[Show abstract][Hide abstract] ABSTRACT: Objective:
To investigate the regulation of calcineurin (CaN) by endoplasmic reticulum stress (ERS) in podocytes in vitro and in vivo at the stage microalbuminuria in diabetic nehropathy (DN).
The urinary albumin excretions of C57BLKS/J (Lepr) db/db and db/m mice at the ages of 6, 9, and 12 weeks were measured. The expressions of CaN and synaptopodin of these mice were observed. In immortalized mouse podocytes, the expression of podocyte CaN incubated with different concentrations of paltimate was quantitatively determined by real-time PCR. The changes of CaN incubated with paltimate with or without ursodeoxy-cholic acid (UDCA) were analyzed by confocal microscopy and Western blotting.
As urine protein increased, the expression of CaN was enhanced and the expression of synaptopodin was reduced in early stage DN db/db mice potocytes. In immortalized mouse podocytes, as the concentrations of palmitate increased, CaN mRNA increased. By confocal microscopy, the fluorescence intensity of CaN increased in palmitate treatment group. After co-incubation with palmitate and UDCA, the fluorescence intensity decreased. The similar results were shown by Western blotting.
At the stage of microalbuminuria in DN, ERS in podocytes up-regulates the expression of CaN.
No preview · Article · Oct 2014 · Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae
[Show abstract][Hide abstract] ABSTRACT: Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum (ER) stress and consequent apoptosis, contributing to the onset of diabetes. We hypothesized that palmitate could induce podocyte apoptosis via ER stress, which initiates or aggravates proteinuria in diabetic nephropathy.
Podocyte apoptosis was detected by 4',6-diamidio-2-phenylindole (DAPI) stained apoptotic cell count and Annexin V-PI stain. The expressions of ER molecule chaperone glucose-regulated protein 78 (GRP78), indicators of ER-associated apoptosis C/EBP homologous protein (CHOP), and Bcl-2 were assayed by Western blotting and real-time PCR. GRP78 and synaptopodin were co-localized by immunofluorescence stain.
Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L (10 hours, 13 hours, and 15 hours compared with 0 hour, P < 0.001) and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours (compared to control, P < 0.001). Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP, and decreased that of Bcl-2. Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to control (P < 0.001), with the maximum concentration being 0.75 mmol/L. Palmitate 0.5 mmol/L loading for 3 hours, 8 hours, and 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to 0 hour (P < 0.001), with the maximum effect at 3 hours. Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control.
Palmitate could induce podocyte apoptosis via ER stress, suggesting podocyte apoptosis and consequent proteinuria caused by lipotoxic free fatty acid could be ameliorated by relief of ER stress.
Preview · Article · Sep 2012 · Chinese medical journal
[Show abstract][Hide abstract] ABSTRACT: To explore the podocyte injury in patients with diabetic nephropathy (DN) and analyze its relationship with glucose regulated protein 78 (GRP78) and proteinuria.
The clinical data of 48 patients diagnosed as DN by renal biopsy were reviewed. All patients were divided into two groups according to proteinuria (>3.5 g/d, n=31 and 3.5 g/d, n=17). The density of podocytes was illustrated by immunohistochemistry staining of Wilms tumor-1 (WT-1), and the immunoflourescence double-staining results of synaptopodin and GRP78 in podocytes were detected.
The podocyte dentistry of urine protein > 3.5 g/d group was significantly lower than that of urine protein>3.5 g/d group urine protein<3.5 g/d group(P=0.003), and it was negatively correlated with proteinuria (P=0.005). The expressions of synaptopodin and GRP78 in podocytes were also negatively correlated with proteinuria (P=0.004 and P=0.001).
The podocyte injury is aggravated with increased proteinuria in DN patients, along with the decrease of the adaptive ability of endoplasmic reticulum to stress.
No preview · Article · Aug 2012 · Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae
[Show abstract][Hide abstract] ABSTRACT: Cyclosporine is effective in treating nephrotic syndrome (NS) with idiopathic membranous nephropathy (IMN) in adults. But high relapse rate remains a major concern. The way to manipulate cyclosporine is inconclusive. The aim of this study was to introduce the way how to titrate the cyclosporine to maintain complete remission without relapse.
Patients with biopsy-proven IMN with NS treated with cyclosporine for at least 1 month from 1996 to 2011 at Peking Union Medical College Hospital were reviewed.
Mean age of the 51 eligible patients was 52 years, with 39 men. Mean proteinuria was (7.47 ± 3.14) g/d, serum albumin (24.50 ± 6.29) g/L, and serum creatinine (82.62 ± 21.18) mmol/L. Cyclosporine was commenced at a mean dose of (3.46 ± 0.63) mg×kg(-1)×d(-1). Oral prednisone (0.40 ± 0.29) mg×kg(-1)×d(-1) was given concomitantly in 38 patients. Cyclosporine was administered for a median of 16 months (range 1 - 93 months) and stopped in non-responders by month six. By month 3 (n = 47), the number in complete remission (CR) and partial remission (PR) was 3 and 24, which shifted to 12 and 17 by month 6 (n = 41). Male gender, heavy proteinuria, low serum albumin level, and high serum creatinine level were significant determinants in poor response by month six (P < 0.05 in all variables compared with responders). There was a significant reversible serum creatinine increase within 25% during month 3 to 12 (P < 0.05 in all variables compared with baseline value). Eleven patients maintained cyclosporine for more than 24 months with a cyclosporine dose of (1.04 ± 1.06) mg×kg(-1)×d(-1). Nine patients were in CR. Renal function, systolic and diastolic blood pressure remained stable. Renal impairment (> 30% rise of serum creatinine), secondary infection, hypertension, gingival hyperplasia and liver impairment occurred in 6, 4, 10, 4, and 1 patients, respectively.
The observation time for cyclosporine to effectively induce CR of NS in IMN adults should be at least six months. Long-term and low-dose of cyclosporine therapy is safe and effective to maintain CR in those responders.
No preview · Article · Nov 2011 · Chinese medical journal
[Show abstract][Hide abstract] ABSTRACT: To investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells.
The in vitro cultured 3T3-L1 cells (preadipocytes) were divided into control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group. Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography. The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay. The mRNA and protein expressions of peroxisome proliferator-activated receptor (PPARgamma) were assayed by quantitative real-time polymerase chain reaction and Western blot.
Oil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation. Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction. The concentrations of free cholesterol in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (12.89 +/- 0.16), (9.84 +/- 0.45), (9.39 +/- 0.46), and (8.61 +/- 0.34) mg/ml, respectively (P < 0.05), and the concentrations of total cholesterol were (12.91 +/- 0.50), (9.94 +/- 0.96), (10.45 +/- 2.51), and (9.53 +/- 0.63) mg/ml, respectively. The leptin concentrations in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (19.02 +/- 0.52), (16.98 +/- 0.11), (15.62 +/- 0.01), and (13.84 +/- 0.66) ng/ml, respectively. The mRNA expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were significantly lower than that in control group (P < 0.05). The protein expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were 80%, 74%, and 61% of that in control group (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the mRNA expression of PPARgamma was (0.60 +/- 0.14), (0.67 +/- 0.03), and (1.30 +/- 0.14) of that in control group (P < 0.05). The protein expression showed a similar trend with mRNA expression (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the expression of leptin in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group was (19.02 +/- 0.52), (15.62 +/- 0.10), and (14.45 +/- 1.01) and (18.07 +/- 0.66) ng/ml, respectively (P < 0.05 compared with the control group).
By downregulating the expression of PPARgamma, rapamycin can decrease cholesterol accumulation in 3T3-L1 cells and inhibit its leptin-secreting capability. This finding may provide a possible explanation for rapamycin-induced hyperlipidemia in clinical practice.
No preview · Article · Oct 2011 · Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae
[Show abstract][Hide abstract] ABSTRACT: To investigate the clinical and pathologic characteristics of anti-glomerular basement membrane(GBM) disease with normal renal function.
The clinical and pathologic data of 6 patients with anti-GBM disease and normal renal function in Peking Union Medical College Hospital were reviewed retrospectively. Furthermore, 29 patients with anti-GBM disease and impaired renal function in the same period in the same hospital were enrolled as the control group. Factors that may influence the prognosis were analyzed.
Six (17.1%) of all 35 patients maintained normal renal function for 12-133 months during follow-up. Five patients had microhematuria and proteinuria, one had pulmonary hemorrhage only, and three manifested as Goodpasture syndrome. Renal biopsies from 4 patients revealed linear deposition of IgG 2+-3+ along the glomerular capillary walls by immunofluorescence. As shown by normal light microscopy, mild mesangial proliferation and crescentic glomerulonephritis with a large amount of fibrinoid necrosis of glomerular capillary walls were observed in different patients; however, most pathological changes were mild. Five of these six patients were treated with immunosuppressive drugs and/or plasma exchange. Compared with the control group, the 6 patients with normal renal function had significantly higher hemoglobin[(77.97±20.62 vs.(99.67±19.80 g/L P=0.024], lower titers of anti-GBM antibody[(224.34 ± 145.79 vs.(80.23 ± 85.73 EU/ml P=0.027], and lower ratio of glomeruli with crescents[(0.58±0.29 vs.(0.17±0.27 ,P=0.005]. These 6 patients with normal renal function were followed up for 12-133 months, among whom 4 patients achieved complete remission and 2 had mild proteinuria and microhematuria.
Anti-GBM disease with normal renal function is not uncommon. Most patients have mild pathologic changes and good prognosis.
No preview · Article · Aug 2011 · Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae
[Show abstract][Hide abstract] ABSTRACT: To determine whether Th17/Treg balance was abnormal in adult patients with minimal change nephrotic syndrome (MCNS), we studied 25 patients with new-onset MCNS and 20 normal persons. The results showed that MCNS patients exhibited a significant increase in Th17 number, Th17-related cytokines (IL-17 and IL-23), and transcription factor (RORγt) levels, as well as an obvious decrease in Treg number, Treg-related cytokines (TGF-β1 and IL-10), and transcription factor (Foxp3) levels. The Th17/Treg ratios increased along with increased proteinuria and decreased albumin levels in patients with MCNS. IL-17 protein expression was also detected in the renal biopsy tissue of MCNS patients, particularly in patients with acute renal failure. Further, Th17/Treg balance returned to normal after effective corticosteroids therapy in 16 MCNS patients. These results indicated that Th17/Treg imbalance existed in MCNS patients, suggesting a potential role of Th17/Treg imbalance in the pathogenesis of MCNS.
No preview · Article · Mar 2011 · Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: Diabetic nephropathy is one of the most common microvascular complications of diabetes mellitus. With an increasing prevalence, its proportion in end-stage renal diseases is ascending. Research on the mechanism of diabetic nephropathy was initially focused on the mesangial matrix and glomerular basement membrane. In recent years, changes in the structure and functions of glomerular filtration barriers, especially podocyte injury, has became new hotspots. Podocyte injury involves the decreases in the density and amount of podocytes, the hypertrophy and degeneration of podocytes, and foot-process effacement, along with changes in some specific protein structure and functions. It is the result of multiple factors and multiple pathways. This articles summarizes the common features of podocyte injury and its role in the development of diabetic nephropathy.
No preview · Article · Oct 2010 · Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae
[Show abstract][Hide abstract] ABSTRACT: To analyze the clinical features of hemodialysis patients complicated by infective endo carditis.
The clinical features of six such patients admitted to Peking Union Medical College Hospital during the year 1990 to 2009 were analyzed. All of them were diagnosed based on Chinese Children Diagnostic Criteria for Infective Endocarditis.
The average age of the six patients was 52.3 +/- 19.3 years old. Four were males. Vascular accesses at the onset of infective endocarditis were as follows: permanent catheters in three, temporary catheters in two, and arteriovenous fistula in one. Three were found with mitral valve involvement, two with aortic valve involvement, and one with both. Five vegetations were found by transthoracic echocardiography, and one by transesophageal echocardiography. Four had positive blood culture results. The catheters were all removed. Four of the patients were improved by antibiotics treatment, in which two were still on hemodialysis in the following 14-24 months and the other two were lost to follow-up. One patient received surgery, but died of heart failure after further hemodialysis for three months. One was well on maintenance hemodialysis for three months after surgery.
Infective endocarditis should be suspected when hemodialysis patients suffer from long-term fever, for which prompt blood culture and transthoracic echocardiography confirmation could be performed. Transesophageal echocardiography could be considered even when transthoracic echocardiography produces negative findings. With catheters removed, full course of appropriate sensitive antibiotics and surgery if indicated could improve the outcome of chronic hemodialysis patients complicated by infective endocarditis.
No preview · Article · Sep 2010 · Chinese Medical Sciences Journal
[Show abstract][Hide abstract] ABSTRACT: To investigate the role of acyl-coenzyme A: cholesterol acyltransferase inhibitor (ACATI) in apoptosis induced by lipids and whether lipids-induced apoptosis is accompanied by increase of free cholesterol in endoplasmic reticulum (ER), in order to further understand the mechanism of lipids-induced apoptosis in advanced atherosclerosis.
Human vascular smooth muscle cells (VSMCs) and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages were used. Tritiated thymidine incorporation was applied to detect cell proliferation. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release. 4',6-diamidino-2-phenylindole (DAPI) staining, caspase-3, -7 assay, and Annexin-V/propidium iodide (PI) staining were used to detect apoptosis. High performance liquid chromatography was used in intracellular free cholesterol and cholesterol ester assay. ER free cholesterol was quantified.
Different lipids had different effects on proliferation and cytotoxicity of VSMCs. 25-hydroxycholesterol (25OHC) had biphasic effects on the proliferation of VSMCs. At low concentration, it stimulated cell proliferation, but turned to proliferation inhibition as concentration reached 15 mug/mL. 25OHC and acetylated low density lipoprotein (AcLDL) could respectively induce apoptosis in human VSMCs and PMA differentiated THP-1 macrophages, which was aggravated by ACATI, accompanied by increase of intracellular free cholesterol content. There was also an increase of cholesterol content in ER with AcLDL-induced apoptosis in THP-1 macrophages.
Lipids could induce apoptosis, accompanied by increase of intracellular free cholesterol content, which could be augmented by ACATI, suggesting that insults resulting in ER free cholesterol rise might be the initiator of apoptosis.
No preview · Article · Jun 2010 · Chinese Medical Sciences Journal
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease. Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum (ER) stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) for five days as an in vitro model, to investigate if acetylated low-density lipoprotein (AcLDL) loading could also induce apoptosis and its underlying mechanisms.
Oil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol (FC) and cholesterol ester (CE) assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed.
Confocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 microg/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER.
ER stress is involved in AcLDL induced apoptosis in THP-1 macrophages with free cholesterol accumulation in the ER.
No preview · Article · Aug 2009 · Chinese medical journal
[Show abstract][Hide abstract] ABSTRACT: To explore the effect of low-flux polysulphone membranes on the serum levels of inflammatory cytokines, hyper-sensitivity C reactive protein (hsCRP), lipoprotein (a) [Lp (a)], and beta2 microglobulin (beta 2MG) in hemodialysis patients.
The blood samples and dialysate samples were collected at pre-dialysis, 120 minutes later during the session, and post-dialysis in 27 stable hemodialysis patients. Variables determined included serum Lp (a), hsCRP, interleukin (IL)-1 beta, IL-6, and beta 2MG. The endotoxin levels were also detected.
There were no significant changes either in endotoxin level of dialysate or in all variables tested during one session (P > 0.05).
Low-flux polysulphone membrane has no effects on serum Lp (a), hsCRP, IL-1 beta, IL-6, and beta 2MG during one session in hemodialysis patients.
No preview · Article · Jul 2009 · Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae
[Show abstract][Hide abstract] ABSTRACT: To evaluate the risk factors of post-renal biopsy bleeding (PBB).
We retrospectively analyzed the clinical data of 1 262 patients who received ultrasound-guided biopsy of native kidney at our hospital from January 2005 to December 2006.
The overall incidence of PBB was 30.3% (383/1,262), among which the incidence of hematoma was 29.4% (371/1,262) (the percentages of less and more than 5cm were 73.9% and 26.1%, respectively) while that of gross hematuria was only 1.3% (17/1,262). The incidences of minor, intermediate, and major bleeding complications were 21.4% (270/1,262), 8.4% (106/1,262), and 0.6% (7/1,262), respectively. In seven patients with major bleeding complications, six had renal disease secondary to rheumatic disease (lupus nephritis, n = 5; scleroderma crisis, n = 1), while the other one had IgA nephropathy (Lee's classification V). The risk of PBB was relatively higher in women and younger patients.
Patients with chronic connective tissue diseases are vulnerable to severe PBB complications. A close monitoring of these patients is necessary.
No preview · Article · Jul 2008 · Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae
[Show abstract][Hide abstract] ABSTRACT: To investigate the clinical and pathological characteristics of lupus nephritis patients complicated with malignant hypertension.
We retrospectively studied 19 patients with lupus nephritis complicated with malignant hypertension who underwent renal biopsy between January 2002 and December 2006.
Of 19 patients, 3 were men and 16 were women, with a mean age of 24.4 +/- 7.7 years old. All had positive antinuclear antibodies and low serum complement was found in 13 patients. All were anemic and 12 of them were thrombocytopenic. Impaired renal function was found in 17 patients with an average serum creatinine of 184.5 +/- 88.9 micromol/L. Severe intrarenal arteriolar lesion was found in all patients. Six patients had lupus vasculopathy, 11 patients had renal thrombotic microangiopathy lesion, 2 had severe arteriosclerosis. All patients received steroids and immunosuppressive drugs, 15 received angiotensin-converting enzyme inhibitor (ACEI)/angiotensin receptor blocker (ARB) with resultant well-controlled blood pressure. Thrombocytopenia and hemolytic anemia resolved remarkably. The renal function improved or recovered in 14 of 17 patients, and 3 developed end-stage renal disease on maintenance dialysis.
Severe intrarenal vascular lesion complicated with renal nephritis parallels clinical manifestation of malignant hypertension. Renal pathology is the key of treatment strategy emphasizing on the significance of renal vascular involvement and type. On the basis of immunosuppressive drugs and steroids to control systemic lupus activity, timely initiation of ACEI/ARB could be of benefit to blood pressure control and long term renal survival.
No preview · Article · Jul 2008 · Chinese Medical Sciences Journal