Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule.

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP-driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped-flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)

Hyaluronan preserves peritoneal membrane transport properties.

BACKGROUND: We have shown that intraperitoneal (i.p.) addition of hyaluronan (HA) in a single dwell study in rat could increase peritoneal fluid removal by decreasing the peritoneal fluid absorption rate. In this study, we investigated the impact of repeated use of HA on peritoneal membrane transport characteristics. METHODS: Twelve male Sprague-Dawley rats received a once-daily i.p. injection of 25 mL 4.25% glucose dialysis solution without (HP group, n = 6) or with 0.025% HA (HA group, n = 6) for 1 week. Forty-eight hours after the last injection, a 4-hour dwell using 25 mL 4.25% glucose dialysis solution with i.p. volume marker and frequent dialysate and blood samplings was performed in each rat as well as in rats that did not receive any injection (control group, n = 8). RESULTS: Although the i.p. volumes were significantly lower in the HP and HA groups compared to the control group, i.p. volume in the HA group was significantly higher than in the HP group. Net ultrafiltration at 4 hours was 5.6 +/- 1.3 mL, 10.2 +/- 1.8 mL, and 13.2 +/- 0.6 mL for the HP, HA, and control group, respectively. The peritoneal fluid absorption rate decreased by 45% in the HA group compared to the HP group. There was no significant difference in peritoneal fluid absorption rate between the HA and the control group. No difference was found in the direct lymphatic absorption rate between the HP and HA groups [0.010 +/- 0.003 mL/minute in the HP group and 0.011 +/- 0.004 mL/min in the HA group] although they were both higher than that of the control group (0.004 +/- 0.001 mL/min). The solute transport rates were in general significantly higher in the HP group compared to the HA and control groups, and there was no significant difference between the latter two groups, except that protein transport rate was significantly lower in the HA group compared to the control group. CONCLUSIONS: The present study suggests that (1) repeated exposure to hypertonic glucose-based dialysis solution results in increased peritoneal solute transport rates, as well as increased peritoneal fluid absorption rates; and (2) these changes, reflecting a highly permeable peritoneal membrane, were ameliorated by repeated i.p. addition of hyaluronan. The similar changes in the direct lymphatic absorption rate in rats that received daily i.p. injection of dialysis solution suggest that direct peritoneal lymphatic absorption was not influenced by hyaluronan.

Effect of dialysate composition on the apoptosis and proliferation of human peritoneal mesothelial cells and protein expression of Fas and c-Myc.

To investigate the effect of dialysate composition on apoptosis and proliferation of human peritoneal mesothelial cells (HPMCs) in vitro, HPMCs were cultured from human omental tissue. The cells were exposed for three days to culture medium containing: (1) 3.86% glucose, (2) 1.36% glucose, (3) 1.75 mmol/L Ca++, (4) 1.25 mmol/L Ca++, or (5) no additions (control). Apoptosis and proliferative cell nuclear antigen (PCNA) were measured by terminal deoxynucleotidyl transferase (TdT)-mediated in situ end-labelling and immunohistochemistry. Protein expression of Fas and of c-Myc were measured by flow cytometry. Apoptotic cells were significantly increased in the high glucose group compared to control (137 +/- 47 cells per field unit vs 63 +/- 30 cells per field unit, p < 0.01), but did not vary in the low glucose and low calcium groups versus control (76 +/- 25 cells per field unit and 77 +/- 31 cells per field unit, respectively, vs 63 +/- 30 cells per field unit, p > 0.05). Cells with positive expression of PCNA were increased in the high calcium group versus control (366 +/- 101 cells per field unit vs 186 +/- 76 cells per field unit, p < 0.01). However, the expression of PCNA was significantly more inhibited in the high glucose group than in the control group (158 +/- 52 cells per field unit vs 186 +/- 76 cells per field unit, p < 0.05). Expression of Fas was stimulated by high glucose (41% +/- 16% vs 25% +/- 8% in the control group, p < 0.05). High glucose and high calcium also up-regulated the expression of c-Myc, but only the high glucose group showed significant difference from control (39% +/- 10% vs 24% +/- 8%, p < 0.05). Our data suggest that: (1) Peritoneal dialysate could induce apoptosis of HPMCs in vitro, the degree of apoptosis depending on concentration of glucose. (2) High calcium stimulates PCNA expression by HPMCs and high glucose inhibits expression of PCNA. (3) Genes associated with apoptosis (Fas, c-Myc) might play an important role in triggering apoptosis of HPMCs.

Influence of peritoneal dialysis on the progression of chronic renal failure.

To determine whether an early start of peritoneal dialysis is beneficial to the preservation of residual renal function, we studied the effect of peritoneal dialysis on the progression of glomerular sclerosis in subtotal nephrectomy rats. Four weeks after subtotal nephrectomy, Wistar rats were treated with peritoneal dialysis (PD) (through a silicone catheter, two exchanges per day with 20 mL 1.5% dextrose solution per exchange for 8 weeks, n = 10). Then the kidneys were checked. The sclerosis index was significantly lower in the PD group than that of the control group, which was 1.12 +/- 0.13, 1.64 +/- 0.19, respectively (p < 0.05). This suggests that peritoneal dialysis may suppress the progression of chronic renal failure. An early start of PD may benefit the preservation of renal function in uremic patients.

Methods to improve the preservation of peritoneal tissues.

Peritoneal mesothelial cells are easily detached during conventional tissue processing, which may result in artifacts in peritoneal tissue examination. Therefore, in the present study, we investigated several methods to improve the preservation of the anterior parietal peritoneal tissues. Peritoneal tissue from the anterior abdominal wall was taken from each of 5 rats killed for the experiment. Tissue samples were immediately treated by one of these methods: (1) fixed with 10% formaldehyde; (2) fixed with Bouin's solution; (3) fixed with Helly's solution. After fixation, the samples were dehydrated with one of (a) ethanol, 1 hour in each step; (b) ethanol, 15 minutes in each step; or (c) tertiarybutyl alcohol. Five sections were taken from each tissue and stained with hematoxylin and eosin. The quality of tissue fixation was evaluated by image analysis. Peritoneal mesothelial cells were well preserved after fixation with Helly's solution or Bouin's solution. With 10% formaldehyde, about 40% of the mesothelial cells were lost. Dehydration with ethanol--especially long-duration dehydration--increased the loss. However, dehydration with tertiarybutyl alcohol avoided the increased loss of mesothelial cells. The submesothelial extracellular matrix was well preserved with Bouin's solution, but not with the other fixatives. Our results suggest that fixation with formaldehyde and dehydration with ethanol results in significant loss of peritoneal mesothelial cells and submesothelial extracellular matrix in peritoneal tissues. Fixation with Bouin's solution and dehydration with tertiary-butyl alcohol may be a better method of preserving peritoneal tissue.

Phospholipids in dialysate and the peritoneal surface layer.

Dialysate concentration of phospholipids has been used to monitor peritoneal membrane status. However, we recently found that the peritoneum has a surface layer in which phospholipids may be the main constituent. Therefore, in this study, we compared the phospholipids composition of peritoneal dialysate and of the peritoneal surface in rats. Eight male Sprague-Dawley rats were used in the study. Five rats received an intraperitoneal injection of 25 mL 4.25% glucose dialysis solution. After four hours, the rats were killed, and the dialysate was drained completely. Then 20 mL of Folch solution was infused into the peritoneal cavity for 30 seconds and drained completely. The other three rats received the Folch solution without dialysis. The effluent and Folch solution were then processed for phospholipids analysis using high performance thin-layer chromatography (HPTLC). The total phospholipids content was ten times higher in the surface layer than in the dialysate effluent. In the effluent, four clearly different components were seen: lysophosphatidylcholine (LPC), sphingomyelin (SM, 29%), phosphatidylcholine (PC, 66%), and phosphatidylinositol (PI, 4.5%). However, in the surface layer, as well as LPC, SM (20.6%), PHC (47%), and PI (6.3%), two additional components were seen, phosphatidylserine (PS, 17.1%) and phosphatidylethanolamine (PE, 8.9%). The quantity of phospholipids in the peritoneal surface of non dialyzed rats was similar to the total quantity of phospholipids (in effluent and in the peritoneal surface) of dialyzed rats. Our results suggest that: (1) a surface layer is present on the peritoneum; this layer could well be extracted by Folch solution; and, with appropriate incubation time, one can separate the surface layer without damaging the mesothelial cells; (2) the composition of phospholipids in the effluent is different from that in the peritoneal surface layer, which contains membrane phospholipids (PS and PE); (3) shielding from the peritoneal surface may be the main reason for the presence of phospholipids in the dialysate.

Enhanced expression of TGF-beta 1 by peritoneal macrophages in CAPD patients.

Peritoneal macrophages are the predominant cells in peritoneal dialysate. To clarify the role of the macrophage in fibrosis of the peritoneum in long-term continuous ambulatory peritoneal dialysis (CAPD) patients, we studied the transforming growth factor beta (TGF-beta 1) mRNA expression by peritoneal macrophages from peritoneal dialysis patients. Macrophages were obtained when the catheter was inserted and one month later. TGF-beta 1 mRNA was measured by slot-blot hybridization using a cDNA probe. The result was that TGF-beta 1 expression was significantly higher in macrophages from peritoneal dialysate than that of macrophages obtained during catheter insertion. This suggests that peritoneal macrophages play a role in the pathogenesis of fibrosis of peritoneum in peritoneal dialysis patients. It also suggests that peritoneal macrophages in dialysate have been somewhat activated.

Effect of glucose on TGF-beta 1 expression in peritoneal mesothelial cells.

The peritoneum's response to peritoneal dialysis is a complex and poorly understood process that can result in fibrosis. The transforming growth factor beta(TGF-beta 1) plays a central role in regulating tissue repair and remodeling after injury. To determine the role of mesothelial cells during peritoneal dialysis in the alteration of the peritoneum, we studied the effect of glucose on TGF-beta 1 expression by mesothelial cells. Human mesothelial cells were cultured in serum-free DMEM with different glucose concentrations for 48 hours. TGF-beta 1 messenger (mRNA) was measured by slot-blot hybridization using a TGF-beta 1 cDNA probe. The result was that glucose stimulates TGF-beta 1 mRNA expression in a dose-dependent way. The expression was more profound in high glucose concentrations. This suggests that TGF-beta 1 mRNA may overexpress by mesothelial cells due to the procedure of dialysis, which may be the most important mechanism of alteration of the peritoneum during peritoneal dialysis.

CD40 ligand expression on macrophages during peritonitis in continuous ambulatory peritoneal dialysis patients.

CD40-CD40 ligand (CD40L) interaction plays an important role in macrophage/monocyte-mediated inflammatory processes by up-regulating cytokine production by macrophages/monocytes and by preventing macrophage apoptosis at the inflammation sites. The present study investigated the possible regulation of CD40L expression in peritonitis during continuous ambulatory peritoneal dialysis (CAPD). We used fluorescence-activated cell sorter (FACS) analysis to detect CD40L expression on macrophages obtained from peritoneal dialysate. Our results showed that CD40L expression on macrophages was significantly increased in a peritonitis group (4.62 +/- 6.54) as compared to a control group (0.76 +/- 0.30, p < 0.01). The CD40L-positive cells were also significantly increased during peritonitis (97.86% +/- 1.67% in the peritonitis group as compared to 73.10% +/- 26.94% in the control group, p < 0.05). After successful treatment, the expression of CD40L was significantly reduced (3.66 +/- 1.12 vs 1.05 +/- 0.02, p < 0.05). We conclude that functionally expressed CD40L on macrophages may take part in acute inflammatory response during peritonitis in CAPD and may play an important role in the local defense against infection in the peritoneal cavity.

[Characterization of proton pump and osmotic water transport in endocytic vesicles from rat kidney proximal tubule].

The characteristics of ATP-driven proton and osmotic water transport were studied in endocytic vesicles isolated from rat kidney proximal tubule labelled in vivo with fluorescein isothiocyanate-dextran (FITC-dextran). ATP-driven proton transport was measured from the time course of endosome pH following addition of external ATP. The rate of endosome acidification and the minimum pH were dependent on the ATP concentration. At an initial endosome pH of 7.4, the final pH values were 7.30, 6.99, 6.68, 6.38 and 6.39 at [ATP] = 0.005, 0.05, 0.5, 5 and 10 mmol/L, respectively. The acidification was inhibited by 97% at 0.5 mmol/L N-ethylmaleimide but was not affected by vanadate and oligomycin. Osmotic water permeability was determined in the same endosomes from the rapid kinetics of FITC-dextran fluorescence following an inward sucrose gradient. The osmotic water permeability coefficient was 0.03 cm/s at 23 degrees C. Water permeability was inhibited by 70% with addition of 0.5 mmol/L mercuric chloride. The inhibition was reversed completely by adding 5 mmol/L mercaptoethanol. These data demonstrate that proximal tubule endosomes contain a proton ATPase and water channel. The endocytic process may be important for regulation of acidification and fluid resorption in the proximal tubule.

[Mechanism of urinary erythrocyte deformity in glomerular diseases].

We developed an in vitro model to produce urinary dysmorphic RBCs and to investigate the influence of pH and osmolality. The pH and osmolality of the RBCs suspension was made to change systematically within a range of pH 5-8 and 200-800 mOsm/kg, and at a flow rate of 0.5 ml/min through a polycarbonate filter (Calif., USA) with pore diameter of 3 mu. Then the samples were examined with phase-contrast microscopy. Our data showed that: (1) The formation of glomerular RBCs depends on their passage through a narrow pore and the presence of certain suspension, which is also present in glomerulonephritis. Suspension of RBCs in urine with the same osmolality and pH, but without filtration did not produce glomerular shapes. (2) Urine facilitated the production of glomerular shapes. Filtration of RBCs suspension in physiological buffer instead of urine produce less dysmorphic shapes than filtration in urine of various pH and osmolality. (3) Certain types of urinary RBCs shapes occurring predominantly at specific conditions of pH and osmolality (< 400 mOsm/kg) produced glomerular shapes most frequently, e.g. doughnut-like RBCs were produced by urine with pH 7. Our data suggested that the mechanism of glomerular RBCs formation might result from the passage of RBCs through narrow defects in the ruptured glomerular capillary wall and a chemical injury to the RBCs in the tubular lumen induced by urinary constituents.

[Comparison of quality of life in continuous ambulatory peritoneal dialysis and hemodialysis patients].

Quality of life is a important outcome of treatment of chronic disease. It's also a major factor to determine the selection of various modes of therapy for end-stage renal diseases (ESRD) patients. Quality of life may be influenced by some case mix factors. 17 continuous ambulatory peritoneal dialysis (CAPD) patients and 17 hemodialysis patients were closely matched on sex, age, initial disease, education, financial support, cormidity and marital status to compare their quality of life. There were no difference of quality of life between these two group. These results suggest that dialysis modality do not likely to exerts an independent effect on quality of life of chronic dialysis patients. It may be warrant to speed up the CAPD program due to its less expensive.

[A new morphological classification of urinary erythrocytes for evaluating the source of hematuria].

For differentiation of glomerular from urological hematuria more accurately, a new morphological classification of urinary erythrocytes was studied in 74 cases of glomerular and 58 cases of urological hematuria. For each urine sample, 100 urinary erythrocytes were observed with phase-contrast microscopy and classified into 5 "glomerular" shapes (G1-G5), 5 "urological" shapes (N1-N5) and an unclassified shape. The result was that occurrence of G1, G2, G3, G4, G5 or unclassified shape group appeared significantly (P < 0.01) higher in glomerular diseases than in urological diseases, and among them G1 was the most specific shape for glomerular hematuria. Using percentage of sigma G (G1 + G2 + G3 + G4 + G5) and setting the cut-off at 20%, the sensitivity and specificity for the diagnosis of glomerular diseases were 95.9%. When percentage of G1 (doughnut-like cell with one or more blebs) was used at a cut-off of 1%, sensitivity and specificity were 75.7% and 96.5% respectively. These results were more satisfactory as compared with those in most of the previous reports. Moreover, this new classification is more objective, accurate and clear.

[Changes in soluble interleukin-2 receptor and its significance in nephrotic syndrome].

Serum and urine soluble interleukin-2 receptor (SIL-2R) levels were determined with ELISA method in 36 cases of nephrotic syndrome in order to investigate the significance of the change of SIL-2R level. The results showed the values in nephrotic stage group (n = 22) exceeded those in nephrotic stage remission group (n = 14, P < 0.01) and those in healthy controls (n = 20, P < 0.01). However, there was no difference of SIL-2R values either in serum or urine between nephrotic stage remission group and healthy controls (P > 0.05). Serum SIL-2R values were correlated with those of serum creatinine (r = 0.44, P < 0.01) and urine SIL-2R values were correlated with those of urine protein excretion (r = 0.48, P < 0.01). The study suggests that increased concentration of SIL-2R can serve as an indicator of nephrotic syndrome activation and/or exacerbation of renal dysfunction.

[Nephrocalcinosis and clinical significance in early chronic renal disease].

Kidney tissue calcium content from 33 diagnostic human renal biopsies was measured to assess tissue calcium concentration early in the course of human renal disease. Renal calcium content was correlated significantly with serum creatinine (r = 0.71, P < 0.001), serum phosphate (r = 0.64, P < 0.001), and serum Ca x P product (r = 0.66, P < 0.001), but not with serum calcium (r = -0.17, P > 0.05). Threefold greater calcium content was measured in biopsied tissue than in normal renal tissue (315 +/- 169 vs 69 +/- 17 mg/kg renal tissue, P < 0.001). The mean level of serum phosphate and Ca x P product was higher in the patients with significant renal function impairment (Ser > 115 mumol/L) than in patients with normal renal function (1.76 +/- 0.33 mmol/L vs 1.29 +/- 0.18 mmol/L, P < 0.001; 43 +/- 4.7 vs 33 +/- 4.5 mmol 2/L2, P < 0.001, respectively). The mean serum calcium levels were not different in the two groups (1.97 +/- 0.24 mmol/L vs 2.07 +/- 0.16 mmol/L, P > 0.05). The results suggest that renal calcium deposition begins early in the course of various renal diseases, and hence may accelerate progression of chronic renal failure the severity of renal calcium deposition is correlated with hyperphosphatemia and with the level of renal impairment. Dietary phosphate restriction may be necessary early in the course of renal disease.

[Treatment of IgA nephropathy by integrated traditional and Western medicine].

45 cases of IgA nephropathy were confirmed by renal biopsy and were divided into 2 groups, the integrated traditional and western medicine (TCM-WM) therapeutic group and the western medicine (WM) therapeutic group. The WM group was treated by conventional western medicine (persantin, vitamin B co.), and the TCM-WM group was treated by the same western medicine plus differential symptomatic treatment with decoction of Chinese crude herbs. In this paper, most patients (30 cases) were present as deficiency of Yin of the Liver and Kidney type, and were treated by Qiju Dihuang pill plus Erzhi pill. All types in TCM-WM group were also used herbs of promoting blood circulation and remove blood stasis in almost every case. After 6 months of such treatment, the total effective rate of TCM-WM group and the WM group were 86.4% and 13.0% respectively. There was very significant difference (P less than 0.01) between them. The results showed that the curative effect of the TCM-WM group was much superior than WM alone for the treatment of IgA nephropathy.

[134 patients with adult primary nephrotic syndrome treated with combined traditional Chinese medicine and Western medicine].

This paper studied the method and effect of adult primary nephrotic syndrome (PNS) with the integrated traditional Chinese and Western medicine (TCM-WM) treatment. 134 patients were divided into WM group (66 cases) and TCM-WM group (68 cases). Both groups were mainly treated with corticosteroid. The latter group was treated with corticosteroid plus Chinese medicinal herbs. The results showed that total effective rate of WM group and TCM-WM group was 56.1% and 85.3% respectively, while the occurrence rate of side effect was 48% and 14.8% respectively. It indicated that corticosteroid plus Chinese medicinal herbs could enhance curative effect and reduce side effect.

[Effects of scorpion injection on interleukin 1 and pathology in experimental glomerulonephritis in situ].

UNLABELLED: The model of experimental glomerulonephritis in situ in rats was established by administration of C-BSA. 24 male Wistar rats were randomly assigned to three groups: C-BSA, control and C-BSA+scorpion (scorpion injection, ip, 1.0 g/kg, once/day). At the end of 4th week, the bioactivity of serum IL-1 was assayed by thymocyte proliferation method and the renal pathology was checked up. Urine sample was collected for 24 hour once/week for protein determination. RESULT: Quantity of proteinuria in scorpion group was reduced significantly in comparing with C-BSA group. Level of IL-1 was significantly lower in scorpion group than that in C-BSA group. Injury of renal tissue also was mild in the treated group. These results suggested that scorpion injection could suppress the activities of IL-1 and ameliorate the pathological injury in this experimental glomerulonephritis in situ.

[Lupus nephritis treated with impact therapy of cyclophosphamide and traditional Chinese medicine].

UNLABELLED: 41 active lupus nephritis patients were treated vigorously with cyclophosphamide (CTX), steroid used as routine, as well as TCM administered according to the various disease stage. 35 cases administered with same western medicine but no TCM was taken as control group. RESULT: After a six-month treatment course, the therapeutical efficacy was significantly higher in treated group than that in control group (P < 0.05). Lupus nephritis usually manifested itself as Liver-Kidney Yin deficiency in TCM. Our study suggested that Chinese herbs might play an important role in the treatment of lupus nephritis.

[Effect of ligustrazine peritoneal dialysate on CD40 expression of rat peritoneal mesothellial cells].

OBJECTIVE: To explore the effect of Ligustrazine (LGZ) affecting the expression of CD40 on mesothelial cells (MC). METHODS: Rat's peritoneal MC isolated from peritoneal cavity were cultured and maintained under the defined conditions in vitro. They were cultured with 4.25% peritoneal dialysis solution (PDS), 4.25% PDS added LGZ (40 mg/L), 4.25% PDS added IFN-gamma (100 u/ml) and 4.25% PDS added IFN-gamma and LGZ for 30 min, the DMEM/F12 cultural medium was taken as control. The expression of CD40 on MC was detected by reverse transcript polymerase chain reaction (RT-PCR) and flow cytometry analysis. RESULTS: MC cultured in vitro expressed CD40 markedly, the expression of CD40 mRNA and its protein was markedly up-regulated following stimulation with 4.25% PDS or IFN-gamma and 4.25% PDS. LGZ (40 mg/L) could reduce the expression of CD40 mRNA and its protein significantly as it was added in 4.25% PDS and 4.25% PDS with IFN-gamma. CONCLUSION: The PDS stimulates the expression of CD40 on MC. LGZ has the function of down-regulating the expression of CD40 on MC. During long-term continuous ambulatory peritoneal dialysis, it might be helpful to alleviate chronic inflammatory reaction by adding LGZ into standard PDS, therefore prevent or retard the occurence and development of peritoneal fibrosis.