Undine Ott

Bone histomorphometry and biochemical markers of bone turnover in patients with chronic kidney disease Stages 3 - 5.

AIMS Precise identification of renal osteodystrophy requires bone histomorphometry. Several markers of bone turnover may be useful to predict classification and severity of renal osteodystrophy, but there are only limited data whether these markers correlate with bone histomorphometry. METHODS In 36 patients with chronic kidney disease (CKD) Stage 3/4 and in 96 patients with CKD Stage 5 bone histomorphometry was performed and renal osteodystrophy was classified according to the standardized international nomenclature. Blood samples were taken at the time of bone biopsy, stored and analyzed at the end of the study. RESULTS Osteitis fibrosa (OF) was the most frequent histomorphometric form, occurred in 47.2% in CKD Stages 3 - 4 and in 61.4% in CKD Stage 5. There was no difference in the frequency of adynamic renal bone disease (ARBD). The correlation coefficients between bone turnover markers and histomorphometric parameters were higher in CKD 5 patients with high bone turnover lesions. The predictive value for high versus low/normal bone turnover status was comparable for alkaline phosphatase (APH), bone alkaline phosphatase (BAP), pyridinoline (Pyd), desoxypyridinoline (Dpyd), tartrat-resistent acid phosphatase (TRAP Vb) and parathyroid hormone (PTH) in CKD Stage 5 patients, but was insufficient for APH and TRAP Vb in CKD Stage 3 - 4 patients. CONCLUSIONS Besides parathyroid hormone, biochemical parameters of bone turnover provide a moderate discrimination and prediction of bone turnover status only in patients with CKD Stage 5. Due to a large variability, they are of limited use in predicting the histomorphometric type of renal osteodystrophy. Bone histology remains necessary for an exact classification of underlying pathology.

Secretion of cytokines by uroepithelial cells stimulated by Escherichia coli and Citrobacter spp.

Urinary tract epithelial cells (T 24/83) are able to express interleukin (IL)-6, IL-8, platelet-derived growth factor (PDGF) and tumour necrosis factor-alpha, but not IL-1 beta, IL-2, IL-4 and IL-10 in response to an infection with uropathogenic bacteria. The process of cytokine secretion is time dependent, with a significant increase in the cytokine activity after 60 min. The expression of virulence factors of the bacteria does not seem to play a role. The interaction between bacterial products (e.g. lipopolysaccharide) and/or bacterial adhesion mediated by adhesins and specific receptor molecules of cell surfaces may be responsible for the activity of mediator protein expression in the epithelial cells. The release of PDGF and IL-8 was found to be higher when due to Escherichia coli HB 101 (rough form) than that caused by other bacterial strains. Citrobacter CB 3009 provoked the highest level of IL-6. The PDGF level correlated significantly with IL-6 and IL-8 values (P<0.001). There was a significant correlation between the time-dependent release of IL-6 and IL-8 (P<0.05). In epithelial cytokine response to bacterial infection, the reaction of the epithelial cells may modify themselves (e.g. internalization of bacteria) and the immuno-regulatory processes that are caused by infection and responsible for parenchymal injury.

Post-transplant diabetes mellitus: risk factors, frequency of transplant rejections, and long-term prognosis

BackgroundEstimates of the incidence of new-onset diabetes after renal transplantation vary between 2% and 54%. It was the aim of the present trial to study the prevalence of post-transplant diabetes mellitus (DM), the risk factors, the frequency of transplant rejections, and the long-term prognosis.MethodsWe studied all consecutive patients with endstage renal disease, but without DM who received kidney transplantation at our center since 1992 (n = 253; age, 52.2 ± 12.6 years; body mass index, 22.0 ± 7.9 kg/m2). Follow up was 3.3 ± 1.6 years (range, 0.1–17.7) years.ResultsIn total, 43/253 patients (17%) developed new-onset DM after transplantation. Patients with new-onset diabetes were significantly older (58.3 ± 11.4 vs 50.9 ± 12.5 years; P < 0.01) and had a tendency to a higher body mass index (24.0 ± 8.5 vs 21.6 ± 7.8 kg/m2; P = 0.077). There were no differences between the groups in respect of blood pressure control (137.7 ± 19.0/81.8 ± 14.2 vs 137.1 ± 21.9/83.9 ± 13.1 mmHg; P = 0.89/0.39), glomerular filtration rate (58.0 ± 28.1 vs 64.1 ± 22.1 ml/min per 1.73 m2; P = 0.13), steroid dosage (4.5 ± 1.2 [n = 21] vs 4.6 ± 2.2 [n = 135] mg/day; P = 0.13), or the frequency and dosage of immunosuppressive drugs such as cyclosporine, tacrolimus, and sirolimus during the follow up. However, more patients with post-transplant diabetes received steroids (83.7% vs 64.3%; P = 0.021) and azathioprine (41.9% vs 24.3%; P = 0.030). Patients with new-onset diabetes had higher serum creatinine values (163.4 ± 67.9 vs 138.7 ± 59.5 µmol/l; P = 0.017). The mean hemoglobin (Hb)A1c in patients with DM was 6.28 ± 1.29% (Tosho HPLC; mean normal, 5.15%). In 18 patients (7.1%) transplant rejections occurred (16 patients without DM [7.6%] vs 2 patients with new-onset DM [4.7%]; P = 0.39). On performing multivariate analysis, the only parameter found to be associated with new-onset DM was the body mass index (R2 = 0.05; β = 0.23; P = 0.02), and the only factor associated with transplant rejection was fasting blood glucose (R2 = 0.07; β = 0.28; P = 0.02). None of the other parameters included in the models (age, duration after transplantation, diabetes duration, immunosuppressive therapy, HbA1c, HLA mismatches) showed any associations.ConclusionsThe prevalence of new-onset DM after renal transplantation was 17%. The most important parameter associated with new-onset diabetes was a higher body mass index, and the most important parameter associated with transplant rejection was an elevated fasting blood glucose level. To prevent transplant rejections and to improve patients’ outcome, in addition to providing optimal immunosuppressive therapy and HLA matching, good blood pressure control and HbA1c, but also near normal fasting blood glucose levels, should be achieved.

Anemia After Renal Transplantation: An Underestimated Problem

In end-stage renal disease patients anemia is known to be an independent risk factor for cardiovascular disease and death. In a monocenter retrospective analysis, we investigated 207 stable patients (68 women/139 men) who underwent a first renal transplantation. Immunosuppressive therapy was performed with either cyclosporine plus mycophenolate mofetil, tacrolimus plus mycophenolate mofetil, or rapamycin plus mycophenolate mofetil; 43.5% of the patients were treated with steroids. Seventy-eight patients (37.7%) displayed anemia, including 8.7% with a severe disorder displaying an average hemoglobin (Hb) level of <6.8 mmo/L in men and <6.2 mmol/L in women. In 8.2% of the cases, we observed moderate anemia (Hb 6.8-7.4 mmol/L in men and 6.2-6.8 mmol/L in women), and in 20.8% (29 men and 14 women), mild anemia (Hb <8.06 mmol/L in men and <7.45 mmol/L in women). Erythropoietin was administered in 55.5% of patients with severe anemia, 53% with moderate anemia, and 11.6% with mild anemia. Serum creatinine level was a significant predictor of anemia (B -0.004; SE 0.001; P < .01). Among patients with creatinine >200 micromol/L, 63% were anemic compared with 22% of those with a serum creatinine level <200 micromol/L (P < .05). No correlation was observed with immunosuppressive medication or treatment with angiotensin-converting enzyme inhibitors/angiotensin-II receptor antagonists. During a 3-year follow-up, both mortality and graft failure rates were significantly greater among anemic patients nonanemic patients (mortality 3.3% vs 0.5%, P < .001; graft failure 4.3% vs 0%, P < .001). We found an unexpectedly high incidence of anemia in patients with well-functioning grafts. Anemia as a risk factor for mortality and graft failure should be treated more intensively among renal transplant patients.

Vitamin B6 Metabolism in Chronic Kidney Disease – Relation to Transsulfuration, Advanced Glycation and Cardiovascular Disease

Background: Vitamin deficiency is common in chronic kidney disease (CKD). Data on B6 supply and possible relationships to cardiovascular events (CVE) in CKD are rare. Pyridoxamine exerts inhibitory effects on the formation of advanced glycation endproducts (AGE) implicated in the pathogenesis of CKD and atherosclerosis. Methods: In 48 CKD patients at stage 2–4, 72 hemodialysis patients (HD), 38 renal transplant recipients (RTR) and 141 healthy controls (mean age 58 ± 13, 61 ± 12, 50 ± 12 and 54 ± 16 years, respectively), plasma and red blood cell (RBC) concentrations of pyridoxal-5′-phosphate (PLP), pyridoxal (PL), 4-pyridoxic acid (PA), pyridoxamine-5′-phosphate (PMP) and of the AGE pentosidine were measured by high-performance liquid chromatography, NΕ-(carboxymethyl)lysine and imidazolone by an ELISA, and total homocysteine and cystathionine by gas chromatography-mass spectrometry. Results: Despite routine low-dose vitamin supplementation in HD, plasma PLP was decreased in HD (79 ± 69 nmol/l) compared with CKD stage 2–4 patients (497 ± 944 nmol/l), RTR (416 ± 604 nmol/l) and controls (159 ± 230 nmol/l; p < 0.001). Plasma PA was significantly increased in HD (11,667 ± 17,871 nmol/l) in comparison with CKD stage 2–4 (435 ± 441 nmol/l), RTR (583 ± 668 nmol/l) and controls (46 ± 49 nmol/l; p < 0.001). B6 forms were significantly affected by renal function (R = 0.792, p < 0.001 for CKD stage 2–4). There was no relation of vitamers with a history of CVE. Relationships between B6 forms and AGE (RBC-PMP with pentosidine in CKD stage 2–4: R = –0.351, p < 0.05) were found. Conclusion: HD patients showed a deficiency in PLP in plasma but not in RBC. Prospective trials are needed to elucidate the potential role of elevated PA on cardiovascular and renal outcome in CKD. Vitamin B6 supplementation might be successful in preventing AGE-related pathologies.

Colocalization of BAX and BCL-2 in small intestine and kidney biopsies with different degrees of DNA fragmentation

Abstract Morphological changes associated with apoptosis are closely correlated with the expression of specific proteins. However, the cause-effect relationships between the expression of these proteins and DNA degradation are barely known. For studying expression of apoptosis-related proteins in relation to different degrees of DNA fragmentation, the small intestine with its spatially organized continuum of proliferation, differentiation and death is a very useful preparation. Enterocytes towards the apex of the villi become increasingly susceptible to apoptosis. Here, this ”apoptotic gradient” is used to demonstrate the presence of BAX and BCL-2 proteins in the cytoplasm of cells at the onset of apoptosis. In semithin serial sections of the small intestine, BAX, BCL-2 and DNA fragmentation were demonstrated. BAX and BCL-2 are always colocalized and only in cells with fragmented DNA. The gradient of BAX or BCL-2 staining is similar to the gradient of DNA fragmentation. Immunoreactivity for BCL-2 or BAX is most intense in cells that are prone to become apoptotic next in the course of cellular turnover but not in cells in an advanced apoptotic state, showing strongly condensed chromatin. When using the same technique on semithin sections of kidney biopsies, containing epithelia with low cellular turnover, we found DNA fragmentation mainly in the epithelial cells of the distal tubules. Similar to the situation in the enterocytes, BAX staining was confined to the cytoplasm of epithelial cells with a moderate degree of DNA fragmentation and reduced in epithelial cells with a high degree of DNA fragmentation. In contrast to the situation in the small intestine, very low levels of BCL-2 were found. The results suggest that expression of BCL-2 and BAX is related to cell damage as indicated by DNA fragmentation but not to advanced stages of cellular death, as indicated by chromatin condensation and cellular shrinkage.

Renal Retransplantation: A Retrospective Monocentric Study

Approximately 10% to 20% of all annual renal transplantations are retransplantations and up to 20% of patients on waiting lists need a repeat kidney because of previous graft failure. The immunological risk is much greater among retransplanted patients than first-time kidney recipients. It is likely that retransplantation will become even more prevalent in the future. However, clinical studies or retrospective data are rare in this patient population. We retrospectively investigated 50 recipients after second or third renal transplantations in our center since 2001. Immunosuppression was performed with corticosteroids, mycophenolate mofetil (MMF), tacrolimus, and induction therapy with either thymoglobulin (2.5 mg/kg body weight; n = 27) or 20 mg basiliximab on days 0 and 4 (n = 22) after renal transplantation; 1 patient was treated with antithymoglobulin Fresenius after combined liver-kidney transplantation. Acute rejection occurred in 12 recipients (44.4%) after thymoglobulin and in 7 recipients (31.8%) after basiliximab induction therapy (P < .05). In 4 (14.8%) thymoglobulin- and 5 (22.7%) basiliximab-treated recipients, vascular rejections were observed (P = NS). Patients with basiliximab treatment showed improved renal function at 1 year after transplantation: serum creatinine 134.3 mumol/L versus 199.6 mumol/L in the thymoglobulin group (P < .05). Over the observation period the renal function remained stable or improved in both groups if rejection treatment was successful. However, allograft failure was higher in the basiliximab-treated group, namely, 18.1% versus 14.8% in thymoglobulin-treated patients, but the difference did not reach statistical significance. In 3 (11.1%) thymoglobulin- and 4 (18.2%) basiliximab-treated patients cytomegalovirus (CMV) infections complicated the follow-up (P = NS). In the follow-up period of 5 years, no malignant diseases were seen in either group. Three basiliximab-treated recipients died in the first year due to sepsis or cardiovascular complications. Two thymoglobulin-treated patients developed BK virus nephropathy in the follow-up period. In conclusion, we observed a high immunological risk and rejection risk among retransplanted kidney recipients in our center. Particularly, severe vascular rejections with a harmful long-term impact on allograft function were observed in this population. Induction treatment seems to be successful to reduce risk and achieve better results. Single-shot thymoglobulin may be preferable to reduce severe vascular rejection and prevent allograft failure than basiliximab with the same infection rate.

A single-center experience with BK virus nephropathy.

BACKGROUND BK virus nephropathy has an increasing role in renal transplant dysfunction, since new, highly potent immunosuppressive drugs have been introduced into therapy following renal transplantation. Diagnosis of acute impairment of renal transplant function is complicated by difficulty in differentiating BK virus nephropathy from acute rejection. PATIENTS AND METHODS We retrospectively described the findings and therapeutic approaches of 6 consecutive patients with BK virus nephropathy in our transplantation center (75 - 80 transplantations/ year). BK virus nephropathy was classified according to Drachenberg et al. [2004]. RESULTS We observed an incidence rate of < 1% for BK nephropathy in our center. Four patients had a pattern B whereas 2 patients revealed a pattern C of BK virus nephropathy. Focal C4d-positive staining of peritubular capillaries were found in 2 of the 6 cases. For earlier detection of BK nephropathy, a diagnostic algorithm for each patient after renal transplantation was established. Urine was continuously monitored by cytology for decoy cells and PCR for BK virus DNA. If PCR was also positive for the BK virus in plasma, biopsy of the renal allograft was performed. Thereby diagnosis could be confirmed sooner. For treatment of BK nephropathy in our center, we reduced immunosuppressive agents and initiated a virustatic treatment with cidofovir in the first 3 cases. However, results were not satisfactory and two allografts were lost. We then reconsidered our therapeutic approach and switched the immunosuppressive treatment to leflunomide with consistent low dose steroids. We use therapeutic drug monitoring for leflunomide and aim at a target level of 40 - 100 microg/ml. We lost no allograft with BK nephropathy since using this therapeutic approach. CONCLUSION In our center, leflunomide therapy, but not cidofovir, was effective in patients with BK virus nephropathy of the renal allograft.

Reflectance enzyme histochemistry (REH): visualization of cerium-based and DAB primary reaction products of phosphatases and oxidases in cryostat sections by confocal laser scanning microscopy

In the present study the reflectance mode of confocal laser scanning microscopy was adapted to detect and to assess semiquantitatively cerium-based primary reaction products of oxidases [Ce(IV) perhydroxide] and phosphatases [Ce(III) hydroxyphosphate converted into Ce(IV) perhydroxyphosphate] as well as of the 3,3′-diaminobenzidine (DAB)-based primary reaction product of cytochromec oxidase in cryostat sections. Confocal laser scanning microscopy offers a unique way of making visible histochemical reaction products which are weakly absorbant but sufficiently reflective. It was easily possible to record simultaneously the reflectance signals at the wavelength of the exciting laser (preferentially 488 nm) and the autofluorescence signals (>580 nm in our set-up) of glutaraldehyde-fixed tissue. The results of an imbibition study of cerium-containing model precipitates indicate that the cerium, generally, should be oxidized prior to observation because the index of refraction of Ce(IV) compounds is considerably higher than that of the corresponding Ce(III) compounds. An attempt at comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is presented. The proposed technique may open new possibilities in enzyme- and immunohistochemistry.

Proteinuria after conversion to sirolimus in kidney transplant recipients: impact of pre-existing proteinuria, graft function, and angiotensin-converting enzyme inhibitors/angiotensin-receptor antagonists

Marx C, Busch M, Ott U, Gerth J, Wolf G. Proteinuria after conversion to sirolimus in kidney transplant recipients: impact of pre‐existing proteinuria, graft function, and angiotensin‐converting enzyme inhibitors/angiotensin‐receptor antagonists.
Clin Transplant 2009 DOI:10.1111/j.1399‐0012.2009.01142.x.
© 2009 John Wiley & Sons A/S.