Lars P. Kihm

Sustained low efficiency dialysis using a single-pass batch system in acute kidney injury - a randomized interventional trial: the REnal Replacement Therapy Study in Intensive Care Unit PatiEnts

Th e authors noticed after the publication of their article [1] an error in their methods. Under “Randomization and treatment assignments”, the Asahi APS 650 membrane was used together with the Octo Nova device (Octo Nova, Diamed, Köln, Germany) and not the Prisma device as indicated in the manuscript. Th is section should therefore read as follows; “Patients randomly assigned to the CVVH-group (Prisma, Gambro Hospal, Lyon, France and Octo Nova, Diamed, Köln, Germany) were treated with 35 ml/kg per hour replacement fl uid in predilution. Treatment was scheduled for 24-h and blood fl ow was maintained between 100 and 120 ml/min. For all CVVH treatments, high-fl ux fi lters (AN69-M100, Gambro Hospal, Lyon, France and Asahi Kasei APS-650, Asahi Kasei Medical Co, Ltd., Japan) were used.” In addition, the funding of this study was erroneously deleted in-house and should read as follows; “Grant of the European Nephrology and Dialysis Institute, Bad Homburg, Germany”.

Increased Infarct Size in Uremic Rats: Reduced Ischemia Tolerance?

In patients with renal failure, myocardial infarction (MI) is more frequent and the rate of death from acute MI is very high. It has been argued that ischemia tolerance of the heart is reduced in uremia, but direct evidence for this hypothesis has not been provided. It was the purpose of this study (1) to ligate the left coronary artery and to measure the nonperfused area (risk area: total infarction plus penumbra) as well as the area of total infarction in subtotally nephrectomized (SNX) rats compared with sham-operated pair-fed control rats and (2) to examine the effects of potential confounders such as BP, sympathetic overactivity, and salt retention. The left coronary artery was ligated for 60 min, followed by reperfusion for 90 min. For visualizing perfused myocardium, lissamine green ink was injected. The nonperfused area (lissamine exclusion) and the area of total infarction (triphenyltetrazolium chloride stain) were assessed in sections of the left ventricle using image analysis. Groups of SNX rats also received: antihypertensive treatment (nadolol plus hydralazine); moxonidine; high salt diet or low salt diet (1.58% versus 0.015%). In surviving animals, the nonperfused area at risk (as the proportion of total left ventricular area), presumably determined by the geometry of vascular supply, was similar in sham-operated and SNX animals (0.38 +/- 0.13 versus 0.45 +/- 0.09; NS). In contrast, the infarcted area, given as a proportion of the nonperfused risk area, was significantly (P < 0.003) higher in SNX (0.68 +/- 0.09) compared with sham-operated (0.51 +/- 0.11) rats and was not altered by any of the above interventions. The finding that a greater proportion of nonperfused myocardium undergoes total necrosis is consistent with the hypothesis of reduced ischemia tolerance of the heart in renal failure. The findings could explain the high rate of death from MI in patients with impaired renal function.

Aquaporin-1 Channel Function Is Positively Regulated by Protein Kinase C

Aquaporin-1 (AQP1) channels contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short term regulation of AQP1 function by other major intracellular signaling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of protein kinase C (PKC) by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites Thr157 and Thr239 (termed AQP1 ΔPKC). AQP1 cationic currents measured with double-electrode voltage clamp were markedly increased after pharmacological activation of PKC by either OAG or phorbol 12-myristate 13-acetate. Deletion of either Thr157 or Thr239 caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 ΔPKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154–168 and 236–250 revealed that both Thr157 and Thr239 are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 ΔPKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1-modulated processes such as endothelial permeability, angiogenesis, and urine concentration.

Benfotiamine Protects against Peritoneal and Kidney Damage in Peritoneal Dialysis

Residual renal function and the integrity of the peritoneal membrane contribute to morbidity and mortality among patients treated with peritoneal dialysis. Glucose and its degradation products likely contribute to the deterioration of the remnant kidney and damage to the peritoneum. Benfotiamine decreases glucose-induced tissue damage, suggesting the potential for benefit in peritoneal dialysis. Here, in a model of peritoneal dialysis in uremic rats, treatment with benfotiamine decreased peritoneal fibrosis, markers of inflammation, and neovascularization, resulting in improved characteristics of peritoneal transport. Furthermore, rats treated with benfotiamine exhibited lower expression of advanced glycation endproducts and their receptor in the peritoneum and the kidney, reduced glomerular and tubulointerstitial damage, and less albuminuria. Increased activity of transketolase in tissue and blood contributed to the protective effects of benfotiamine. In primary human peritoneal mesothelial cells, the addition of benfotiamine led to enhanced transketolase activity and decreased expression of advanced glycation endproducts and their receptor. Taken together, these data suggest that benfotiamine protects the peritoneal membrane and remnant kidney in a rat model of peritoneal dialysis and uremia.

Renal toxicity mediated by glucose degradation products in a rat model of advanced renal failure

Background  In peritoneal dialysis (PD) residual renal function contributes to improved patient survival and quality of life. Glucose degradation products (GDP) generated by heat sterilization of PD fluids do not only impair the peritoneal membrane, but also appear in the systemic circulation with the potential for organ toxicity. Here we show that in a rat model of advanced renal failure, GDP affect the structure and function of the remnant kidney.

RAGE expression in the human peritoneal membrane

BACKGROUND Experimental animal models have demonstrated that the interaction of advanced glycation end-products (AGE) with their receptor RAGE is, at least in part, responsible for peritoneal damage. This study investigates the in vivo expression of RAGE in the peritoneal membrane of uraemic human patients. METHODS Peritoneal biopsies of 89 subjects (48 uraemic and 41 healthy age-matched patients) were examined. The expression of CD3, IL-6, activated NFkappaBp65, VEGF, transforming growth factor (TGF)-beta1, smooth-muscle actin (SMA), methylglyoxal (MGO) and RAGE was analysed immunohistochemically. Additionally, in 4 of the 48 uraemic patients, peritoneal biopsies were repeated after 15 months at the time of catheter removal to analyse the above parameters and the extent of NFkappaB-binding activity determined by electrophoretic mobility shift assay (EMSA) in the long-term follow-up. RESULTS In comparison to the healthy controls, uraemic patients showed a significant increase in fibrosis, angiogenesis, submesothelial thickness, MGO-derived protein adducts, RAGE, IL-6, VEGF, TGF-beta1, SMA and NFkappaBp65 in their peritonea. Four patients, followed up longitudinally from peritoneal dialysis (PD) catheter insertion to removal, demonstrated further significant increase in the above parameters, particularly in RAGE expression and NFkappaB activation. CONCLUSIONS Along with a higher expression of several indicators for inflammation, angiogenesis, fibrosis and AGE accumulation, the peritoneal membrane of the uraemic patients showed an increased submesothelial thickness and a marked induction of RAGE expression and NFkappaB-binding activity, which both further increased after PD treatment. These findings in human peritoneum support the concept of the AGE-RAGE interaction being crucial in peritoneal damage due to uraemia and PD.

Nanotube Action between Human Mesothelial Cells Reveals Novel Aspects of Inflammatory Responses

A well-known role of human peritoneal mesothelial cells (HPMCs), the resident cells of the peritoneal cavity, is the generation of an immune response during peritonitis by activation of T-cells via antigen presentation. Recent findings have shown that intercellular nanotubes (NTs) mediate functional connectivity between various cell types including immune cells - such as T-cells, natural killer (NK) cells or macrophages - by facilitating a spectrum of long range cell-cell interactions. Although of medical interest, the relevance of NT-related findings for human medical conditions and treatment, e.g. in relation to inflammatory processes, remains elusive, particularly due to a lack of appropriate in vivo data. Here, we show for the first time that primary cultures of patient derived HPMCs are functionally connected via membranous nanotubes. NT formation appears to be actin cytoskeleton dependent, mediated by the action of filopodia. Importantly, significant variances in NT numbers between different donors as a consequence of pathophysiological alterations were observable. Furthermore, we show that TNF-α induces nanotube formation and demonstrate a strong correlation of NT connectivity in accordance with the cellular cholesterol level and distribution, pointing to a complex involvement of NTs in inflammatory processes with potential impact for clinical treatment.

Contrast Enhanced Sonography Shows Superior Microvascular Renal Allograft Perfusion in Patients Switched From Cyclosporine A to Everolimus

Background. Real-time contrast enhanced sonography (CES) provides quantitative information on microvascular tissue perfusion in renal allografts. In contrast to calcineurin inhibitors, mammalian target of rapamycin inhibitors may have beneficial effects on renal microvascular tissue perfusion. There is no information on the microperfusion of renal allografts in patients receiving either mammalian target of rapamycin inhibitor or calcineurin inhibitor. Methods. In a prospective randomized, clinical trial, renal parenchymal tissue perfusion of 24 stable renal allograft recipients was evaluated with CES. Eleven patients were kept on cyclosporine A (CsA); 13 were converted to everolimus (EVR). Measurements were made at the time of the switch from CsA to EVR, 8.21±6.36 months posttransplantation, and 21.2±6.57 months posttransplantation. In addition to laboratory and clinical parameters, Doppler indices and estimated glomerular filtration rate (eGFR) were measured. Results. After the switch from CsA to EVR, microvascular perfusion in the EVR-treated patients (A×β value at baseline 9.23±7.44 dB/sec, A×β value at time of follow-up 19.6±13.0 dB/sec, P=0.03) and the estimated GFR (81.2±20.3 and 96.9±22.6 mL/min, P=0.001) improved significantly. Microvascular perfusion (A×β 7.04±5.32 dB/sec and A×β 8.66±9.01 dB/sec, P=0.34) and the eGFR of the group continuing CsA treatment remained stable (78.5±25.9 and 73.2±37.3 mL/min, P=0.1). Conclusion. The study demonstrates that renal microperfusion visualized by CES based on microbubble contrast agent and concomitantly kidney function, improved significantly after the switch from CsA to EVR.

Cellular Effects of Everolimus and Sirolimus on Podocytes

Everolimus (EVL) and Sirolimus (SRL) are potent immunosuppressant agents belonging to the group of mammalian target of rapamycin (mTOR) inhibitors used to prevent transplant rejection. However, some patients develop proteinuria following a switch from a calcineurin inhibitor regimen to mTOR inhibitors. Whether different mTOR inhibitors show similar effects on podocytes is still unknown. To analyze this, human podocytes were incubated with different doses of EVL and SRL. After incubation with EVL or SRL, podocytes revealed a reduced expression of total mTOR. Phosphorylation of p70S6K and Akt was diminished, whereas pAkt expression was more reduced in the SRL group. In both groups actin cytoskeletal reorganization was increased. Synaptopodin and podocin expression was reduced as well as nephrin protein, particularly in the SRL group. NFκB activation and IL-6 levels were lower in EVL and SRL, and even lower in SRL. Apoptosis was more increased in SRL than in the EVL group. Our data suggests that mTOR inhibitors affect podocyte integrity with respect to podocyte proteins, cytoskeleton, inflammation, and apoptosis. Our study is the first to analyze both mTOR inhibitors, EVL and SRL, in parallel in podocytes. Partially, the impact of EVL and SRL on podocytes differs. Nevertheless, it still remains unclear whether these differences are of relevance regarding to proteinuria in transplant patients.

Uremia aggravates left ventricular remodeling after myocardial infarction.

Background: Renal failure is a well-established cardiovascular risk factor. We hypothesized that uremia negatively affects post-myocardial infarction (MI) remodeling and left ventricular (LV) function and examined the pathohistological correlations. Methods: Subtotally nephrectomized rats (SNX) and controls with MI only (MIC) were examined 1, 4 and 8 weeks after MI. MI size, ejection fraction (EF), cardiac fibrosis, vascular density and cardiomyocyte density were studied. Results: The extension of MI was 0.08 ± 0.02 in SNX versus 0.06 ± 0.02 in MIC rats (p < 0.031). Prior to MI, EF was comparable in SNX and MIC (74 ± 3 vs. 72 ± 2%, n.s.). Despite a relatively small infarct size EF in SNX decreased to 58 ± 4% 1 week after infarction and progressively worsened to 51 ± 4% after 8 weeks. In MIC animals EF only slightly decreased 1 week after MI (70 ± 3%) and remained unchanged at follow-up. In SNX animals LV end-diastolic diameter continuously increased following MI throughout the study period indicating accelerated remodeling. Furthermore, accelerated myocardial fibrosis was already notable 1 week after MI in SNX animals and the volume density of capillaries and cardiomyocytes was significantly lower in SNX rats. Conclusion: MI in experimental uremia is associated with progressive impairment of LV function, LV dilatation and accelerated myocardial fibrosis.