[Standardized histomorphological processing of peritoneal biopsies as part of the German Peritoneal Biopsy Registry (GRIP, German registry in PD)]. / Standardisierte histomorphologische Aufarbeitung von Peritonealbiopsien im Rahmen des Deutschen Peritonealbiopsieregisters (GRIP, German Registry In PD).

The peritoneal lining of the abdominal cavity consists of a parietal and visceral sheet. The serosa is an interesting organ, which in medical practice is particularly important in the context of chronic peritoneal dialysis (PD). This method of renal replacement therapy utilizes the semipermeability of the peritoneal surface by applying PD solutions of differing osmolarity to eliminate toxic substances and regulate fluid and electrolyte equilibrium. This method is an ideal approach especially for younger patients and is very effective at least for some time. Pre-existing injury to the peritoneum, for example as a consequence of chronic renal insufficiency or associated comorbidities and inflammatory changes that develop during PD, results in a structural remodelling of the serosa. As a consequence, the filtering function of the serosa is lost and PD has to be replaced by another renal replacement therapy. Thorough knowledge of the morphology of peritoneal changes as well as of the risk factors is of paramount importance for therapeutic management and prognosis of PD patients. In order to take this into account, the German Registry In Peritoneal Dialysis (Deutsches Peritonealbiopsieregister, GRIP) was founded a few years ago, which now includes roughly 1700 biopsies, of which detailed clinical and histomorphological information was systematically acquired and collected.

Podoplanin-positive cells are a hallmark of encapsulating peritoneal sclerosis.

BACKGROUND: Encapsulating peritoneal sclerosis (EPS) and simple peritoneal sclerosis are important complications of long-term peritoneal dialysis (PD). Podoplanin is expressed by mesothelial cells and lymphatic vessels, which are involved in inflammatory reactions in the peritoneal cavity. METHODS: We studied 69 peritoneal biopsies from patients on PD (n = 16), patients with EPS (n = 18) and control biopsies taken at the time of hernia repair (n = 15) or appendectomy (n = 20). Immunohistochemistry was performed to localize podoplanin. Additionally, markers of endothelial cells, mesothelial cells, myofibroblasts (smooth muscle actin), proliferating cells, and double labelling for smooth muscle actin/podoplanin were used on selected biopsies. RESULTS: Podoplanin was present on the endothelium of lymphatic vessels in the submesothelial fibrous tissue and on mesothelial cells. In patients on PD and in biopsies with appendicitis, the mesothelial cells demonstrated a cuboidal appearance and circumferential podoplanin staining, with gaps between the cells. The number of lymphatic vessels was variable, but prominent at sites of fibrosis. In patients with EPS, a diffuse infiltration of podoplanin-positive cells with a fibroblastic appearance was present in 15 out of 18 biopsies. This pattern was focally present in 3 out of 16 on PD and none in the 35 controls. The podoplanin-positive cells did not express the endothelial marker or the mesothelial marker (calretinin). CONCLUSIONS: EPS is characterized by a population of podoplanin and smooth muscle actin double-positive cells. Podoplanin might be a suitable morphological marker supporting the diagnosis and might be involved in the pathogenesis of EPS.

Improved estimation of glomerular filtration rate by serum cystatin C in preventing contrast induced nephropathy by N-acetylcysteine or zinc--preliminary results.

BACKGROUND: Prevention of contrast media (CM) induced nephropathy (CIN) by prophylaxis (e.g. N-acetylcysteine; NAC) is controversially discussed. Up to now, assessment of kidney function has been based on measurements of serum creatinine, although this biomarker has several limitations. We investigated NAC and zinc (Zn) for the prevention of CIN by monitoring creatinine and cystatin C. METHODS: In a prospective, placebo-controlled, double blind trial, patients with moderately impaired kidney function receiving low-osmolar, non-ionic CM were randomly assigned to an oral treatment for 2 days with 1.2 g/day of NAC (n = 19), for 1 day with 60 mg/day of Zn (n = 18) or placebo (n = 17). All patients received peri-procedurally 1 ml/kg/h of 0.45% saline for 24 h. At baseline, prior to exposure of CM, 2 and 6 days after CM, creatinine and cystatin C were measured. RESULTS: There was no difference in the incidence of CIN, but a significant drop in creatinine (P < 0.05) was observed in all patients during volume expansion. Creatinine showed no increase after CM and it was normalized to the baseline values in all groups at the study end. In contrast, 2 days after CM there was a significant rise in cystatin C in the Zn (P = 0.012) and the placebo (P = 0.041) group, whereas NAC prevented this deterioration of kidney function. CONCLUSIONS: Cystatin C seems to reflect CM-induced changes in kidney function better than creatinine. NAC and Zn have no effect in preventing CIN by the standard definition, but based on cystatin C we can confirm a preventive effect of NAC. It appears mandatory to assess kidney function by cystatin C in CIN intervention trials, because relying on creatinine can be misleading.

Peritoneal mast cells in peritoneal dialysis patients, particularly in encapsulating peritoneal sclerosis patients.

BACKGROUND: We assumed that increased mast cell numbers contribute substantially to the fibrosis often seen in the peritoneum of peritoneal dialysis (PD) patients, particularly those with encapsulating peritoneal fibrosis (EPS). Therefore, we investigated mast cells in different pathological conditions of the peritoneum. METHODS: One hundred fifteen tissue probes with different peritoneal pathological states were selected (normal, n = 20; chronic appendicitis, n = 25; herniotomy, n = 24; fibrosis, n = 11; PD, n = 26; and EPS, n = 9). For staining of mast cells, we used alpha-naphtol-AS-d-chloracetate-esterase and mast cell tryptase. Next, we counted numbers of mast cells per square millimeter. Tryptase was measured by using image analysis. RESULTS: Measurements by means of both methods correlated well (r = 0.812). Numbers of mast cells per square millimeter were as follows: normal, 26 +/- 16; chronic appendicitis, 241 +/- 217; herniotomy, 115 +/- 88; fibrosis, 99 +/- 66; PD, 81 +/- 64, and EPS, 24 +/- 23 (P = 0.00006). Amounts of tryptase present were 2.900 +/- 0.118, 2.871 +/- 0.150, 2.733 +/- 0.183, 3.041 +/- 0.176, 2.780 +/- 0.184, and 2.609 +/- 0.234, respectively (P = 0.00002). CONCLUSION: We found upregulation of mast cells in specimens of chronic inflammatory diseases of the peritoneum. This also was true for PD patients, with the exclusion of patients with EPS. Therefore, loss-of-control functions of mast cells may contribute to the ill-understood disease entity of EPS.

Unchanged serum levels of advanced glycation endproducts in patients with liver disease.

Advanced glycation end products (AGEs), e.g., carboxymethyllysine (CML) or imidazolone are involved in several age-related disorders. Concerning their accumulation, the importance of hepatic and renal function is controversially discussed. To test whether impairment of hepatic or renal function will affect their accumulation, both AGEs have been measured in various populations, such as 52 patients with liver disease [viral hepatitis C without (n = 19) and with (n = 10) fatty liver; nonalcoholic fatty liver (n = 13), nonalcoholic steatohepatitis (n = 10)]. Serum concentrations of both AGEs have been compared to those in 20 healthy controls and 24 patients with moderate renal impairment (creatinine clearance 23-55 ml/min). Concerning CML (95% C.I. 803-1200 ng/ml), no differences between the various groups could be observed. Likewise, serum levels of imidazolone (95% C.I. 1.3-5.6 units) were similar in all populations. In conclusion, moderate impairment in hepatic or in renal function did not affect serum levels of CML and imidazolone. Apparently, any increase observed in severe cirrhosis or renal failure seems to be rather a consequence than a cause of both disorders.

The expression patterns of peritoneal defensins.

BACKGROUND: Local defense mechanisms are important for the integrity of the peritoneum, but few details are known about the expression patterns of antimicrobial proteins such as human defensin in normal and damaged peritoneum. METHODS: Part A: The expression of different defensins in normal (n = 12), inflamed (n = 5), and metastatic peritoneum (n = 4) and in cultured human peritoneal mesothelial cells was analyzed using mRNA and immunohistochemistry. Part B: Using immunohistochemistry the expression of different defensins was analyzed in different subgroups: healthy controls (n = 25), patients with chronic appendicitis (n = 25) or acute appendicitis (n = 10), and end-stage renal disease patients (n = 25, with 15 on peritoneal dialysis). RESULTS: Part A: Human neutrophil peptides (HNP) 1 and 3 and human beta-defensins (HBD) 1 to 3 mRNA were detected in peritoneal specimens. In addition, HNP1,3, HBD1, HBD2, and HBD3 proteins were detected using immunohistochemistry. Part B: HBD1 showed a constitutive expression in mesothelium, while HBD2 and HNP1,3 were associated with inflammation. Decreased expressions of HNP1,3 were observed in end-stage renal disease patients and in patients on peritoneal dialysis. CONCLUSIONS: For the first time, the expression patterns of defensins in normal and damaged peritoneum have been described. The reduced expression of some defensins in end-stage renal disease is of potential clinical interest against the background of the frequent infective complications seen in peritoneal dialysis.

Metallothionein and dendritic cells in skin of end-stage renal disease patients not on dialysis, or on hemodialysis or peritoneal dialysis.

OBJECTIVE: Renal failure leads to a variety of defects in immune function. The skin, as a major player in the immune system network, also exhibits multiple derangements. The pathogenesis of these defects and derangements are poorly understood; therefore, we studied immune competent cells, dermal dendrocytes (DC), and a special proinflammatory protein, metallothionein (MT), in the skin of these patients. DESIGN: 22 patients with end-stage renal disease (ESRD) but not on dialysis, 18 patients on hemodialysis (HD), 14 patients on peritoneal dialysis (PD), and 35 healthy controls were included in the study. Immunohistochemical staining of skin biopsies for DC and MT was performed with the following antibodies: for DC, antibody against factor XIIIa; and for MT, Dako-MT, E9 (Dako, Carpinteria, California, USA). Measurements were made by counting stained DC per square millimeter, and by optical density (OD) for MT (mean SEM). RESULTS: Metallothionein was increased in the skin of HD (OD 0.42 +/- 0.05, p < 0.01) and PD patients (OD 0.33 +/- 0.04, p < 0.05) compared to controls (OD 0.23 +/- 0.02) and ESRD patients not on dialysis (OD 0.22 +/- 0.05). In contrast, numbers of DC were reduced in patients on PD compared to controls (59 +/- 13 vs 96 +/- 59 DC/mm2, p < 0.01) and increased in patients with ESRD prior to dialysis (141 +/- 13 DC/mm2, p < 0.05). Patients on HD were in-between (105 +/- 20 DC/mm2), with a significant difference versus patients on PD (p < 0.05). CONCLUSIONS: Our data show that the mode of dialysis influences the number of antigen-presenting cells in the dermis. However, in both dialysis modes, a proinflammatory immune status of the skin (MT) was present and, therefore, other regulatory elements for dermal dendrocytes apart from proinflammation exist.

Metallothionein in liver-biopsies from patients with different diseases.

Metallothioneins (MT) are ubiquitous found in eukaryotic organism. MT have a potential for metal-storage and protect the cells against stress. On the genomic level, proinflammatory cytokines like interleukin-6 and transition metals like copper cause induction of MT. Therefore, an estimation of MT in liver-biopsies from patients with different diseases probably could help in identifying acute-phase reactions and processes which lead to increased copper. We investigated paraffin embedded liver biopsies from 170 patients and 13 control biopsies from cases of sudden death. Tissue was stained with a primary antibody against MT and a peroxidase technique was used to make results visible. A grading was performed using an immunoreactive score (IRS from 0-24) and by computer-aided measurement of the optical density (OD) of the stained tissue slides. Patients with cholestasis (IRS: 12.1 +/- 2.8, n = 11), autoimmune (10.6 +/- 3.1, n = 7) or inflammatory bowel diseases (IBD) (13.3 +/- 5.1, n = 4) and lymphoma (9.8 +/- 5.8, n = 21) showed marked increases in MT compared to the controls (5.2 +/- 2.8, n = 13). Patients with chronic hepatitis B or C or chronic alcoholic abuse had no elevation of MT. Furthermore, no correlation was found between histological damage and amount of MT except in cases of cholestasis, in which increased MT was observed. Results by OD confirmed the findings. In summary, we were able to demonstrate a clear increase of MT content in liver-biopsies in proinflammatory and cholestatic conditions. Marked elevation in patients with systemic diseases (like autoimmune-, IBD and lymphoma) seems to be best explained by an acute-phase induction of MT by proinflammatory cytokines. This could help in identifying these conditions in liver biopsies.

Gastrointestinal involvement in granulomatosis with polyangiitis and microscopic polyangiitis: histological features and outcome.

AIM: Gastrointestinal (GI) involvement in patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) is rare. METHOD: Medical charts of seven patients with GPA and MPA and GI involvement were reviewed regarding clinical presentation, outcome, diagnostic tools and therapy. Second, the cellular composition of the inflammatory infiltrate associated with the vascular lesions in histological samples (ileum, colon, rectum, duodenum) were investigated to identify possible treatment targets. Immunohistochemistry was done with antibodies against CD20, CD3 and CD34. Samples from a healthy control group (n = 15) were used for comparison. RESULTS: Mean age at onset of the first symptoms of vasculitis was 48 ± 21.3 years. At time of diagnosis GI symptoms were present in five out of seven patients (71%) and occurred during relapse of the vasculitis in two patients (29%). All patients had abdominal pain, four of seven (57%) had an acute kidney injury and three patients required renal replacement therapy. At the time of diagnosis five of seven patients (71%) required surgery and mean Birmingham Vasculitis Activity Score (BVAS) on admission was high (26.3 ± 7.7). Regarding outcome, one patient died due to gastrointestinal bleeding. Histological analysis showed significantly higher expression of CD3 in this patient compared to the control group (P = 0.02). Analysis of expression of CD20 and CD34 showed no statistically significant differences between patients with GPA and MPA with GI involvement compared to the control group. CONCLUSIONS: GI involvement in GPA and MPA is rare. Therapy directed at T cells might be an alternative treatment option.

Induction of metallothionein in mesothelial cells by zinc.

Patients on peritoneal dialysis (PD) are exposed to peritoneal dialysis fluids with unphysiological properties. Local defense systems are of importance. In this respect, metallothionein (MT) might play an important role. Because nothing is known about the achievability of MT induction in peritoneum by zinc, we performed the following study. We investigated human peritoneal mesothelial cells (HPMC) from omentum and a mesothelioma cell (MTC) line after addition of zinc in concentrations from 35 to 350 microM. Measurements of MT-mRNA and protein (by immuncytochemistry [IHC], Western blots, and dot blots) were performed. Zinc caused a clear and highly significant fourfold increase of RNA in MTC and to a lower extent in HPMC (1.6-fold, P < 0.001). IHC demonstrated a clear induction in HPMC and MTC. Western and dot blots confirmed this and showed an increase of MT from 112-mg/g total protein (TP) to 410-mg/g TP. Zinc was able to upregulate MT significantly in HPMC and MTC on the RNA and protein level. Fourfold increases of MT were achievable.

The role of micro-inflammation in the pathogenesis of uraemic pruritus in haemodialysis patients.

BACKGROUND: Uraemic pruritus (UP) is still one of the most vexing and disabling symptoms in chronic renal failure. The pathogenesis of UP is obscure and effective therapeutic strategies are elusive. Deduced from partial successful treatment modalities, there is evidence that an alteration of the immune system with a pro-inflammatory pattern along with a deranged T-helper-cell differentiation may be involved in the pathogenesis of UP. We, therefore, investigated whether UP is related to an augmented Th1-differentiation as measured by determination of intracytoplasmatic (i.c.) cytokines and expression of chemokine receptors. Additionally, pro-inflammatory cytokines were determined in serum. METHODS: In a multicentre study, 171 patients on haemodialysis (HD) were screened for UP. Finally, 13 HD patients with and 13 HD patients without UP, as well as 15 healthy controls were enrolled in the study. Peripheral blood mononuclear cells were isolated and the proportion of Th1- and Th2-cells was determined by flow cytometry. The expression of chemokine receptors on CD4 cells (CXCR3 preferentially on Th1 and CCR4 on Th2) and i.c. cytokines (IFNgamma for Th1 and IL4 for Th2) were measured after in vitro stimulation. Serum cytokine levels (IL6 and TNFalpha) and CRP were measured by ELISA. RESULTS: Compared to HD patients without UP, those complaining of UP showed a significantly enhanced proportion of Th1-cells as measured by both techniques. Additionally, serum CRP and IL6 levels were significantly higher in HD patients with UP, compared to HD patients without UP. CONCLUSIONS: These results point to a central role of inflammation in the pathogenesis of UP in HD patients.

Induction of metallothionein in proximal tubular cells by zinc and its potential as an endogenous antioxidant.

BACKGROUND: This study was undertaken to gain further insights into the expression of metallothionein (MT) in kidney, to define the necessary dosage of a metal (zinc) to achieve induction of MT and to evaluate the antioxidative potential of MT in comparison to other more common antioxidative therapeutics, like N-acetyl-L-cysteine (NAC), and endogenous molecules, like glutathione. METHODS: MT was measured in renal specimens from cadaver kidneys from patients with chronic diseases (n = 76) and controls (n = 21) by immunohistochemistry. In addition, induction experiments were performed in cell cultures of proximal tubular cells (LCC-PK1) and MT measured on the RNA and protein level (immunohistochemistry, Western and dot blotting). Antioxidative potential of MT was compared to NAC and glutathione. RESULTS: MT was restricted to tubular cells with no differences between controls and patients. Zn caused a dose-dependent increase of MT on the RNA as well as on the protein level (RNA (ratio MT/histone 3.3): control 0.34 +/- 0.12; Zn 17 microM 0.65 +/- 0.26; Zn 35 microM 1.25 +/- 0.43 (p < 0.05), Zn 52 microM 1.35 +/- 0.46 (p < 0.05), and protein: 5.8-fold increase from 47 +/- 13 mg/g total protein (n = 6) to 272 +/- 140 mg/g total protein (n = 6)). The antioxidative effect of MT was equal to NAC and glutathione. CONCLUSIONS: Induction of renal MT by zinc is easily achievable and might be an interesting therapeutic and preventive tool against oxidative stress.

Apoptosis of mesothelial cells caused by unphysiological characteristics of peritoneal dialysis fluids.

There is an ongoing debate as to which peritoneal dialysis fluids (PDFs) provide the best preservation of peritoneal cells. To investigate this topic further, we measured apoptosis and necrosis of cultured mesothelial cells (MCs) after exposure to different single unphysiological features of PDFs and PDFs for whole. MCs were incubated in buffers containing plasticizers, high osmolarity by sodium chloride, low pH, and high glucose for 0.5, 4, and 24 h. The same procedure was repeated with different PDFs. Apoptosis and necrosis were measured by FACS-analysis (annexin-FITC and propidium iodide). We found that plasticizers were clearly able to induce apoptosis after 24 h (18 +/- 4%). The same result was observed with high osmolarity by sodium chloride (17 +/- 5%), but not for high glucose (9 +/- 8%). All fluids with low pH (5.2) caused severe and almost complete necrosis (after 4 and 24 h). Incubation in neutral, two-compartment PDFs (glucose 4.25%) without plasticizers for 4 h showed no significant necrosis (3%), but after 24 h apoptosis was detectable in 10 +/- 9% and necrosis in 29 +/- 8% of MCs. In conclusion, after improving PDFs and introducing neutral fluids, further attention should be drawn to inducers of apoptosis. Apoptosis can be detected quite early (24 h) and is caused by plasticizers and high osmolarity.

Osteoarthritis of the knee--clinical assessments and inflammatory markers.

OBJECTIVE: The present cross sectional study was performed to test the hypothesis that in osteoarthritis (OA) of the knee severity of this disease is related to local levels of inflammatory metabolites and their corresponding enzymes. METHODS: From 41 patients with OA of the knee (age range 45-79 years) undergoing arthroscopy blood, synovial fluid (SF) and synovial membrane (SM) were collected. Clinical conditions were primarily assessed by the WOMAC-index and radiographic grading (K&L-grade). Concentrations of PGE(2), TxB(2)and NO(2/3)and that of IL-6, IL-1 alpha, IL-1 beta, TNF alpha, COX-2 and iNOS were determined in SF and SM, respectively. RESULTS: With advancing age K&L-grade and COX-2 in SM increased significantly (P=0.005 and P=0.01, respectively). TNF alpha and IL-1 alpha were not detectable in SM samples. Apart from a correlation between PGE(2)and WOMAC-index (r=0.36, P=0.035) no significant relationships could be found between the various inflammatory parameters and any of the assessed clinical signs. CONCLUSIONS: Apparently no direct relationships exist between the measured markers of inflammation (e.g. PGE(2), NO(2/3)) or the involved enzymes (e.g. COX-2, iNOS) and the severity of OA of the knee. The degenerative condition of this disease might be due to the more local, mainly mechanical injury with little systemic upset. However, further longitudinal studies are needed to clarify whether the assessed biochemical markers could serve as predictors for the progression of OA.