Phosphoinositide 3-kinase is required for high glucose-induced hypertrophy and p21WAF1 expression in LLC-PK1 cells.

Transforming growth factor-beta (TGF-beta), Smads, and the cyclin-dependent kinase (cdk) inhibitor p21(WAF1) are important in the pathogenesis of diabetic tubular hypertrophy. Phosphoinositide 3 kinase (PI3K)/Akt kinase activity is increased in diabetic glomerular hypertrophy. Thus, we studied the role of PI3K in high glucose (30 mM)-induced p21(WAF1), Smad2/3, and cell cycle-dependent hypertrophy in LLC-PK1 cells. We found that high glucose time-dependently (1-48 h) increased PI3K/Akt kinase activity. LY294002 (a PI3K inhibitor) attenuated high glucose-induced cell cycle-dependent (G(0)/G(1) phase) hypertrophy at 72 h while attenuating high glucose-induced p21(WAF1) gene transcription and protein expression at 36-48 h. LY294002 also attenuated high glucose-induced binding of p21(WAF1) to the cyclin E/cdk2 complex, whereas attenuating high glucose-induced TGF-beta bioactivity, Smad2/3 phosphorylation, and Smad2/3 DNA-binding activity at 36-48 h. We concluded that PI3K is required for high glucose-induced cell cycle-dependent hypertrophy, p21(WAF1) transcription and expression, p21(WAF1) binding to the cyclin E/cdk2 complex, TGF-beta bioactivity, and Smad2/3 activity in LLC-PK1 cells.

Increased glomerular and extracellular malondialdehyde levels in patients and rats with focal segmental glomerulosclerosis.

BACKGROUND: Evidence suggests an increase in oxidative stress in patients with chronic kidney disease, as glomerulosclerosis is the prerequisite for chronic kidney disease; whether the oxidative stress already exists early on is not known. MATERIALS AND METHODS: In this study we measured the plasma and urinary levels of malondialdehyde (MDA), the end product of lipid peroxidation, and assessed the immunoreactivity of MDA and superoxide dismutase (SOD) in glomeruli of patients and rats with primary focal segmental glomerulosclerosis (FSGS), and compared our findings with those of minimal change disease (MCD) and normal controls (NC). RESULTS: Our results showed that plasma MDA level was significantly increased in patients with FSGS compared with both patients with MCD and normal controls. The urinary MDA level was also significantly increased and was significantly correlated with plasma MDA level in patients with FSGS. The immunostaining for glomerular MDA and SOD was significantly higher in the patients with FSGS than in either the patients with MCD or NC, and was also significantly higher in rats with puromycin aminonucleoside (PAN)-induced FSGS than in rats with MCD. Glomerular MDA level was significantly correlated with the degree of glomerulosclerosis in the patients with FSGS. CONCLUSIONS: Our data suggest that oxidative stress occurs early on before the onset of renal failure, and may play an important role in the pathogenesis of glomerulosclerosis.

Advanced glycation end product-induced proliferation in NRK-49F cells is dependent on the JAK2/STAT5 pathway and cyclin D1.

Advanced glycation end products (AGEs) are important in the pathogenesis of diabetic nephropathy, which leads to renal fibrosis. Previously, we found that the janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway is necessary for AGE-induced cellular proliferation in normal rat kidney interstitial fibroblast (NRK-49F) cells. However, a direct link between JAK/STAT and cell-cycle progression has not been well established. In this regard, STAT5 has been found to induce cyclin D1 and proliferation in hematopoietic cells. Therefore, we examined effects of AGE on STAT5 and cell-cycle-dependent mitogenesis in NRK-49F cells. We found that AGE increased cyclin D1 expression and cyclin-dependent kinase (cdk)4 activity while decreasing p21(WAF1/CIP1) expression. We also found that AGE (100 microg/mL) induced STAT5 tyrosine phosphorylation. Meanwhile, AGE induced STAT5 protein-DNA binding activity, which was reversed by AG-490 (a specific JAK2 inhibitor) and STAT5 decoy oligodeoxynucleotide (ODN). In addition, STAT5 decoy ODN reversed AGE-induced cell-cycle-dependent cellular proliferation and cyclin D1 protein expression. We concluded that AGE induced cell-cycle-dependent cellular proliferation by inducing the JAK2-STAT5-cyclin D1 and cdk4 pathways in NRK-49F cells.

Differential effects of FMLP-activated neutrophils from patients with IgA nephropathy enhanced endothelin 1 production of glomerular mesangial cells.

BACKGROUND: Neutrophil infiltration in the glomeruli is common in patients with IgA nephropathy (IgAN). The pathogenetic roles of the infiltrated neutrophils and their relationship with glomerular mesangial cells, however, are not clear. METHODS: We examined the effects of coculture with N-formyl-methionyl-leucyl-phenylalanine (FMLP) activated neutrophils on the viability, endothelin 1 (ET-1) production, and ET-1 mRNA expression of rat glomerular mesangial cells. Neutrophils were isolated from 15 IgAN patients, from 13 patients with non-IgA mesangial proliferative glomerulonephritis (MsPGN), and from 10 normal controls. RESULTS: The ET-1 production by mesangial cells was significantly higher after stimulation with FMLP-activated neutrophils from IgAN patients than that of MsPGN patients and normal controls, and this effect was significantly abolished by pretreating mesangial cells with superoxide dismutase and partly abolished by catalase. The ET-I mRNA expression of mesangial cells showed a parallel increase with ET-1 protein. The trypan blue exclusion test showed significant mesangial cell death after stimulation with FMLP-activated neutrophils as compared with quiescent neutrophils, and the cell death was also prevented by superoxide dismutase but not catalase. The FMLP-activated neutrophils from IgAN patients produced more superoxide than those of MsPGN patients and normal controls. CONCLUSION: The FMLP-activated neutrophils from patients with IgAN have differential effects in enhancing the cell death and the ET-1 production of glomerular mesangial cells through the release of superoxide.

Differential effects of circulating IgA isolated from patients with IgA nephropathy on superoxide and fibronectin production of mesangial cells.

BACKGROUND: IgA nephropathy (IgAN) is characterized by predominant deposition of IgA in the glomerular mesangium. Serum IgA is often elevated in patients with IgAN, and it has been postulated that it is responsible for the mesangial lesions. However, the direct effect of circulating IgA on mesangial cells is not clear. METHODS: We investigated the effects of sera and IgA which were isolated from patients with IgAN on thymidine uptake, superoxide and fibronectin production and fibronectin mRNA expression of cultured rat mesangial cells, and we compared the findings to the effects of IgA isolated from patients with non-IgA mesangial proliferative glomerulonephritis (MsPGN) and normal controls. IgA was isolated with affinity chromatography using cyanogen bromide activated Sepharose 4B coupled to sheep antihuman IgA antiserum. RESULTS: Our results demonstrated that both sera and IgA from patients with IgAN dose-dependently increased mitogenesis of mesangial cells as measured by (3)H-labeled thymidine uptake. The thymidine uptake by sera and IgA isolated from patients with IgAN was significantly higher than that of sera and IgA isolated from patients with MsPGN and normal controls. Sera and IgA from patients with IgAN significantly enhanced superoxide and fibronectin production and fibronectin mRNA expression of mesangial cells. The superoxide and fibronectin production was also significantly higher as compared with patients with MsPGN and normal controls. CONCLUSIONS: Our results indicate that circulating IgA isolated from patients with IgAN is different from that of patients with MsPGN and normal controls and may potentially induce oxidative injury and production of extracellular matrix of glomerular mesangial cells in IgAN.

Plasma and urinary endothelin-1 in focal segmental glomerulosclerosis.

The kidney is an important site of endothelin-1 (ET-1) production and is particularly susceptible to ET-1 action. Infusion of ET-1 in rats induces both functional and morphological alterations in the kidneys. Increased plasma level of ET-1 has been reported in patients with chronic renal failure. However, there are still no reports on the plasma and urinary ET-1 levels in patients with focal segmental glomerulosclerosis (FSGS). In the present study, we have measured the plasma concentration and urinary excretion rate of ET-1 in 15 patients with nephrotic syndrome due to FSGS, and observed the serial changes of plasma and urinary ET-1 in nephrotic rats with FSGS, induced by repeated injection with puromycin aminonucleoside (PAN). ET-1 was measured with radioimmunoassay. The results showed that plasma ET-1 concentration in FSGS patients was significantly higher than in normal controls (P < 0.05), and that urinary ET-1 excretion rate was also significantly higher in FSGS patients than in normal controls (P < 0.01). In FSGS patients, the plasma and urinary ET-1 was significantly correlated (P < 0.05), and the urinary ET-1 excretion rate was significantly correlated with the amount of proteinuria (P < 0.05) and the glomerular sclerosing score (P < 0.01). In the ten rats with PAN-induced FSGS, serial examination showed a significant increase in plasma ET-1 after 8 weeks of injections, while the urinary ET-1 excretion rate showed a biphasic increase that showed a peak after 4 to 6 weeks. The same changes in plasma and urinary ET-1 levels were not observed in control rats injected with normal saline at the same frequency. Our results suggest that ET-1 may be involved in the pathogenesis of FSGS in both humans and rats.

Alterations of glomerular and extracellular glutathione peroxidase levels in patients and rats with focal segmental glomerulosclerosis.

Glutathione peroxidase (GPX) is an important antioxidant that effectively scavenges hydrogen peroxide. Recent studies have revealed that plasma GPX activity is decreased in patients with chronic kidney failure. However, there have been no reports on renal and urinary GPX levels in patients with kidney disorders. Therefore in this study we have measured the plasma and urinary GPX levels and glomerular GPX immunostaining in patients with focal segmental glomerulosclerosis (FSGS) and normal kidney function as compared with those in patients with minimal change disease (MCD) and those in normal control subjects. Rats with puromycin aminonucleoside-induced FSGS were also studied. The results showed that the plasma GPX level was significantly lower in FSGS patients than in either MCD patients or normal control subjects (both P <.01). The urinary GPX level was also significantly lower in FSGS patients than in either MCD patients (P <.05) or normal control subjects (P <.01). The immunostaining score of glomerular GPX was significantly lower in FSGS patients than in normal control subjects (both P <.05). Serial examinations of glomerular GPX immunostaining in FSGS rats also demonstrate a decrease in the score with the progression of disease. Our results indicate that all the plasma, urinary, and glomerular GPX levels are decreased in FSGS patients, indicating a decreased antioxidant defense in the early stages of chronic glomerular diseases.

Significance of salivary epidermal growth factor in peptic ulcer disease in hemodialysis patients.

BACKGROUND/AIMS: Hemodialysis (HD) patients are prone to developing peptic ulcers. However, of all the risk factors associated with peptic ulcers, none have been shown to be more prevalent in HD patients than in the general population. However, salivary epidermal growth factor (EGF) may play a role in peptic ulcer diseases. METHODS: Salivary EGF levels and bioactivities were assayed in 47 maintenance HD patients and 30 normal controls, and the molecular weights of EGF were assessed using high-performance liquid chromatography (HPLC). RESULTS: Salivary EGF levels were not different between both groups of subjects (4.2 +/- 0.34 vs. 5 +/- 0.54 ng/mg protein, NS), and HPLC revealed that salivary EGF in both groups had similar molecular weights. However, salivary EGF bioactivity was significantly depressed in the HD patients as compared to the normal controls (0.59 +/- 0.08 vs. 1.55 +/- 0.15 ng/mg protein, p < 0.01). Stepwise multiple regression showed that the low salivary EGF levels were associated with female gender (p < 0.05), while low salivary EGF bioactivity was associated with HD per se (p < 0.05). In the 22 HD patients who underwent gastric endoscopy, salivary EGF bioactivity was significantly lower in those with peptic ulcers than in those without (0.38 +/- 0.08 vs. 0.69 +/- 0.08 ng/mg protein, p < 0.05). CONCLUSION: Decreased salivary EGF bioactivity may contribute to peptic ulcer disease among maintenance HD patients.

Role of receptor for advanced glycation end-product (RAGE) and the JAK/STAT-signaling pathway in AGE-induced collagen production in NRK-49F cells.

Advanced glycation end-product (AGE) is important in the pathogenesis of diabetic nephropathy (DN), and captopril (an angiotensin converting enzyme inhibitor) is effective in treating this disorder. We have shown that the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) cascade is responsible for AGE-induced mitogenesis in NRK-49F (normal rat kidney fibroblast) cells, but its role in renal fibrosis in DN remains unknown. Therefore, we have sought to determine whether JAK/STAT is involved in AGE-regulated collagen production in NRK-49F cells. We found that AGE time (1-7 days) and dose (10-200 microg/ml)-dependently increased collagen production in these cells. Additionally, AGE increased RAGE (receptor for AGE) protein expression. AGE-induced RAGE expression was dose-dependently inhibited by antisense RAGE oligodeoxynucleotide (ODN) and captopril. AGE-induced type I collagen production and JAK2-STAT1/STAT3 activation were decreased by AG-490 (a specific JAK2 inhibitor), antisense RAGE ODN and captopril. Meanwhile, STAT1 and STAT3 decoy ODNs also suppressed the induction of collagen by AGE. We concluded that RAGE and the JAK2-STAT1/STAT3 pathway were involved in AGE-induced collagen production in NRK-49F cells. Furthermore, captopril was found to reverse AGE-induced collagen production, probably by attenuating RAGE expression and JAK2-STAT1/STAT3 activities.

Insulin and heparin suppress superoxide production in diabetic rat glomeruli stimulated with low-density lipoprotein.

Patients with diabetic nephropathy frequently show increased levels of circulating low-density lipoprotein (LDL) and oxidized LDL, which have been reported to be related to the generation of oxygen-free radicals. In the present study, we evaluated the effects of insulin and heparin on the superoxide production of glomeruli, which were isolated from rats with streptozotocin-induced diabetes for one week, one month, and three months, respectively, and the glomeruli were stimulated with native and oxidized LDL. LDL was isolated from normal subjects with normolipidemia, and the superoxide was measured by using a spectrophotometer. The results demonstrated that the poorly controlled diabetic rat glomeruli showed a significantly higher production of superoxide than normal glomeruli under basal status and after stimulation, and this production increased further with the progression of diabetes. Insulin suppressed both the basal and stimulated production of superoxide in diabetic glomeruli, but not in normal glomeruli. Heparin suppressed superoxide production of diabetic glomeruli stimulated by either native or oxidized LDL, and it also partly suppressed superoxide production of normal glomeruli stimulated by oxidized LDL. Our results suggest that glomerular injury in diabetics with hyperlipidemia may be mediated through enhanced generation of oxygen-free radicals, which can be partially attenuated by insulin and heparin.

Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes.

The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.

Neuronal and endothelial nitric oxide synthase expression in outer medulla of streptozotocin-induced diabetic rat kidney.

AIMS/HYPOTHESIS: Several investigations have shown that the renal medulla has a greater capacity to generate nitric oxide than the renal cortex. To further evaluate the changes of nitric oxide synthesis in the kidney, particularly in the outer medulla, in disorders involving fluid and electrolyte imbalances, we sought to determine renal nitric oxide synthase expression in the diabetic rats. METHODS: We determined renal nitric oxide synthase mRNA and urinary nitrite/nitrate excretion in 12 normal and 12 streptozotocin-induced diabetic rats by reverse transcription-polymerase chain reaction with Southern blot hybridization and with Griess reaction, respectively. Nitric oxide synthase immunoreactivity was detected by immunohistochemistry in four normal and four diabetic rats. RESULTS: Neuronal and endothelial nitric oxide synthase mRNA were 3.5-fold and 1.8-fold increased in the outer medulla of 12 diabetic rats with no difference found in the cortex and inner medulla when compared with 12 normal rats. Urinary nitrite/nitrate excretion was significantly increased from the first week after diabetic induction. In normal rats, immunohistochemical studies showed positive neuronal and endothelial nitric oxide synthase immunostaining in almost all segments of renal tubules. Diabetic rats had the greatest enhancement of immunostaining for neuronal and endothelial nitric oxide synthase in the proximal straight tubule and medullary thick ascending limb. CONCLUSION/INTERPRETATION: Our results indicate that increases in neuronal and endothelial nitric oxide synthase synthesis in the kidney, particularly in the outer medulla, possibly play an important part in the adaptation of renal function to hyperglycaemia and hyperosmolality in diabetes.

Reactive oxygen species enhances endothelin-1 production of diabetic rat glomeruli in vitro and in vivo.

Both reactive oxygen species (ROS) and endothelin-1 (ET- 1) have been implicated in the pathophysiology of diabetic nephropathy. The interrelationship between them, however, has not been documented in this disease. To determine whether ROS regulates ET-1 production in diabetic kidneys, we examined the in vitro and in vivo effects of ROS donors and scavengers on ET-1 production of diabetic rat glomeruli. For in vitro study, the glomeruli were isolated with a sieving method from streptozotocin-induced diabetic rats and killed at 1 week, 1 month, and 3 months, respectively. Superoxide was measured by a spectrophotometer, and ET-1 was measured by radioimmunoassay. The results demonstrated that the basal production levels of superoxide and ET-1 were higher in diabetic glomeruli than in normal glomeruli in vitro. There was a positive correlation between the production of superoxide and ET-1 in diabetic glomeruli. The basal ET-1 production was markedly attenuated by ROS scavengers including superoxide dismutase, catalase, dimethyl sulfoxide, and deferoxamine in diabetic glomeruli. Exogenous ROS generated by xanthine/xanthine oxidase significantly enhanced ET-1 generation by both diabetic and normal glomeruli. A high glucose concentration (500 mg/dL) in vitro increased ET-1 production by normal glomeruli but not diabetic glomeruli, and insulin partly suppressed ET- 1 production by diabetic glomeruli. The in vivo study demonstrated that when diabetic rats were injected daily with superoxide dismutase or catalase after diabetes was induced, the basal production of ET-1 was markedly attenuated after 1 week and 1 month, respectively. These results indicate that exogenously or endogenously derived ROS can enhance ET-1 production by diabetic rat glomeruli and that ROS scavengers suppress ET- 1 production both in vitro and in vivo. The effects of ROS on ET-1 production of diabetic glomeruli may be partly caused by the effect of hyperglycemia or insulin deficiency.

Native and oxidized low-density lipoproteins enhance superoxide production from diabetic rat glomeruli.

Oxygen free radicals have been implicated in mediating diabetic complications, and patients with diabetic nephropathy frequently show increased levels of circulating and oxidized low-density lipoproteins (LDL). In the present study, we measured the superoxide production of glomeruli isolated from poorly controlled diabetic (streptozotocin) rats sacrificed 1 week and 1, and 3 months after the induction of diabetes. The animals were stimulated with native and oxidized LDL isolated from normal humans with normolipidemia. The superoxide ion was measured by using a spectrophotometer. The results demonstrated that the poorly controlled diabetic rat glomeruli showed a significantly higher production of superoxide than normal glomeruli under basal conditions, and this production increased further with the progression of diabetes. Stimulation with either LDL or oxidized LDL enhanced superoxide production by diabetic glomeruli, with oxidized LDL being more potent than LDL. Our results suggest that oxidized LDL may play important roles in the pathogenesis of diabetic nephropathy through enhanced generation of oxygen free radicals.

Fatal outcome after ingestion of star fruit (Averrhoa carambola) in uremic patients.

Clinical outcome of dialysis patients after eating star fruit (Averrhoa carambola) varies, but it may be fatal. In the past 10 years, 20 such patients were treated in our hospital when they developed clinical symptoms after eating the fruit or drinking star fruit juice. Their initial presentations included sudden-onset limb numbness, muscle weakness, intractable hiccups, consciousness disturbance of various degrees, and seizure. No other major events that might be responsible for these symptoms could be identified. Eight patients died, including one patient with a serum creatinine level of 6.4 mg/dL who had not yet begun dialysis. The clinical manifestations of the survivors were similar to those who died except for consciousness disturbance and seizure. Death occurred within 5 days despite emergent hemodialysis and intensive medical care. The survivors' symptoms usually became less severe after supportive treatment, and these patients subsequently recovered without obvious sequelae. The purpose of this article is to report that patients with renal failure who ingest star fruit may develop neurological symptoms and also run the risk for death in severe cases. Mortality may also occur in patients with chronic renal failure not yet undergoing dialysis.

Induction of heat shock protein 70 protects mesangial cells against oxidative injury.

The heat shock response is an immediate cellular response to elevated temperatures and other types of injury that consists of the synthesis of so-called heat shock protein (hsp). This study was designed to investigate the production and the protective role of the 70 kDa hsp (hsp70) in cultured rat mesangial cells. When mesangial cells undergo thermal (45 degrees C, 15 min) stimulation, they express hsp70 mRNA expression and increased hsp70 protein production. Following this, Northern blots show an enhanced gene expression of hsp70 at one hour that reached a maximum by 12 hours after heat shock. The hsp70 protein production, estimated by Western blots, was detectable 12 hours after heat shock and reached a maximum by 36 hours. Oxidative injury generated by xanthine and xanthine oxidase inhibited cell survival and cellular proliferation, as measured by trypan blue exclusion and [3H]-labeled thymidine uptake. It did not affect hsp70 mRNA expression. Furthermore, when mesangial cells were preconditioned by heat shock, subsequent oxidative injury caused less inhibition of cell survival and cellular proliferation. Pretreatment of cells with quercetin, a transcription inhibitor, abolished the protective effect of heat shock on subsequent oxidative injury. We conclude that heat shock, not oxidative injury, induces hsp70 in mesangial cells, and this induction of hsp70 protects mesangial cells against subsequent oxidative injury.

Role of the Janus kinase (JAK)/signal transducters and activators of transcription (STAT) cascade in advanced glycation end-product-induced cellular mitogenesis in NRK-49F cells.

Advanced glycation end product (AGE) is important in the pathogenesis of diabetic nephropathy, which is characterized by cellular hypertrophy/hyperplasia leading to renal fibrosis. However, the signal transduction pathways of AGE remain poorly understood. The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway has been associated with cellular proliferation in some extra-renal cells. Because interstitial fibroblast proliferation might be important in renal fibrosis, we studied the role of the JAK/STAT pathway in NRK-49F (normal rat kidney fibroblast) cells cultured in AGE/BSA and non-glycated BSA. We showed that AGE dose-dependently (10-200 microgram/ml) increased cellular mitogenesis in NRK-49F cells at 5 and 7 days. However, cellular mitogenesis was unaffected by the simultaneous presence of BSA. Regarding the JAK/STAT pathway, AGE (100 microgram/ml) induced tyrosine phosphorylation of JAK2 (but not JAK1, JAK3 or TYK2) at 15-60 min; it also induced the tyrosine phosphorylation of STAT1 and STAT3 at 1-2 h and 0.5-4 h respectively. Being a transcription factor, AGE also increased the DNA-binding activities of STAT1 and STAT3 AG-490 (a specific JAK2 inhibitor) (5 microM) inhibited tyrosine phosphorylation of JAK2 and the DNA-binding activities of STAT1 and STAT3. The same results were obtained by using specific 'decoy' oligodeoxynucleotides (ODNs) that prevented STAT1 and STAT3 from binding to DNA. Meanwhile, the STAT1 or STAT3 decoy ODN and AG-490 were effective in reversing AGE-induced cellular mitogenesis. We concluded that the JAK2-STAT1/STAT3 signal transduction pathway is necessary for AGE-induced cellular mitogenesis in NRK-49F cells.

Interaction between high glucose and TGF-beta in cell cycle protein regulations in MDCK cells.

Transforming growth factor-beta (TGF-beta) may mediate high glucose effects in renal cells. Thus, Madin-Darby canine kidney cells were studied for the modulation of cell cycle regulatory proteins by high glucose (27.5 mM) and TGF-beta1. We showed that unlike other renal cells, TGF-beta1 mRNA and its bioactivity were not induced by high-glucose culture. Furthermore, high glucose per se increased cellular proliferation without alterations in cell size. High glucose also increased the percentage of cells in the G2/M phase while decreasing cells in the G0/G1 phase of the cell cycle. In contrast, TGF-beta1 dose dependently (1 to 4 ng/ml) decreased cellular mitogenesis while increasing hypertrophy in the cells, especially in the presence of high glucose. TGF-beta1 also increased the percentage of cells arrested in the G0/G1 phase while decreasing cells in the G2/M phase of the cell cycle. Regarding two of the cell cycle regulatory proteins, high glucose increased cdc2 kinase activity and retinoblastoma protein (pRb) phosphorylation. In contrast, TGF-beta1 decreased cdc2 kinase activity and pRb phosphorylation, especially in the presence of high glucose. Additionally, glucose dose dependently (5.5, 16.5, 27.5, and 38.5 mM) increased type I and II TGF-beta receptor protein expression. In conclusion, changes in cdc2 kinase activity and pRb phosphorylation were correlated with high glucose and TGF-beta1-induced growth effects in a cell cycle-dependent manner in the Madin-Darby canine kidney cells. Furthermore, high glucose may potentiate TGF-beta1-induced effects by enhancing TGF-beta receptor protein expression.

Prediction of equilibrated postdialysis BUN by an artificial neural network in high-efficiency hemodialysis.

In urea kinetic modeling, postdialysis blood urea nitrogen (BUN) is usually underestimated with an overestimation of the Kt/V especially in high-efficiency hemodialysis (HD). Thus, an artificial neural network (ANN) was used to predict the equilibrated BUN (Ceq) and equilibrated Kt/V (eKt/V60) by using both predialysis, postdialysis, and low-flow postdialysis BUN. The results were compared to a Smye formula to predict Ceq and a Daugirdas' formula (eKt/V30) to predict eKt/V60. Seventy-four patients on high-efficiency or high-flux HD were recruited. Their mean urea rebound was 28.6+/-2%. Patients were divided into a "training" set (n = 40) and a validation set (n = 34) for the ANN. Their status was exchanged later, and the two results were pooled. In the prediction of Ceq, both Smye formula and low-flow ANN were equally highly accurate. In patients with a high urea rebound (>30%), although Smye formula lost its accuracy, low-flow ANN remained accurate. In the prediction of eKt/V60, both Daugirdas' formula and low-flow ANN were equally accurate, although the Smye formula was not so accurate. In patients with a high urea rebound, although both Smye and Daugirdas' formulas lost their accuracy, low-flow ANN remained accurate. We concluded that low-flow ANN can accurately predict both Ceq and eKt/V60 regardless of the degree of urea rebound.