Diltiazem suppresses collagen synthesis and IL-1beta-induced TGF-beta1 production on human peritoneal mesothelial cells.

BACKGROUND: After long-term treatment with continuous ambulatory peritoneal dialysis (CAPD), some patients may develop peritoneal fibrosis. Peritoneal mesothelial cells (PMCs) participate in the inflammatory reactions in the peritoneal cavity, and transforming growth factor-beta1 (TGF-beta1) and interleukin-1beta (IL-1beta) are involved in peritoneal fibrosis. Diltiazem is used frequently in patients with CAPD to treat hypertension. The objectives of this study were to examine the effects of diltiazem on collagen- and IL-1beta-induced TGF-beta1 production on human PMCs and the signalling pathway of diltiazem in this induction. METHODS: Human PMCs were cultured from the enzymatic disaggregation of human omentum. Collagen synthesis was measured by [3H]proline incorporation into pepsin-resistant, salt-precipitated collagen. The expression of collagen I and III, and TGF-beta1 mRNA was evaluated by northern blotting. The production of TGF-beta1 by human PMCs was measured by immunoassay. The changes of intracellular calcium level after adding Fura-2-AM were measured by fluorescence spectrophotometry. Western blotting was used to assess mitogen-activated protein kinase (MAPK) signalling proteins. RESULTS: We found that diltiazem (<0.2 mM) inhibited collagen I and III mRNA expression and collagen syntheses on a dose-dependent basis. Diltiazem (0.2 mM) suppressed IL-1beta- (5 ng/ml) induced TGF-beta1 production on human PMCs at both the protein and mRNA levels. Diltiazem (0.2 mM) also inhibited IL-1beta- (5 ng/ml) induced collagen I and III mRNA expression. Intracellular calcium levels did not change after the treatment with diltiazem, IL-1beta or both. The IL-1beta-treated human PMCs increased phospho-JNK (stress-activated c-Jun N-terminal kinase) and phospho-p38 MAPK expression, while diltiazem could suppress this phenomenon. CONCLUSIONS: Diltiazem suppressed collagen synthesis of human PMCs and inhibited IL-1beta-induced TGF-beta1 production on human PMCs. This signalling transduction may be through p38 MAPK and JNK pathways instead of intracellular calcium. These results suggest diltiazem to be a potential therapeutic regimen in preventing peritoneal fibrosis and support further in vivo studies.     

Lingual amyloidosis--a rare complication of long-term haemodialysis.

Case A 54 year-old male was diagnosed with polycystic kidney diseasein uraemic stage, and had been dialysed for 25 years, sincethe age of 29 years. He received haemodialysis by artificialkidneys with unsubstituted cellulose membrane for the first16 years. Carpal tunnel syndrome (CTS) developed soon     

Failure of a daily haemofiltration programme using a highly permeable membrane to return beta 2-microglobulin concentrations to normal in haemodialysis patients.

In an attempt to return to normal serum beta 2-microglobulin levels in a group of seven ESRD patients, a programme of daily HF with highly permeable AN69 membranes was undertaken. Pre-HF beta 2-M serum levels stabilized after 4 days at 20 mg/l, only 40% lower than the initial concentration. A total of 985 +/- 20 mg beta 2-M was removed over the week. The beta 2-M release rate averaged 97 micrograms/min with a broad range of values (63-128 micrograms/min). beta 2-M release peaked at 602 micrograms/min 1 h after the end of the HF session before returning to baseline by 12 h post-HF. We conclude that a return to normal blood beta 2-M concentrations in ESRD patients seems quite unrealistic despite a highly intensive extracorporeal therapy. Therefore other therapeutic alternatives have to be designed to prevent or cure beta 2-M amyloidosis.     

Immunoelectron microscopic study on type I, II and III TGF-beta receptors on visceral glomerular epithelial cells in relation to glomerular basement membrane alterations in proteinuric rats.

BACKGROUND: Transforming growth factor (TGF)-beta is a regulator of extracellular matrix accumulation. Both TGF-beta receptors, type I (TbetaRI) and type II (TbetaRII), may be required for signal transduction in the TGF-beta pathway. The aim of this study was to investigate the relationship between the TGF-beta pathways and glomerular basement membrane (GBM) accumulation in vivo. METHODS: We examined TbetaRI, II, and III protein expression on visceral glomerular epithelial cells (GEP) in relation to GBM alterations in passive Heymann nephritis (PHN), anti-GBM nephritis and anti-thymocyte serum (ATS) nephritis. Renal tissues were examined by pre-embedding immunoelectron microscopy 3, 7 and 14 days after induction of nephritis in rats. RESULTS: In normal control rats TbetaRI was not detected on GEP, TbetaRII expression was very occasionally found on GEP and TbetaRIII was seen in the cytoplasm of the GEP. TbetaRI, TbetaRII, and TbetaRIII were constitutively expressed on glomerular endothelial cells. By day 3 of anti-GBM nephritis and PHN, expression of TbetaRI, TbetaRII, and TbetaRIII was still similar to that of normal control rats, and GBM alterations in both models were not prominent except for deposit formation in PHN. From day 7 onwards, in both models, expression of TbetaRI and TbetaRII on GEP increased in association with GBM thickening. Expression of TbetaRIII in the cytoplasm of the GEP was increased, with occasional positive staining being seen on the urinary surface of the GEP from day 7 onwards. On the other hand, at day 3 of ATS nephritis, increased expression of TbetaRI and TbetaRII on GEP was noted, but from day 7 onwards, expression of TbetaR II on GEP dramatically decreased. Expression of TbetaRIII in the cytoplasm of the GEP also transiently increased at day 3. GBM thickening was not noted in ATS nephritis. CONCLUSIONS: The results suggest that persistent upregulation of expression of TbetaRI, TbetaRII and possibly TbetaRIII on GEP may contribute to GBM matrix accumulation in vivo.     

Intrarenal localization of interleukin-1{beta} mRNA in crescentic glomerulonephritis

II-1ß is a potent proinflammatory peptide, and inducesexpression of other cytokines which are involved in the immuneresponse. Kidney biopsies from nine patients with crescenticGN were studied by in-situ hybridization to determine the siteof expression of II-1ß mRNA. Biopsies from nine patientswith non-proliferative renal disease were studied as negativecontrols, and tonsillar tissue was studied as a positive control.An II-1ß cDNA probe was ³²P-labelled by random primersand hybridized to paraffin-embedded tissue sections after de-waxing.II-1ß mRNA was expressed in tonsil, but not in negativecontrols. Positive mRNA expression was seen in four of the ninecrescentic biopsies. This was observed in interstitial cellswith morphological characteristics of macrophages adjacent totubular cells, in cells within the glomerular tuft, and in tubularepithelial cells. II-1ß mRNA is expressed in renaltissue in crescentic GN. Tubular and interstitial expressionof II-1ß mRNA appears of equal prominence to glomerularexpression. Intrarenal cytokine synthesis may be involved inthe pathogenesis of crescentic glomerulonephritis.     

NADPH oxidase subunits (NOX-1, p22phox, Rac-1) and tacrolimus-induced nephrotoxicity in a rat renal transplant model.

BACKGROUND: TGF-beta and oxidative stress are known mediators of renal injury. However, the precise mechanisms by which TGF-beta and oxidative stress may be involved in the development of nephrotoxicity are not known. We examined whether anti-TGF-beta antibody limits nephrotoxicity produced by tacrolimus (TAC) and whether this altered genes that regulate oxidative stress. METHODS: Renal transplants were performed in Wistar-Furth and Lewis rat strains. Groups included: isograft controls; untreated allografts; allografts treated with 0.25 mg/kg TAC till 90 days with or without 1.0 mg/kg anti-TGF-beta antibody or control antibody. Serum creatinine and BUN levels and renal histology were determined. Real time PCR and western analysis were used to quantify mRNA and protein expression. RESULTS: BUN and creatinine were elevated in TAC-treated rats. TAC increased expression of TGF-beta (37-fold) and NADPH oxidase subunits, NOX-1 (18-fold), p22(phox) (31-fold) and Rac-1 mRNA (20-fold), respectively. Contrariwise, expression of antioxidant genes, superoxide dismutase (SOD) and thioredoxin (TRX) was decreased. Anti-TGF-beta antibody but not control antibody reversed the TAC-induced changes in gene expression, renal histology and function. CONCLUSIONS: Our findings suggest a potential for anti-TGF-beta antibody as a novel adjunct therapeutic tool to prevent TAC-induced nephrotoxicity in transplant recipients. The mechanism of protection involves suppression of TGF-beta and the expression of genes that regulate oxidative stress. Moreover, the specific up-regulation of NOX-1, a non-phagocytic NADPH oxidase subunit and its reversal by anti-TGF-beta antibody strongly implicates for the first time the up-regulation of renal parenchymal cell NADPH oxidase in the aetiology of immunosuppression-induced nephrotoxicity.     

New strategies in haemodiafiltration (HDF): prospective comparative analysis between on-line mixed HDF and mid-dilution HDF.

BACKGROUND: Improvement in the uraemic toxicity profile obtained with the application of convective and mixed dialysis techniques has stimulated the development of more efficient strategies. Our study was a prospective randomized evaluation of the clinical and technical characteristics of two new haemodiafiltration (HDF) strategies, mixed HDF and mid-dilution HDF, which have recently been proposed with the aim of increasing efficiency and safety with respect to the standard traditional HDF infusion modes. METHODS: Ten stable patients on renal replacement therapy (mean age 64.7 +/- 8.2 years) were submitted in randomized sequence to one mid-week session of mid-dilution HDF and one of mixed HDF with trans-membrane pressure feedback control. All sessions were carried out under similar operating conditions and involved monitoring pressure within the internal dialyser compartments and calculating the rheological and hydraulic indexes. Efficiency in removing urea, phosphate and beta2-microglobulin (beta2-m) was tested. RESULTS: In mixed HDF, safer and more effective flux/pressure conditions resulted in better preservation of the hydraulic and solute membrane permeability (mean in vivo ultrafiltration coefficient 36.9 +/- 3.9 vs 20.1 +/- 3.3 ml/h/mmHg) and ensured higher volume exchange (38.7 +/- 4.2 vs 35.3 +/- 6.5 l/session, P = 0.02) and greater efficiency in removing small and middle molecules (mean urea clearance: 274 +/- 42 vs 264 +/- 47 ml/min, P = 0.028; eKt/V: 1.78 +/- 0.22 vs 1.71 +/- 0.26, P = 0.036; mean phosphate clearance: 138 +/- 16 vs 116 +/- 45 ml/min, P = 0.2; mean beta2-m clearance: 81 +/- 13 vs 59 +/- 13 ml/min, P = 0.001). CONCLUSIONS: Mixed HDF was the most efficient technique in the highest range of safe operating conditions. In mid-dilution HDF, high pressures generated inside the dialyser compromised membrane permeability and limited the total infusion rate, resulting in an overall reduction in solute removal.     

Renal handling of prednisolone/prednisone: effect of steroid dose and ll{beta}-hydroxysteroid dehydrogenase

The purposes of this study were: (1) to determine under steady-stateconditions whether the renal clearance of prednisolone is concentrationdependent, and (2) to establish whether the urinary excretionof prednisolone and its biologically inactive 11-dehydro metaboliteprednisone depend upon the activity of 11ß-hydroxysteroiddehydrogenase (11ß-OHSD). For that purpose 10 healthyvolunteers were infused to steady state over a 13-h period eitherat a low (11 µg/h x kg) or a high (70 µg/h x kg)rate with prednisolone on two occasions, once without and oncewith administration of glycyrrhetinic acid, an inhibitor of11ß-OHSD. Prednisolone and prednisone were measuredby high-pressure liquid chromatography. Mean renal clearancevalues of total or unbound prednisolone were several times higherduring the high than the low infusion rate. The fractional renalclearance of unbound prednisolone during the high, but not duringthe low infusion rate exceeded 1. This indicates that in additionto unbound prednisolone, protein-bound prednisolone is excretedin urine at high plasma concentrations. Inhibition of 11ß-OHSDincreased the urinary ratios of prednisolone/prednisone in allsubjects. Conclusions: (1) The renal clearance of prednisoloneis concentration dependent; (2) there must be tubular secretionand/or glomerular filtration of prednisolone bound to plasmaproteins; (3) the urinary excretion of prednisolone/prednisoneis modulated by the activity of 11 ß-OHSD.     

Biochemical characterization of serum and urinary beta 2 microglobulin in end-stage renal disease patients.

Since the identification of beta 2 microglobulin (beta 2-M) in haemodialysis-associated amyloidosis, the biochemical characterization of the different forms of beta 2-M has been sought by several groups. New beta 2-M isoforms (pI 5.1 and lower) have been identified in amyloid deposits, and it has been suggested that they are of pathogenetic importance. The finding of N-terminal proteolysed beta 2-M in amyloid deposits prompted the hypothesis that proteolysis would render beta 2-M more amyloidogenic. Finally, a 'novel beta 2-M' (pI 5.2) with a single amino acid replacement (Asn by Asp at position 17) has been reported as possibly specific for patients with dialysis associated amyloidosis, and consequently proposed as 'the amyloidogenic' form. We purified beta 2-M from serum of a newly haemodialysed patient and from urine of a transplanted patient in the early recovery period. Both patients were clinically amyloid free. Three pure isoforms were obtained from serum (pI 5.7, 5.3, and 5.1) and only two from urine (5.7 and 5.3). Further purification of each isoform was obtained by HPLC in a C4 column. Sequence analysis showed that all isoforms had an intact N-terminus. Tryptic digestion of the serum isoforms was performed after alkylation with iodoacetic acid and the peptides were isolated by HPLC in a C18 column. The 5.3 and 5.1 isoforms had identical peptide patterns with the appearance of an early peak missing in the 5.7 form. The sequence of this peptide showed a replacement of the D 42 (Asp 42) by N (Asn) after K41 (Lys 41).(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term efficiency, biocompatibility, and clinical safety of combined simultaneous LDL-apheresis and haemodialysis in patients with hypercholesterolaemia and end-stage renal failure

Three hypercholesterolaemic patients on maintenance haemodialysiswith angiographically proven coronary artery disease were treatedin a once-a-week schedule by combined, synchronous lipid apheresis(using heparin-induced extracorporeal LDL precipitation) andhaemodialysis (HELP/HD) for 65–104 weeks. Clinical side-effectswere few and mostly related to high ultrafiltration rates inpatients with low compliance regarding interdialytic fluid restriction.Biocompatibility of the procedure was shown to be good and bloodcell losses, leukocyte (elastase release) and thrombocyte (ß-thromboglobulinextrusion) as well as complement (C3a formation) activationwere minimal. Interestingly, most of the C3a generated in theextracorporeal HELP circuit was immediately removed again inthe precipitate filter. In the pseudo-steady-state after 3 monthsof regular therapy, acute haematocrit-corrected reduction ofplasma components after the session compared to pre values wereabout 55% for the risk factors LDL cholesterol (LDL-C), lipoprotein(a)(Lp(a)), and fibrinogen (FIB) with good recovery of HDL-C andother proteins. Urea, creatinine, and phosphate eliminationwas similar to normal haemodialysis. Mean interapheresis values of risk factors after one (n=2) andtwo (n=1) years of treatment were crucially dependent upon ultrafiltration(UF); thus, in two patients with high UF LDL-C concentrationsamounting to 185 and 220mg/dl at baseline and were reduced toabout 135mg/dl LDL-C, while in the patient with low UF the reductionwas from 231 mg/dl to 80 mg/dl. The atherogenic index (LDL-C/HDL-C)was reduced from 6.4 and 5.1 to about 4.3 in patients with highUF, from 6.1 to 3.3 in the patient with low UF. Fibrinogen andLp(a) were normalized in all patients. In summary, the combined HELP/HD treatment in hypercholesterolaemicdialysis patients proved to be a safe, effective, and selectiveprocedure for lipoprotein and fibrinogen normalization withexcellent biocompatibility and good clinical patient tolerance,providing a tool ready for future atherosclerosis regressionstudies in ESRD patients.     

The impact of short-term ciclosporin A treatment on insulin secretion and insulin sensitivity in man

Background. The objectives of the present study were to investigatethe possible adverse effects of ciclosporin A (CsA, SandimmunNeoral®) on insulin secretion and insulin sensitivity (IS)in man. Methods. A total of 11 Caucasian non-diabetic haemodialysis(HD) patients were recruited from the Norwegian transplant waitinglist to participate in this study. The patients underwent twoconsecutive 3 h hyperglycaemic glucose clamp procedures, beforeand following 2 weeks of oral CsA treatment. Statistical analysesincluded nine patients (7M/2F, mean age 61 ± 14 years)as two patients were withdrawn due to side effects and poorcompliance. First and second phase insulin secretion (Secr_1.phaseand Secr_2.phase) were estimated as area under the insulin serumconcentration vs time curve (AUC) during the first 10 min andthe last hour of the clamp, respectively. The IS index (ISI)was calculated as the glucose disposal rate corrected for insulinlevels during the last 60 min of the procedure. Results. Secr_2.phase decreased significantly (30%) followingCsA treatment (P = 0.045). In contrast, no significant changewas observed in the average Secr_1.phase or ISI, although relativelylarge inter-individual differences were present. Calculationbased on C-peptide concentrations gave the same results. Nosignificant changes in body weight, dialysis status, patientmedication or safety parameters were observed. Conclusions. Short-term treatment with CsA at doses used followingtransplantation seems to impair Secr_2.phase, but has no significanteffect on Secr_1.phase, in Caucasian HD patients. The mechanismbehind these findings and their possible clinical implicationsneed further study.