbesartan, a new antagonist of the type 1 angiotensin II receptor, has been proven to be renal protective in both diabetic and non-diabetic nephropathy, but its exact mechanism is still uncertain. Here we investigated the influence of irbesartan on the expression of the integrin-linked kinase (ILK) and its relationship with epithelial-mesenchymal transition (EMT) in mice with unilateral ureteral obstruction (UUO). METHODS: The mice were randomly divided into 3 groups: sham operation (C, n=20), UUO (n=40), and UUO with irbesartan treatment (UUO+irbesartan, n=40). Irbesartan was given at a dose of 50 mg/kg body weight per day by gavage. The experimental animals in the control group received the same volume of vehicle (0.9% saline solution). The animals were sacrificed at d 1, 3, 7, and 14, respectively, after the surgery. RESULTS: The expression of the ILK at mRNA and protein levels were significantly increased in the UUO group 1 d after the surgery, which was significantly decreased by treatment with irbesartan (P<0.01, respectively). The expression of alpha-smooth muscle actin (alpha-SMA) was significantly increased, while E-cadherin was decreased in mice with UUO at d 3 after the surgery. Treatment with irbesartan significantly abrogated such effects (P<0.01). The immunohistochemistry analysis indicated that the protein expression of the ILK was positively correlated with alpha-SMA, but negatively with E-cadherin. CONCLUSION: These results suggested that irbesartan attenuated renal tubulointerstitial fibrosis in UUO mice, which may be related to the inhibition of ILK expression, subsequently preventing the tubular EMT.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Compuestos de Bifenilo/farmacología , Transdiferenciación Celular/efectos de los fármacos , Fibrosis/prevención & control , Riñón/efectos de los fármacos , Riñón/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Tetrazoles/farmacología , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Actinas/metabolismo , Animales , Cadherinas/metabolismo , Regulación hacia Abajo , Células Epiteliales , Irbesartán , Riñón/patología , Masculino , Ratones , Nefritis Intersticial/prevención & control , ARN Mensajero/metabolismo , Distribución AleatoriaRESUMEN
OBJECTIVE: To investigate the expression of integrin-linked kinase (ILK) in kidneys of mice with unilateral ureteral obstruction and its relevance with the epithelial-mesenchymal transition. METHODS: Mice were randomly divided into two groups, sham operation (C, n = 20) and unilateral ureteral obstruction (UUO, n = 40). The animals were sacrificed at day 1, 3, 7 and 14 respectively after the surgery. Tubulointerstitial fibrosis (TIF) was graded according to Masson staining. The protein level of ILK was examined by Western blot. Tissue/cytological expression for ILK, alpha-SMA and E-cadherin were investigated by immunohistochemistry. The mRNA levels of ILK, alpha-SMA and E-cadherin were analyzed by quantitative real-time PCR. RESULTS: In the control animals (group C), weak staining for ILK was detected mainly in the podocytes. Significant increase of staining for ILK in the experimental mice (UUO group) was detected from day 1 onward (t = 16.5, P < 0.01), reaching the peak at day 7. The protein expression of E-cadherin was continuously down-regulated from day 3 onward after surgery (t = 21.0, P < 0.01), while expression for alpha-SMA was up-regulated. From day 1 to day 7, the protein expression of ILK was positively correlated with alpha-SMA (R = 0.88, P < 0.01), but negatively correlated with E-cadherin (R = -0.87, P < 0.01). The mRNA expression of ILK and alpha-SMA analyzed by real-time PCR increased from postoperative day 1 and 3 respectively, but the mRNA expression of E-cadherin decreased from day 3 onward. CONCLUSION: Increasing expression of ILK occurs in the early phase of UUO mouse and may play an important role in the process of TIF through mediating the epithelial-mesenchymal transition.
Asunto(s)
Túbulos Renales/patología , Proteínas Serina-Treonina Quinasas/biosíntesis , Obstrucción Ureteral/patología , Actinas/biosíntesis , Actinas/genética , Animales , Western Blotting , Cadherinas/biosíntesis , Cadherinas/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibrosis , Inmunohistoquímica , Túbulos Renales/metabolismo , Masculino , Mesodermo/metabolismo , Mesodermo/patología , Ratones , Músculo Liso/química , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Obstrucción Ureteral/genética , Obstrucción Ureteral/metabolismoRESUMEN
AIMS: Emerging evidence suggests that the urinary excretion of cytokines is associated with the progression of chronic kidney disease (CKD). However, detection of urinary cytokines in high throughput is still a problem in clinical practice. In this cross-sectional study, we applied a novel proteomic technology, antibody array, to analyze urinary cytokine profiles in patients with CKD. METHODS: A total of 10 subjects including 7 CKD patients and 3 normal controls were studied. These patients with CKD were divided into two groups according to the levels of estimated glomerular filtration rate (eGFR): group A (eGFR >or=80 ml/min/1.73 m(2), n = 3) and group B (eGFR Asunto(s)
Anticuerpos/análisis
, Citocinas/inmunología
, Citocinas/orina
, Enfermedades Renales/orina
, Análisis por Matrices de Proteínas
, Adulto
, Enfermedad Crónica
, Estudios Transversales
, Citocinas/biosíntesis
, Ensayo de Inmunoadsorción Enzimática
, Femenino
, Humanos
, Inmunoensayo
, Enfermedades Renales/fisiopatología
, Masculino
, Persona de Mediana Edad
RESUMEN
BACKGROUND: Renal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats. METHODS: Seventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n = 32) and diabetic nephropathy (group DN, n = 40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed. RESULTS: There was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P < 0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7 +/- 103.0) nm vs (121.8 +/- 19.1) nm, P < 0.01] and TBM [(767.7 +/- 331.1) nm vs (293.0 +/- 110.5) nm, P < 0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P < 0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r = 0.92, P < 0.05), VG (r = 0.86, P < 0.05), AT (r = 0.94, P < 0.01) and positively correlated with the expression of p27kip1 (r = 0.96, P < 0.01). CONCLUSION: The expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.
Asunto(s)
Diabetes Mellitus Experimental/patología , Proteínas Inmediatas-Precoces/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Riñón/patología , Albuminuria/etiología , Animales , Factor de Crecimiento del Tejido Conjuntivo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Hipertrofia , Proteínas Inmediatas-Precoces/análisis , Péptidos y Proteínas de Señalización Intercelular/análisis , Masculino , Nefrectomía , Ratas , Ratas Sprague-Dawley , EstreptozocinaRESUMEN
BACKGROUND: Cellular hypertrophy is an early, important pathological feature of renal diseases such as diabetic nephropathy and remnant kidney. Recent studies have demonstrated that angiotensin II (AngII) plays a key role in mediating cell hypertrophy. The aim of our work was to explore the role of connective tissue growth factor (CTGF) in mediating AngII-induced tubular cell hypertrophy in vivoandin vitro. METHODS: In an in vivo study, male Sprague-Dawley rats were randomly divided into three groups: control rats, diabetic rats and diabetic rats treated with irbesartan (IRB). The index of kidney hypertrophy (kidney weight/body weight, KW/BW), glomerular tuft area (AG), glomerular tuft volume (VG) and proximal tubular area (AT) were determined. Renal expression for CTGF was detected by immunohistochemical staining. In an in vitro study, the influence of CTGF antisense oligonucleotide (CTGF AS) on AngII-induced CTGF expression and cell hypertrophy was also investigated. RESULTS: In an in vivo study, diabetic rats showed a significant increase of KW/BW, AG, VG, and AT from week 1 onwards compared to normal control, which could be significantly inhibited by using IRB. Furthermore, there was a significantly increasing expression of CTGF in both glomeruli and tubuli in diabetic rats compared to control, and the extent of CTGF expression closely correlated with the severity of renal hypertrophy. Treatment with IRB could markedly inhibit the renal expression of CTGF. In an in vitro study, AngII stimulated the expression of CTGF mRNA and CTGF protein. AngII significantly increased the total protein content in HK2 cells, which was markedly inhibited by co-treatment with CTGF AS. The average cellular diameter determined by scanning electronic microscope showed that the increase of cell size induced by AngII could be significantly inhibited by CTGF AS. Furthermore, flow cytometer study showed that AngII arrested the cell cycle in the G0-G1 phase, which was significantly reversed by treatment with CTGF AS. CONCLUSION: Our data provide both in vivo and in vitroevidence that CTGF is involved in mediating AngII-induced renal hypertrophy.