Burkhard Kreft

The kidney as a second site of human C-reactive protein formation in vivo.

C‐reactive protein (CRP) is the main acute phase reactant in humans. Its production is presumably restricted to the liver but extrahepatic expression by inflamed tissue has not been studied indetail. By real‐time PCR and immunohistochemistry we here show that renal cortical tubular epithelial cells (TEC) express CRP mRNA and protein within 6 h after stimulation with conditioned medium (CM) or IL‐6, but not IL‐1α or TNF‐α. Western blot analysis with monoclonal anti‐CRP antibody that recognizes native CRP revealed protein secretion into supernatants of CM‐stimulated TEC cultures. While hepatoma‐derived Hep3B cells could be induced similarly, peripheral blood mononuclear cells could not. CRP mRNA transcripts were observed in nephrectomized renal allografts with severe acute rejection but not with chronic allograft nephropathy (CAN). Of 19 needle biopsies of acutely rejecting kidney transplants, 15 demonstrated CRP mRNA production with the relative expression levels increasing with the severity of rejection. On the other hand, none of 7 graft biopsies with acute tubular necrosis (ATN) or CAN showed CRP mRNA expression. By using monoclonal anti‐CRP antibody, cortical tubules as well as glomerular cells were shown to locally express CRP in rejecting, but not in ATN kidneys. We conclude that inflamed kidneys represent a so far unknown site of CRP formation in vivo. These data shed new light on the acute phase reaction not merely representing a systemic inflammatory pathway but probably being part of the local immune response.

The impaired immune response to diphtheria vaccination in elderly chronic hemodialysis patients is related to zinc deficiency

Zinc deficiency causes abnormalities of the immuneresponse. In chronic hemodialysis therapyabnormalities in zinc metabolism as well as animpaired immune response to vaccination have beenreported. Therefore we performed a vaccination studyagainst diphtheria and hypothesized that the responseto diphtheria vaccination is related to serum zincdeficiency in hemodialysis patients. Serum zincconcentrations were assayed in 16 chronic hemodialysispatients (10 male, 6 female; mean age 65 years)without a documented vaccination history againstdiphtheria. Nine of these patients were tripleimmunized against diphtheria while seven received asingle vaccination. The response to diphtheriavaccination was measured by ELISA detecting specificantibodies to diphtheria-toxoid. Seroconversion 6 and12 months after vaccination was defined as thedoubling of antibody titers in patients ≥ 0.1IU/ml prior to vaccination or as titers %gt; 0.1 IU/mlin all other patients. Only 6/16 hemodialysispatients responded to immunization against diphtheriaby specific antibody production (> 0.1 IU/ml).Twelve months after the single injection 3/7 patientsseroconverted while six months after the triplevaccination 3/9 patients responded to immunization.This was not age-dependent, whereas in non-responderswe detected significantly decreased serum zinc levels.In contrast, responders showed similar serum zinclevels as age-matched controls. Furthermore, wemeasured a decreased α2-macroglobulinconcentration only in the responders amongst thehemodialysis patients. Protection against diphtheriaand the immune response to diphtheria vaccination inhemodialysis patients is poor. The failure to respondto active diphtheria vaccination is related to asignificantly decreased serum zinc concentration inhemodialysis patients.

Bactericidal Activity of Renal Tubular Cells: The Putative Role of Human β-Defensins

Renal tubular epithelial cells (RTC) form a barrier between the host and ascending microbes in upper urinary tract infection. Previous studies have shown the ability of the kidney to produce defensins – antimicrobial peptides that play a pivotal role in unspecific host defense. To further clarify the role of renal epithelium for direct antibacterial activity we investigated the expression, regulation and production of antimicrobial peptides by cultured human RTC. Cell culture supernatants of RTC exert strong bactericidal activity against Escherichia coli and Klebsiella pneumoniae, two of the most important pathogens in urinary tract infections. The antimicrobial effect depends on salt concentration, a typical feature of human defensins. RT-PCR of RNA from cultured proximal and distal RTC showed constitutive expression of human β-defensin 1 (hbd-1) and human β-defensin 2 (hbd-2) whereas only hbd-1 expression could be detected in RNA preparation from renal biopsy material. Hbd-2 expression of RTC was induced by inflammatory processes as shown by semiquantitative competitive RT-PCR. Coincubation of the cultured cells with IL-1α or E. coli promote the strongest hbd-2 induction whereas TNF-α and LPS lead to a weaker or no (IL-6) hbd-2 induction. This is the first evidence that human RTC are able to produce antibacterial substances in a biologically relevant amount and that β-defensins are candidate proteins responsible for this effect.

Interleukin-8 Secretion of Cortical Tubular Epithelial Cells Is Directed to the Basolateral Environment and Is Not Enhanced by Apical Exposure to Escherichia coli

ABSTRACT In upper urinary tract infections, tubular epithelial cells (TEC) may play a pivotal role in the initiation of the renal inflammatory response. They exert crucial immunological functions such as processing and presentation of foreign antigen, secretion of proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha) and chemokines (IL-8, MCP-1, ENA-78, and RANTES). Since monolayer cultures are a limited model for polarized tubular epithelial cells, we studied the side-dependent IL-8 secretion of TEC by using cell culture inserts as a basement membrane imitation. Primary cultures of proximal TEC were stimulated with differently fimbriated mutants of Escherichia coli, E. coli LPS, S-fimbria isolates, and IL-1α. IL-8 protein was measured by enzyme-linked immunosorbent assay, and IL-8-like biological activity was tested by measuring elastase release from polymorphonuclear cells in supernatants of the upper and lower compartments. IL-8 mRNA was compared by competitive PCR. IL-8 secretion by TEC into the basolateral environment was significantly higher than secretion into the apical compartment, representing the tubular lumen. However, stimulation of IL-8 secretion by TEC was restricted to IL-1α and was not inducible by E. coli mutants, S fimbriae, or lipopolysaccharide. With this in vitro model of polarized TEC, we show that luminal contact of TEC with uropathogenic E. coli does not result in enhanced IL-8 secretion. The basolaterally directed production of the neutrophil chemotactic factor IL-8 by TEC after stimulation with IL-1α might play an important role in the initiation of inflammatory cell influx into the renal parenchyma.

VEGF Production by Primary Human Renal Proximal Tubular Cells: Requirement of HIF-1, PI3-Kinase and MAPKK-1 Signaling

Renal proximal tubular epithelial cells (PTEC) respond to hypoxia exposure or interleukin-1β (IL-1β) treatment with increased vascular endothelial growth factor (VEGF) production. With respect to O2 deprivation, the hypoxia-inducible factor 1α/ β (HIF-1) is the most important transcription factor driving VEGF mRNA expression. HIF-1 is also activated by IL-1β and may thus be involved in the stimulation of VEGF production by this cytokine. However, the molecular mechanisms of HIF-1 dependent VEGF synthesis are poorly understood. Herein, human PTEC in primary culture were challenged by hypoxic incubation and/or IL-1β treatment in absence or presence of specific phosphatidylinositol 3-kinase (PI3K) or mitogen activated protein kinase kinase-1 (MAPKK-1) inhibitors for assay of VEGF protein, VEGF mRNA and detection of HIF-1α by Western Blotting, EMSA and fluorescence microscopy. In addition, the activities of PI3K and MAPKK-1 were studied following hypoxia and IL-1β treatment of the cultures. The study shows that PI3K but not MAPKK-1 inhibition resulted in the loss of hypoxic and IL-1β induced HIF-1α accumulation, whereas VEGF synthesis was reduced by either intervention. Thus, PI3K signaling is required for HIF-1α accumulation and VEGF synthesis, whereas MAPKK-1 signaling is required for VEGF synthesis only. Furthermore, hypoxia alone was sufficient to activate PI3K in PTEC in contrast to MAPKK-1, whose activity was lowered in hypoxia.

Darmbrand (Enteritis necroticans) Eine historische und aktuelle Übersicht

ZusammenfassungIn den Jahren 1946 bis 1948 trat in Lübeck und anderen Städten Norddeutschlands eine epidemisch verlaufende Infektion auf, die bis dahin nicht bekannt war. Die Erkrankung wurde aufgrund ihrer Morphologie als Darmbrand (Enteritis necroticans) bezeichnet.Untersuchungen ergaben, dass es sich bei dem Infektionserreger um Clostridium perfringens Typ C handelte. In der Folge konnten die Pathogenese der Enteritis necroticans und eine krankheitsauslösende Bedeutung des β-Toxins von Clostridium perfringens Typ C gesichert werden. Entsprechend konnte durch die Immunisierung gegen dieses Toxin die Inzidenz der Enteritis necroticans in Papua-Neuguinea, einem Land, in dem die Erkrankung in bestimmten Regionen noch heute endemisch ist, deutlich gesenkt werden. Wenngleich der Darmbrand in Europa eine kaum noch auftretende Erkrankung ist, so ist mit seinem epidemischen Auftreten besonders in Notstandsgebieten zu rechnen.Die vorliegende Arbeit soll an die in Lübeck aufgetretene Darmbrandepidemie erinnern, eine Darstellung der Epidemiologie, Pathogenese und Therapie der Erkrankung unter Berücksichtigung inzwischen gewonnener Erkenntnisse geben und andere durch Clostridium perfringens induzierte Erkrankungen abgrenzen.AbstractEnteritis necroticans, locally called “Darmbrand”, is a severe and life threatening infectious disease which was epidemic in Northern Germany after World War II. Darmbrand had a limited appearance, occurring only for a few years. In Lübeck many cases were diagnosed in 1946/1948 and the book “Darmbrand, Enteritis necroticans” was published in 1949 by clinicians and pathologists.Enteritis necroticans is also known as a tropical cause of bloody diarrhea and is caused by Clostridium perfringens Type C (type β-toxin). The disease is related to pig feasts in Papua New Guinea. Although necrotizing enterocolitis is now a rather rare disease we must be aware of the appearance of this fulminant entity.This paper represents a review on the historic and current aspects of enteritis necroticans and discusses the epidemiology, pathogenesis and treatment of this disease.

Measurement of Fluid Volume Shifts during Hemodialysis by A-Mode Ultrasonography

Background: The rate of intercompartmental fluid volume changes during hemodialysis (HD) is a major determinant of dialysis-induced hypotension and lacks direct monitoring. The aim of the study was to evaluate the feasibility of tissue thickness (TT) measurement in monitoring the mobilization of interstitial fluids during HD. Methods: We studied the intradialytic changes in forehead TT and inferior vena cava diameter (IVCD) in 20 patients. Plasma refilling was calculated from changes in hematocrit (Hct) and ultrafiltration rates. Results: During ultrafiltration of 2,437 ± 117 ml (mean ± SEM), Hct increased significantly from 27.9 ± 0.7 to 30.0 ± 0.9%. IVCD decreased significantly from 9.7 ± 0.2 to 6.1 ± 0.4 mm/m2. We found a simultaneously pronounced reduction in TT from 4.46 ± 0.12 to 3.78 ± 0.12 mm (≥15.3%) with a significant correlation to plasma refilling (0.613). Conclusion: Volume changes in the peripheral shell tissues during HD can be monitored directly and noninvasively by A-mode ultrasound.

Urodilatin and Pentoxifylline Prevent the Early Onset of Escherichia coli-Induced Acute Renal Failure in a Model of Isolated Perfused Rat Kidney

Background/Aims: Raised cytokine levels and a hypoperfusion-associated decrease in glomerular filtration rate (GFR) are hallmarks of the genesis of septic acute renal failure (ARF). Therefore, anti-inflammatory as well as renal vasodilating therapeutic strategies may afford renal protection during septic ARF. The present study was designed to determine the effects of administration of urodilatin, pentoxifylline and theophylline to improve renal function in an ex-vivo model of ‘septic renal injury’. Methods: Eight series of experiments were performed: no intervention, perfusion with a suspension containing Escherichia coli bacteria (strain 536/21); E. coli + 10 μg/l urodilatin, E. coli + 20 μg/l urodilatin, E. coli + 100 μM theophylline, E. coli + 100 μM pentoxifylline and E. coli + URO 20 μg/l given 90 min after start of perfusion. Renal vascular and glomerular functional parameters as well as TNF-α release were analyzed up to 180 min. Results: Perfusion with E. coli caused an acute deterioration of renal vascular and glomerular function. URO 20 μg/l and PTX decreased renal vascular resistance (RVR) from 83.7 ± 18.4 to 9.2 ± 1.1 and 8.6 ± 2.2 mm Hg/ml/min/g kidney and increased renal perfusion flow rate (PFR) from 8.2 ± 1.5 to 14.6 ± 0.8 and 14.1 ± 2.2 ml/min/g kidney. As a result, GFR improved from 102.1 ± 15.6 to 442 ± 48.3 and 525.8 ± 57 μl/min/g kidney during treatment with URO 20 μg/l and PTX, respectively. Renal TNF-α release was significantly reduced by URO 20 μg/l (from 178 ± 23 to 45.2 ± 2 and 47 ± 3 pg/ml) in the E. coli + URO 20 μg/l and by PTX in the E. coli + PTX group if added to the perfusion medium upon start of perfusion. Interestingly, URO 20 μg/l also decreased RVR significantly from 62.2 ± 6.1 to 35.9 ± 6.0 mm Hg/ml/min/g kidney, improved PFR from 5.4 ± 1.0 to 8.7 ± 1.0 ml/min/g kidney, increased GFR from 160 ± 43.3 to 280.7 ± 27.9 μl/min/g kidney, and decreased TNF-α release to 122 ± 18 pg/ml if applied 90 min after induction of septic ARF. In contrast, URO 10 μg/l did not significantly increase urine flow and did not appear to significantly improve renal perfusion. Theophylline showed no beneficial effects at all. Conclusion: This suggests that urodilatin and pentoxifylline might be useful to protect renal function if given before a septic renal insult. Additionally, treatment with urodilatin is capable of restoring renal function in early Gram-negative sepsis-induced ARF even if given after the septic insult.

Absence of Cytokine Response to Bacterial Challenge in Human Tubuloepithelial Cells

In acute bacterial renal infections, which are most frequently caused by Escherichia coli, tubuloepithelial cells are involved with respect to bacterial adherence, invasion and cytotoxicity. In addition, cytokines expressed by tubuloepithelial cells may be relevant for the recruitment of inflammatory cells and tissue damage in bacterial interstitial nephritis. We asked which inflammatory cytokines are produced by primary human tubuloepithelial cells following in vitro exposure to E. coli and found no release of IL-6, IL-8 and TNF-α by tubular cells challenged by bacteria. Purified virulence factors (fimbriae, lipopolysaccharide) from E. coli were also without effects on cytokine release by tubular cells. Since lymphocytic infiltration is a characteristic feature in the chronic form of interstitial nephritis, MHC class II expression by tubular cells in response to bacterial coincubation was analyzed. Exposure to both IFN-γ and E. coli enhanced MHC class II expression on tubuloepithelial cells. In conclusion, tubuloepithelial cells may play an active role in the local defense against bacteria, e.g. by expressing MHC class II molecules. However, in vitro inflammatory cytokines are not induced by E. coli in this cell population.