The effects of wine-processing on ascending and descending: The distribution of flavonoids in rat tissues after oral administration of crude and wine-processed Radix scutellariae.

ETHNOPHARMACOLOGICAL RELEVANCE: Ascending and descending theory is a core principle of traditional Chinese medicine (TCM) theories. It plays an essential role in TCM clinical applications. Some TCM medicine has specific properties, which could alter the inclination and direction of their actions. The properties of the ascending and floating process of one herbal medicine are affected by means of herb processing. Wine-processing, which is sautéing with rice wine, is one of the most popular technologies of herb processing. Wine-processing increases the inclination and direction of its actions, thereby producing or strengthening their efficacy in cleaning the upper-energizer heat. Radix scutellariae, the dried roots of Scutellaria baicalensis Georgi, is a well-known TCM used for the treatment of inflammation, pyrexia, jaundice, etc. Recently, wine-processed Radix scutellariae was normally applied in clinical studies for the treatment of upper-energizer syndrome. In order to investigate the effects of wine-processing on ascending and descending of Radix scutellariae, the comparative study of distribution of flavonoids in rat tissues of triple energizers (SanJiao-upper, middle, lower jiao) after oral administration of crude and wine-processed Radix scutellariae aqueous extracts was carried out. MATERIALS AND METHODS: The rats were randomly assigned to two groups and orally administered with crude and wine-processed Radix scutellariae aqueous extracts, respectively. At different pre-determined time points after administration, the concentrations of compounds in rat tissue homogenate were determined, and the main tissue pharmacokinetic parameters were investigated. Tissue pharmacokinetic parameters including AUC0-t, t1/2, Tmax and Cmax were calculated using DAS 2.0. An unpaired Student t-test was used to compare the differences in tissue pharmacokinetic parameters between the two groups. All the results were expressed as arithmetic mean±S.D. RESULTS: The parameters of Cmax and AUC0-t of some flavonoids in wine-processed Radix scutellariae were remarkably increased (p<0.05, p<0.01, p<0.001) in the rat upper-energizer tissues (lung and heart) compared with those of the crude group. However, in the rat middle- and lower-energizer tissues (spleen, liver and kidney), the Cmax and AUC0-t of some flavonoids were significantly decreased (p<0.05, p<0.01) compared with the crude group. The main explanation for these differences seems to the effects of wine-processing on ascending and descending theory. CONCLUSIONS: All of these differences in the distribution of triple energizers after oral administration of crude and wine-processed Radix scutellariae aqueous extracts may lead to the increase of efficacy on the upper-energizer tissues and were in compliance with the ascending and descending theory. Therefore, wine-processing was recommended when Radix scutellariae was used for cleaning the upper-energizer heat and humidity. The obtained knowledge can be used to evaluate the impact of these differences on the efficacy of both the drugs in clinical applications and might be helpful in explaining the effects of wine-processing on ascending and descending theory.

Synergistic effects of leflunomide and benazepril in streptozotocin-induced diabetic nephropathy.

BACKGROUND: Leflunomide (LEF) and benazepril have renoprotective effects on diabetic nephropathy (DN) through their anti-inflammatory and anti-fibrotic activities. This study investigated whether combined treatment using LEF and benazepril affords superior protection compared with the respective monotherapies. METHODS: Diabetes was induced with streptozotocin (STZ, 65 mg/kg) by intraperitoneal injection in male Wistar rats. Two weeks after STZ injection, diabetic rats were treated daily for 12 weeks with LEF (10 mg/kg), benazepril (10 mg/kg), or a combination of both. Basic parameters (body weight, fasting blood glucose level, and 24 h urinary protein excretion), histopathology, inflammatory [inflammatory cell infiltration (ED-1), monocyte chemoattractant protein-1 (MCP-1), and Toll-like receptor-2 (TLR-2)] and glomerulosclerotic factors [transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF)], and oxidative stress (8-hydroxy-2'-deoxyguanosine, 8-OHdG) were studied. RESULTS: Benazepril or LEF treatment significantly prevented body weight loss and 24 h urinary protein excretion induced by diabetes; combined treatment with LEF and benazepril further improved these parameters compared with giving each drug alone (all p < 0.01). Increased expression of inflammatory (MCP-1 and TLR-2) and glomerulosclerotic (TGF-ß1 and CTGF) factors in diabetic rat kidney was reduced by treatment with either LEF or benazepril and was further reduced by the combined administration of the two drugs (p < 0.01). These effects were accompanied by suppression of urinary 8-OHdG excretion. There was no significant between-group difference in blood glucose level. CONCLUSIONS: LEF treatment lessens DN, and combined treatment with LEF and benazepril provides synergistic effects in preventing DN.

The monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is involved in peritoneal dialysis-related epithelial-mesenchymal transition of peritoneal mesothelial cells.

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) has a role in the process of peritoneal fibrosis (PF), a serious complication in peritoneal dialysis (PD) patients. Even though monocyte chemoattractant protein-1 (MCP-1) was demonstrated to directly increase extracellular matrix (ECM) synthesis, the role of the MCP-1/CCR2 system in PD-related EMT and ECM synthesis in cultured human PMCs (HPMCs) and in an animal model of PD has never been elucidated. In vitro, HPMCs were exposed to 5.6 mM glucose (NG), NG+MCP-1 (10 ng/ml) (NG+MCP-1), or 100 mM glucose (HG) with or without CCR2 inhibitor (RS102895) (CCR2i) or a dominant-negative mutant MCP-1-expressing lentivirus (LV-mMCP-1). In vivo, PD catheters were inserted into 60 Sprague-Dawley rats, and saline (Control, C) (N=30) or 4.25% PD solution (PD) (N=30) was infused for 4 weeks. Twenty rats from each group were treated with empty LV or LV-mMCP-1 intraperitoneally. Snail, E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin protein expression in HPMCs and the peritoneum was evaluated by western blot analysis. Compared with NG cells, Snail, α-SMA, and fibronectin expression was significantly increased, while E-cadherin expression was significantly decreased in HPMCs exposed to HG and NG+MCP-1, and these changes were significantly abrogated by CCR2i (P<0.05). In addition, MCP-1-induced EMT was significantly attenuated by anti-TGF-ß1 antibody. In PD rats, Snail and fibronectin expression was significantly increased in the peritoneum, whereas the ratios of E-cadherin/α-SMA protein expression were significantly decreased (P<0.05). The thickness of the peritoneum and the intensity of Masson's trichrome staining in the peritoneum were also significantly higher in PD rats than in C rats (P<0.05). These changes in PD rats were significantly abrogated by LV-mMCP-1. These findings suggest that the MCP-1/CCR2 system is directly involved in PD-related EMT and ECM synthesis and that this is mediated, at least in part, via TGF-ß1.

The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions.

Previous studies have demonstrated the importance of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of diabetic nephropathy in terms of inflammation, but the direct role of the MCP-1/CCR2 system on podocyte apoptosis under diabetic conditions has never been explored. In vitro, mouse podocytes were exposed to a medium containing 30 mM glucose (HG) with or without CCR2 siRNA or CCR2 inhibitor (RS102895). Podocytes were also treated with MCP-1 or TGF-ß1 with or without anti-TGF-ß1 antibody, CCR2 siRNA, or CCR2 inhibitor. In vivo, 20 db/m and 20 db/db mice were divided into two groups, and ten mice from each group were treated with RS102895. Western blot and Hoechst 33342 or TUNEL staining were performed to identify apoptosis. HG-induced apoptosis and TGF-ß1 levels were significantly abrogated by CCR2 inhibition. In addition, treatment with MCP-1 directly induced apoptosis via CCR2. Moreover, TGF-ß1- and MCP-1-induced apoptosis were significantly ameliorated by the inhibition of CCR2 and anti-TGF-ß1 antibody, respectively. Glomerular expression of cleaved caspase-3 and apoptotic cells within glomeruli were also significantly increased in db/db mice compared to db/m mice, and these increases were significantly attenuated in db/db + RS102895 mice. These results suggest that interactions between the MCP-1/CCR2 system and TGF-ß1 may contribute to podocyte apoptosis under diabetic conditions.

Apoptosis occurs differentially according to glomerular size in diabetic kidney disease.

BACKGROUND: Apoptosis, which is involved in the process of mesangial cell and podocyte loss in diabetic nephropathy, is known to be regulated by protein kinase B/Akt (Akt). A number of studies have therefore investigated the activity of Akt under diabetic conditions, but the results have not been consistent. In this study, we hypothesized that apoptosis may occur differentially and that Akt may be differentially activated according to glomerular size in diabetic kidney disease. METHODS: Fifty male Sprague-Dawley rats were injected intraperitoneally with diluent (C, n = 25) or streptozotocin (DM, n = 25). After 3 months, glomeruli were isolated using sieves with pore sizes of 250, 150, 125 and 75 µm and then classified into large glomeruli (on the 125-µm sieve, LG) and small glomeruli (on the 75-µm sieve, SG) groups. Western blot analyses for phospho-Akt, apoptosis-related molecules (Bax, Bcl-2, active fragments of Caspase-3 and phospho-p53) and cyclin-dependent kinase inhibitors were performed. CONCLUSIONS: The numbers of total cells and podocytes in isolated glomeruli were determined using transmission electron microscopy. Akt phosphorylation was significantly decreased in DM-LG, while it was significantly increased in DM-SG (P < 0.05). The ratio of Bax/Bcl-2 protein expression and active fragments of Caspase-3 and phospho-p53 protein expression were significantly increased in DM-LG compared to DM-SG and C-SG (P < 0.001 and P < 0.01, respectively). In contrast, the expression of p27(Kip1) and p21(Cip1) was significantly increased in DM-SG compared to DM-LG and C-SG (P < 0.05). The numbers of total glomerular cells and podocytes were significantly decreased in DM-LG (P < 0.05). In conclusion, these data show differential expression of Akt activity and apoptosis-related molecules according to glomerular size in diabetic nephropathy, suggesting that apoptosis may be more operative in more hypertrophic glomeruli, resulting in fewer glomerular cells and podocytes in diabetic nephropathy.

Local kallikrein-kinin system is involved in podocyte apoptosis under diabetic conditions.

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.

Effects of an oral adsorbent on oxidative stress and fibronectin expression in experimental diabetic nephropathy.

BACKGROUND: Previous studies have demonstrated that AST-120 (Kremezin((R))), a well-known oral adsorbent, inhibits the progression of diabetic (DM) and non-DM chronic kidney disease along with a decrease in oxidative stress. This study was undertaken to investigate whether AST-120 could reduce oxidative stress and ameliorate the development of nephropathy in experimental DM rats with normal renal function. METHODS: Rats were injected with diluent (C, n = 16) or 65 mg/kg streptozotocin intraperitoneally (DM, n = 16), and eight rats from each group were treated with chow containing 5% AST-120. After 3 months, plasma advanced oxidation protein products (AOPP) and total malondialdehyde (MDA) levels, 24-h urinary albumin excretion, and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion were determined by ELISA. Glomerular endothelial nitric oxide synthase (eNOS), subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (gp91phox, p47phox and p22phox), and fibronectin (FN) mRNA and protein expressions were determined by real-time PCR and western blot, respectively. In addition, dichlorodihydrofluorescein diacetate (DCF-DA) staining was performed to detect glomerular reactive oxygen species (ROS) production. RESULTS: Compared to the C group, 24-h urinary albumin excretion was significantly higher in the DM group (P < 0.01), and AST-120 treatment significantly reduced albuminuria in DM rats (P < 0.05). Glomerular eNOS, gp91phox, p47phox and FN expression were significantly increased in DM rats compared to C rats, and these increases in DM glomeruli were significantly abrogated by AST-120 treatment (P < 0.05). The increases in plasma AOPP and MDA levels as well as renal oxidative stress in DM rats, assessed by DCF-DA staining and urinary 8-OHdG excretion rates, were also significantly attenuated by AST-120 treatment (P < 0.05). CONCLUSION: In conclusion, the renoprotective effects of AST-120 in DM nephropathy seem to be associated with the amelioration of enhanced oxidative stress and FN expression under diabetic conditions.

Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions.

Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10(-7) M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.

Induction of heme oxygenase-1 protects against podocyte apoptosis under diabetic conditions.

Heme oxygenase-1 (HO-1) is an anti-oxidant enzyme normally upregulated in response to oxidant injury. Here we determined the role of HO-1 in podocyte apoptosis in glomeruli of streptozotocin-treated rats and in immortalized mouse podocytes cultured in media containing normal or high glucose. HO-1 expression, its activity, the ratio of Bax/Bcl-2 protein, and active caspase-3 fragments were all significantly higher in isolated glomeruli of diabetic rats and in high glucose-treated podocytes. These increases were inhibited by zinc protoporphyrin treatment of the rats or by HO-1 siRNA treatment of the podocytes in culture. The number of apoptotic cells was also significantly increased in the glomeruli of diabetic rats and in high glucose-treated podocytes. Inhibition of HO-1 accentuated the increase in apoptotic cells both in vivo and in vitro. Our findings suggest that HO-1 expression protects against podocyte apoptosis under diabetic conditions.

Colchicine attenuates inflammatory cell infiltration and extracellular matrix accumulation in diabetic nephropathy.

Recent studies have demonstrated that an inflammatory mechanism contributes to the pathogenesis of diabetic nephropathy (DN). It is also known that colchicine (Col) can prevent various renal injuries via its anti-inflammatory action. However, the effect of colchicine on DN has never been explored. This study was undertaken to elucidate the effect of colchicine on inflammation and extracellular matrix accumulation in DN. In vivo, 64 rats were injected with diluent (C; n = 32) or streptozotocin intraperitoneally (DM, n = 32). Sixteen rats from each group were treated with Col. In vitro, rat mesangial cells and NRK-52E cells were cultured in media with 5.6 mM glucose (NG) or 30 mM glucose (HG) with or without 10(-8) M Col. Monocyte chemotactic protein-1 (MCP-1) mRNA expression was determined by real-time PCR (RT-PCR), and the levels of MCP-1 in renal tissue and culture media were measured by ELISA. RT-PCR and Western blotting were also performed for intercellular adhesion molecule-1 (ICAM-1) and fibronectin (FN) mRNA and protein expression, respectively, and immunohistochemical staining (IHC) for ICAM-1, FN, and ED-1 with renal tissue. Twenty-four-hour urinary albumin excretion at 6 wk and 3 mo were significantly higher in DM compared with C rats (P < 0.05), and colchicine treatment significantly reduced albuminuria in DM rats (P < 0.05). Col significantly inhibited the increase in MCP-1 mRNA expression and protein levels under diabetic conditions both in vivo and in vitro. ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. IHC revealed that the number of ED-1(+) cells were significantly higher in DM compared with C kidney (P < 0.005), and this increase was significantly attenuated by Col treatment (P < 0.01). In conclusion, Col prevents not only inflammatory cell infiltration via inhibition of enhanced MCP-1 and ICAM-1 expression but also ECM accumulation in DN. These findings provide a new perspective on the renoprotective effects of Col in DN.

FR167653 inhibits fibronectin expression and apoptosis in diabetic glomeruli and in high-glucose-stimulated mesangial cells.

Previous in vitro studies suggest that the p38 MAPK pathway may be involved in the pathogenesis of diabetic nephropathy, but the consequences of the inhibition of the p38 MAPK pathway have not been well elucidated in diabetic (DM) glomeruli. This study was undertaken to investigate the effect of p38 MAPK inhibitor, FR167653, on fibronectin expression and apoptosis in DM glomeruli and in high-glucose-stimulated mesangial cells (MC). In vivo, 32 Sprague-Dawley rats were injected with diluent (control, N = 16) or streptozotocin intraperitoneally (DM, N = 16). Eight rats from each group were treated with FR167653 for 3 mo. In vitro, rat MC were exposed to medium containing 5.6 mM glucose or 30 mM glucose [high glucose (HG)] with or without 10(-6) M FR167653 for 24 h. Fibronectin mRNA and protein expression were determined by real-time PCR and Western blot, respectively. Western blot for apoptosis-related molecules, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, and Hoechst 33342 staining were performed to determine apoptosis. FR167653 ameliorated the increases in fibronectin-to-GAPDH mRNA ratio and protein expression in DM glomeruli by 89 and 79% and in HG-stimulated MC by 70 and 91%, respectively (P < 0.05). Under diabetic conditions, Bcl-2 protein expression was decreased, whereas cleaved caspase-3 protein expression was increased (P < 0.05), and these changes were inhibited by FR167653 treatment. Apoptotic cells were also significantly increased in DM glomeruli and in HG-stimulated MC (P < 0.05), and FR167653 ameliorated these increases in apoptotic cells, both in vivo and in vitro. In conclusion, these findings suggest that the inhibition of the p38 MAPK pathway has a beneficial effect on the development of diabetic nephropathy by inhibiting the increase in fibronectin expression and apoptosis.

MCP-1/CCR2 system is involved in high glucose-induced fibronectin and type IV collagen expression in cultured mesangial cells.

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that plays an important role in the recruitment of macrophages. Although previous studies have demonstrated the importance of MCP-1 in the pathogenesis of diabetic nephropathy (DN) in terms of inflammation, the role of MCP-1 and its receptor (C-C chemokine receptor 2; CCR2) in extracellular matrix (ECM) accumulation under diabetic conditions has been largely unexplored. This study was undertaken to investigate the functional role of the MCP-1/CCR2 system in high glucose-induced ECM (fibronectin and type IV collagen) protein expression in cultured mesangial cells (MCs). Mouse MCs were exposed to medium containing 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), or 30 mM glucose (HG) with or without mutant MCP-1 (mMCP-1), CCR2 small interfering (si)RNA, or CCR2 inhibitor (RS102895). To examine the relationship between MCP-1 and transforming growth factor (TGF)-beta1, MCs were also treated with TGF-beta1 (2 ng/ml) with or without mMCP-1 or CCR2 siRNA. Transient transfection was performed with Lipofectamine 2000 for 24 h. Cell viability was determined by an MTT assay, mouse and human MCP-1 and TGF-beta1 levels by ELISA, and CCR2 and ECM protein expression by Western blotting. Transfections of mMCP-1 and CCR2 siRNA increased human MCP-1 levels and inhibited CCR2 expression, respectively. HG-induced ECM protein expression and TGF-beta1 levels were significantly attenuated by mMCP-1, CCR2 siRNA, and RS102895 (P < 0.05). MCP-1 directly increased ECM protein expression, and this increase was inhibited by an anti-TGF-beta1 antibody. In addition, TGF-beta1-induced ECM protein expression was significantly abrogated by the inhibition of the MCP-1/CCR2 system (P < 0.05). These results suggest that an interaction between the MCP-1/CCR2 system and TGF-beta1 may contribute to ECM accumulation in DN.

Gene expression patterns in glucose-stimulated podocytes.

To explore the mechanisms of podocyte injury under diabetic conditions, we performed an expression profile in glucose-stimulated podocytes. Differential gene expression profiles between conditionally immortalized mouse podocytes cultured in medium containing 5.6 and 30 mM glucose were measured with oligonucleotide microarrays. Of the genes identified, heme oxygenase-1, vascular endothelial growth factor-A, and thrombospondin-1 showed a consistently increased pattern, whereas angiotensin-converting enzyme-2 and peroxisomal proliferator activator receptor-gamma were down-regulated. These results were validated using real-time PCR and western blotting in podocytes, and with immunohistochemistry on renal tissues from streptozotocin-induced diabetic rats. Not only is this the first report of gene expression profiling of podocyte injury under diabetic conditions, but the identified genes are promising targets for future diabetes research.

High glucose decreases collagenase expression and increases TIMP expression in cultured human peritoneal mesothelial cells.

BACKGROUND: Peritoneal fibrosis (PF), a serious problem in long-term continuous ambulatory peritoneal dialysis (CAPD) patients, is characterized by extracellular matrix (ECM) accumulation which results from an imbalance between the synthesis and the degradation of ECM components. Previous studies have demonstrated that ECM synthesis is increased in human peritoneal mesothelial cells (HPMCs) under high glucose conditions, but the effects of high glucose on degradative pathways have not been fully explored. This study was undertaken to elucidate the effects of high glucose on these proteolytic processes in cultured HMPCs. METHODS: HPMCs were isolated from human omentum and were exposed to 5.6 mM glucose (NG), 5.6 mM glucose +34.4 mM mannitol (NG + M), or 40 mM glucose (HG) with or without PKC inhibitor (PKCi). Real-time PCR and western blot were performed to determine collagenases (MMP-1, -8 and -13) and TIMPs (TIMP-1 and -2) mRNA and protein expression, respectively. The individual activities of collagenases in culture media were determined by ELISA. RESULTS: Types I and III collagen protein expression were significantly increased in HG-conditioned media compared to NG media (P < 0.05). The MMP-1, -8 and -13/GAPDH mRNA ratios were significantly lower in HPMCs exposed to HG medium compared to NG cells by 64, 52 and 37%, respectively, and their protein expression by 76, 42 and 49%, respectively, in HG- vs NG-conditioned media. The activities of collagenases in HG-conditioned media were also significantly lower than those in NG media (P < 0.05). In contrast, HG significantly increased TIMPs mRNA ratios and protein expression in HPMCs. These changes in collagenase and TIMP expression induced by HG were abrogated upon pre-treatment with PKCi. CONCLUSION: In conclusion, impaired matrix degradation may contribute to ECM accumulation in PF.

Glomerular glucocorticoid receptor expression is reduced in late responders to steroids in adult-onset minimal change disease.

BACKGROUND: Compared to children, adult patients with minimal change disease (MCD) tend to have a slower response to steroids, but little is known about the factors influencing the steroid responsiveness in these patients. In this study, we investigated the difference in the expression of the glomerular glucocorticoid receptor (GCR) according to steroid responsiveness in 28 adult-onset MCD patients. METHODS: Based on the response to steroid treatment, the patients were divided into early responders (ER, n=20) and late responders (LR, n=8) according to the response to steroids on the basis of 4 weeks of treatment. The clinical and laboratory findings, and the glomerular mRNA and protein expression of GCR and nephrin, assessed by real-time polymerase chain reaction and immunohistochemistry, respectively, were compared between the ER and LR groups. Ten microscopic haematuric patients in whom renal biopsy was performed and revealed no histological abnormalities were included for control (C). RESULTS: The mRNA expression of GCR was significantly lower in the LR than that in the ER group (P<0.01), whereas it was comparable between the C and ER groups. GCR protein expression was also decreased in the LR compared with the C and ER groups. In contrast, there was no significant difference in nephrin mRNA expression among the three groups. On the other hand, the GCR mRNA expression correlated inversely with the time to complete remission (r= -0.49, P<0.05), but not with the amount of proteinuria at presentation. CONCLUSION: In conclusion, the levels of glomerular GCR expression may be a useful predictor of steroid responsiveness in adult-onset MCD patients.

Differential expression of nephrin according to glomerular size in early diabetic kidney disease.

Diabetic nephropathy (DN) is clinically characterized by proteinuria. Many studies tried to demonstrate a relationship between proteinuria and changes in nephrin in various forms of glomerular diseases including DN, but the results are not consistent. Glomerular hypertrophy occurs in DN, yet hypertrophy does not develop in all glomeruli concurrently. For investigation of the differences in nephrin expression according to glomerular size, glomeruli were isolated from 10 control and 10 streptozotocin-induced diabetic rats at 6 wk after the induction of diabetes by a sieving technique using sieves with pore sizes of 250, 150, 125, and 75 microm. Glomeruli then were classified into large glomeruli (LG; on the 125-microm sieve) and small glomeruli (SG; on the 75-microm sieve) groups. Glomerular volumes were determined using an image analyzer, and mRNA and protein expression was determined by real-time PCR and Western blot, respectively. The mean volumes of diabetic LG (1.51 +/- 0.06 x 10(6) microm(3)) and control LG (1.37 +/- 0.05 x 10(6) microm(3)) were significantly higher than those of diabetic SG (0.94 +/- 0.03 x 10(6) microm(3)) and control SG (0.87 +/- 0.03 x 10(6) microm(3); P < 0.01). Nephrin mRNA expression was significantly reduced in the diabetic LG group compared with the diabetic SG and control glomeruli groups (P < 0.05). In contrast, nephrin mRNA expression was significantly higher in the diabetic SG group compared with the diabetic LG and control glomeruli groups (P < 0.05). Even after correction for 18s rRNA and Wilms' tumor-1 mRNA expression, the differences in nephrin mRNA expression remained significant. The expression of nephrin protein showed a similar pattern to the mRNA expression. In conclusion, these data suggest that the nephrin gene is differentially expressed according to glomerular size. Furthermore, more hypertrophied glomeruli with lesser nephrin expression may be responsible for albuminuria in the early stage of DN.