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Die Pravalenz von Frailty ist bei Patienten mit chronischer Niereninsuffizienz recht hoch und nimmt mit deren Schweregrad zu. Eine Reihe mit einer chronischen Niereninsuffizienz assoziierter Faktoren begunstigt offenbar das haufige Auftreten von Frailty in dieser Patientengruppe. Neben ihrem ungunstigen Einfluss auf Lebensqualitat, Morbiditat und Mortalitat ist Frailty ein bedeutsames Kriterium bei der Festlegung der Therapieziele bei einer chronischen Nierenerkrankung, bei der Entscheidung fur eine Dialysebehandlung oder fur eine konservative Behandlung ohne Dialyse und bei der Listung eines Patienten fur eine Nierentransplantation. Uber die Masnahmen zur Besserung der Auswirkungen von Frailty bei Patienten mit chronischer Niereninsuffizienz herrscht noch Unsicherheit. Je nach Stadium und Verlauf der Nierenerkrankung und den auseren Umstanden konnen diese recht unterschiedlich sein. Anhand eines geriatrischen Assessments, einschlieslich einer Sozialanamnese, lassen sich individuelle physische, psychische oder emotionale Probleme erkennen. Dieses bildet die Grundlage fur bedarfsgerechte Masnahmen wie beispielsweise Schulungen zum Verhalten bei Nierenerkrankung, Trainingstherapie und Rehabilitation. Von Letzterer scheinen Patienten mit Niereninsuffizienz in ahnlichem Mas zu profitieren wie Nierengesunde. So konnte die fruhzeitige Abklarung Frailty-assoziierter Probleme bei Patienten mit chronischer Niereninsuffizienz dazu beitragen, Defizite baldmoglichst zu erkennen und durch situationsgerecht angepasste Masnahmen eine Verbesserung von Lebensqualitat und/oder Prognose bei diesen Patienten zu erreichen.

Background: Severe hypertriglyceridemia (HTG) is associated with major complications such as acute or relapsing pancreatitis (AP) and atherosclerotic cardiovascular disease (ASCVD). Rapid elimination of triglyceride (TG)-rich lipoproteins (LP) with double filtration plasmapheresis (DFPP) without need for substitution has been found to be effective for the acute, short-term treatment of HTG-induced AP. Data on the long-term use of DFPP to prevent HTG-associated complications are scarce. Objectives: To evaluate the use and efficacy of regular DFPP treatment in clinical practice for preventing recurrence of HTG-associated complications in thera­py refractory patients. Methods: Retrospective multicenter study in patients with severe symptomatic drug and diet refractory HTG with regular DFPP treatment. Patients’ incidence of HTG-associated pancreatic or cardiovascular complications was compared before treatment and with regular DFPP treatment. Results: Ten patients (3 female) were identified with baseline maximal TG concentrations of 2,587–28,090 mg/dL (median 5,487 mg/dL; interquartile range [IQR] 4,340–12,636). The mean observation period was 3.9 ± 3.4 years before and 3.8 ± 3.0 years after commencement of DFPP. In 5 patients, severe HTG was related to chylomicronemia, 2 patients had familial partial lipodystrophy Dunnigan, and 1 patient had additional LP(a)-hyperlipoproteinemia. The main HTG-associated complication was recurrent AP in 8 patients, including 1 patient treated during pregnancy. Two patients presented severe progressive ASCVD. With long-term DFPP treatment, the annual rate of HTG-associa­ted pancreatic or cardiovascular complications declined from median 1.4 (IQR 0.7–2.6) to 0 (IQR 0.0–0.4; p < 0.005). The absolute number of events was reduced by 77%. In 6 patients (60%) episodes of AP did not occur, nor was progression of ASCVD detected clinically or by routine imaging techniques. DFPP was effective in the elimination of TG-rich LP from plasma, and was safe and well-tolerated. Conclusion: Long-term, regular DFPP treatment resulted in stabilization of patients with severe HTG and related recurrent AP or progression of ASCVD, who were refractory to conventional dietary and drug therapy.

Background Due to rising vascular comorbidities of patients undergoing dialysis, the prevalence of permanent hemodialysis catheters as hemodialysis access is increasing. However, infection is a major complication of these catheters. Therefore, identification of potential predicting risk factors leading to early infection related complications is valuable, in particular the significance the CRP (C-reactive protein)-value is of interest. Methods In this retrospective study 151 permanent hemodialysis catheters implanted in 130 patients were examined. The following data were collected at the time of catheter implantation: CRP-value, history of catheter-related infection, microbiological status, immunosuppression and diabetes mellitus. The primary outcomes were recorded over the 3 months following the implantation: catheter-related infection, days of hospital stay and death. Catheter removal or revision, rehospitalization and use of antibiotics were identified as secondary outcomes. Results We identified a total of 27 (17.9%) infections (systemic infection: 2.26 episodes/ 1000 catheter days, local infection: 0.6 episodes/ 1000 catheter days). The development of an infection was independent of the CRP-value (p = 0.66) as well as the presence of diabetes mellitus (p = 0.64) or immunosuppression (p = 0.71). Univariate analysis revealed that infection was more frequent in patients with MRSA-carriage (p

Mineralocorticoid receptor antagonists have beneficial effects on left ventricular remodeling, cardiac fibrosis, and arrhythmia in heart failure, but efficacy and safety in dialysis patients is less clear. We evaluated the effect of spironolactone on left ventricular mass (LVM), an independent predictor of all-cause and cardiovascular mortality, in hemodialysis patients. In this placebo-controlled, parallel-group trial, 97 hemodialysis patients (23% female; mean age 60.3 years) were randomized to spironolactone 50 mg once daily (n=50) or placebo (n=47). The primary efficacy endpoint was change in LVM index (LVMi) from baseline to 40 weeks as determined by cardiac magnetic resonance imaging. Safety endpoints were development of hyperkalemia and change in residual renal function. There was no significant change in LVMi in participants randomized to spironolactone compared to placebo (-2.86±11.87 vs. 0.41±10.84 g/m2). There was also no difference in the secondary outcomes of mean 24-hour systolic or diastolic ambulatory blood pressure, left ventricular ejection fraction, 6-minute walk test distance, or New York Heart Association functional class. Moderate hyperkalemia (pre-dialysis potassium levels of 6.0-6.5 mmol/L) was more frequent with spironolactone treatment (155 vs. 80 events), but severe hyperkalemia (≥6.5 mmol/L) was not (14 vs. 24 events). Changes in residual urine volume and measured glomerular filtration rate did not differ between groups. There were no deaths in the spironolactone group and 4 deaths in the placebo group. Thus, treatment with 50 mg spironolactone did not change left ventricular mass index, cardiac function, or blood pressure in hemodialysis patients. Spironolactone increased the frequency of moderate hyperkalemia, but did not increase severe hyperkalemia.

The prevalence of frailty is very high in patients with chronic renal insufficiency and increases with its severity. A number of factors associated with chronic renal insufficiency appear to favor the frequent occurrence of frailty in these patients. In addition to its unfavorable impact on the quality of life, morbidity and mortality, frailty is an important criterion in setting treatment goals for chronic kidney disease as well as in the decision whether to undergo dialysis treatment or conservative treatment without dialysis and also in listing a patient for a kidney transplantation. There is still uncertainty about the measures to improve the effects of frailty in patients with chronic kidney insufficiency. These can vary considerably depending on the stage and course of the kidney disease and external circumstances. Individual physical, psychological or emotional problems can be identified on the basis of a geriatric assessment including a social anamnesis. This forms the foundation for tailored measures, such as advice on how to behave when suffering from kidney disease, training therapy and rehabilitation. Patients with renal insufficiency seem to benefit from the latter to a similar extent as patients without kidney disease. Thus, an early assessment of frailty-associated problems in patients with chronic renal insufficiency could help to identify deficits as soon as possible and, by measures adapted to the situation, to achieve an improvement in the quality of life and/or prognosis of these patients.Die Prävalenz von Frailty ist bei Patienten mit chronischer Niereninsuffizienz recht hoch und nimmt mit deren Schweregrad zu. Eine Reihe mit einer chronischen Niereninsuffizienz assoziierter Faktoren begünstigt offenbar das häufige Auftreten von Frailty in dieser Patientengruppe. Neben ihrem ungünstigen Einfluss auf Lebensqualität, Morbidität und Mortalität ist Frailty ein bedeutsames Kriterium bei der Festlegung der Therapieziele bei einer chronischen Nierenerkrankung, bei der Entscheidung für eine Dialysebehandlung oder für eine konservative Behandlung ohne Dialyse und bei der Listung eines Patienten für eine Nierentransplantation. Über die Maßnahmen zur Besserung der Auswirkungen von Frailty bei Patienten mit chronischer Niereninsuffizienz herrscht noch Unsicherheit. Je nach Stadium und Verlauf der Nierenerkrankung und den äußeren Umständen können diese recht unterschiedlich sein. Anhand eines geriatrischen Assessments, einschließlich einer Sozialanamnese, lassen sich individuelle physische, psychische oder emotionale Probleme erkennen. Dieses bildet die Grundlage für bedarfsgerechte Maßnahmen wie beispielsweise Schulungen zum Verhalten bei Nierenerkrankung, Trainingstherapie und Rehabilitation. Von Letzterer scheinen Patienten mit Niereninsuffizienz in ähnlichem Maß zu profitieren wie Nierengesunde. So könnte die frühzeitige Abklärung Frailty-assoziierter Probleme bei Patienten mit chronischer Niereninsuffizienz dazu beitragen, Defizite baldmöglichst zu erkennen und durch situationsgerecht angepasste Maßnahmen eine Verbesserung von Lebensqualität und/oder Prognose bei diesen Patienten zu erreichen.

Was ist neu? Ursachen Bei der akuten Form der tubulointerstitiellen Nephritis (TIN) sind meist allergische Reaktionen auf Medikamente die Ursache, neben Antibiotika zunehmend auch Protonenpumpenhemmer. Bei den infektiösen Formen war zuletzt eine Zunahme von Hanta-Virus-Infektionen zu beobachten. Relativ neu beschrieben ist die IgG4-assoziierte Form. Diagnostik Eine definitive Diagnose der TIN ist nur mittels Nierenbiopsie möglich. Diese wird selten gewonnen, sodass von einer hohen Dunkelziffer auszugehen ist. Die IgG4-assoziierte Form weist spezifische histologische Veränderungen auf. Essenziell ist eine sorgfältige Anamnese. Laborchemisch typisch, aber wenig spezifisch ist ein Anstieg der Retentionsparameter bei geringgradiger Proteinurie und nicht glomerulärer Hämaturie. Definitiv gesicherte Marker für das Vorliegen einer TIN sind bislang nicht validiert. Therapie Die wichtigste therapeutische Maßnahme besteht im Ausschalten eines potenziellen Auslösers. Medikamentöse Therapie der ersten Wahl sind Glukokortikoide, obgleich die studienbasierte Evidenz sehr schmal ist und überwiegend auf älteren Daten beruht.

Bei der akuten Form der tubulointerstitiellen Nephritis (TIN) sind meist allergische Reaktionen auf Medikamente die Ursache, neben Antibiotika zunehmend auch Protonenpumpenhemmer. Bei den infektiösen Formen war zuletzt eine Zunahme von Hanta-Virus-Infektionen zu beobachten. Relativ neu beschrieben ist die IgG4-assoziierte Form.Eine definitive Diagnose der TIN ist nur mittels Nierenbiopsie möglich. Diese wird selten gewonnen, sodass von einer hohen Dunkelziffer auszugehen ist. Die IgG4-assoziierte Form weist spezifische histologische Veränderungen auf. Essenziell ist eine sorgfältige Anamnese. Laborchemisch typisch, aber wenig spezifisch ist ein Anstieg der Retentionsparameter bei geringgradiger Proteinurie und nicht glomerulärer Hämaturie. Definitiv gesicherte Marker für das Vorliegen einer TIN sind bislang nicht validiert.Die wichtigste therapeutische Maßnahme besteht im Ausschalten eines potenziellen Auslösers. Medikamentöse Therapie der ersten Wahl sind Glukokortikoide, obgleich die studienbasierte Evidenz sehr schmal ist und überwiegend auf älteren Daten beruht.

Detection of renal artery stenosis (RAS) using Doppler is difficult to evaluate, particularly under conditions such as bilateral RAS or difficultly accessible renal arteries (RA). The objective of the present study was to assess the utility of splenic arterial compared to renal flow as an additional parameter in the Doppler evaluation of RAS. The difference between the resistive indices (RI) determined in renal and splenic parenchymal arteries (ΔRI(K−S)) was evaluated in 181 hypertensive subjects without any evidence of RAS. Subsequently 47 RA in 24 patients with suspected RAS were angiographically assessed. A ΔRI(K−S) of 0.055 (median) was determined in the population without any evidence of RAS similar to RA with angiographically excluded stenosis (ΔRI(K−S) 0.068). In contrast, in angiographic proven RAS, ΔRI(K−S) was significantly lower (−0.050; P

ObjectiveThrombophilic risk factors (TRFs) occur rather frequently in hemodialysis (HD) patients. However, little is known about their significance in HD patients, besides their potential impact on arteriovenous (AV) access failure, with varying results. We examined the effects of a wide variety of TRFs on both early AV fistula occlusion and survival among HD patients in long-term follow-up.MethodsIn this single-center, observational study, 70 consecutive HD patients from our dialysis center were examined with respect to shunt occlusion within the first 2 years after fistula creation and patient survival in a long-term follow-up (at least 16 years). We examined the presence of factor V, prothrombin, and MTHFR mutations using real-time fluorescence polymerase chain reaction. Furthermore, antithrombin (AT), protein C, protein S, and antiphospholipid antibodies (APL-Abs) were assessed.ResultsAmong the 70 patients, 32 had MTHFR mutations, 10 had heterozygous factor V Leiden mutations, 4 had prothrombin mutations, 4 had protein S deficiency, 2 had protein C deficiency, 9 had AT deficiency, and 14 had APL-Abs. 40 patients had shunt occlusion. TRFs were associated with a significantly increased risk for shunt thrombosis (PConclusionsTRFs in hemodialysis patients have a strong impact on patient survival and early AV fistula failure; however, patient survival is not significantly affected by early shunt occlusion.

The mechanisms underlying the impaired immune response in hemodialysis (HD) patients are not completely understood. The α-defensins human neutrophil peptides-1, 2, and 3 are low molecular weight peptides with antimicrobial activity and important effector molecules of innate immune responses. We now examined the expression of these peptides in HD patients. Seventy-six patients on chronic HD treatment (mean time on HD 5.8 years; mean age 70 years) were studied and compared with 38 healthy volunteers and 20 patients with infections and normal renal function. Expression of α-defensins was analyzed semiquantitatively in leukocytes on the messenger RNA (mRNA) level by reverse transcriptase polymerase chain reaction; the α-defensin protein levels in serum were detected by enzyme-linked immunosorbent assay. α-Defensin concentrations (140 ± 10.5 ng/mL; mean ± standard error of the mean) as well as mRNA levels in leukocytes (82.9 ± 7.9 arbitrary units [a.u.]) in HD patients were not significantly different from those in healthy volunteers (156 ± 15.2 ng/mL; 81.4 ± 11.3 a.u.). Defensin levels were independent of the time of the patient on HD and their age. During infection periods (mean increase of the C-reactive protein to 161 ± 17.3 mg/L), defensin serum levels increased to 321 ± 65 ng/mL (P < 0.005) and mRNA expression in leukocytes to 159 ± 19.2 a.u. (P < 0.05). These increases were not significantly different from those in patients with normal renal function (298 ± 46.8 ng/mL and 128 ± 9.1 a.u., respectively) suffering from infections (C-reactive protein 222 ± 26.6 mg/L). Our results suggest that the impaired immune defense in dialysis patients is not due to a deficiency in α-defensins in these patients as neither basal levels nor expression during infections were reduced compared with subjects with normal renal function.

The risk of infection in patients with rheumatic diseases is elevated, but a clear marker to differentiate the cause of the systemic inflammation is missing. We assessed the ability urinary immunoglobulin free light chains (FLCs) to indicate the presence of infection in patients with rheumatic disease. We performed a retrospective analysis of patients with rheumatic disease attending the Georg-August University Hospital in Goettingen, Germany, from January 2011 to December 2013. Subjects were included if they had urine levels of κ and λ FLCs available. A reference group of patients without autoimmune disease, but with documented infection, was constructed. A total of 1500 patients had their urinary FLCs quantified during the study period. Of the 382 patients with rheumatic disease, 172 (45%) displayed no systemic inflammation, 162 (42%) had inflammation due to the underlying disease activity, and 48 (13%) had inflammation due to a confirmed infection. Urinary FLC concentrations were much higher in patients with rheumatic diseases and infection (κ 68.8 ± 81.8 mg/L, λ 31.4 ± 53.5 mg/L) compared to those with inflammation due to rheumatic disease activity (κ 22.7 ± 26.3 mg/L, λ 8.1 ± 9.1 mg/L, κ p

In patients with rheumatic diseases, reliable markers for determining disease activity are scarce. One potential parameter is the level of immunoglobulin free light chains (FLCs), which is known to be elevated in the blood of patients with certain rheumatic diseases. Few studies have quantified FLCs in urine, a convenient source of test sample, in patients with different rheumatic diseases. We carried out a retrospective analysis of patients with rheumatic disease attending the University hospital of Goettingen, Germany. Subjects were included if they had urine levels of both κ and λ FLCs available and did not have myeloma. Data regarding systemic inflammation and kidney function were recorded, and FLC levels were correlated with inflammatory markers. Of the 382 patients with rheumatic disease, 40.1 % had chronic polyarthritis, 21.2 % connective tissue disease, 18.6 % spondyloarthritis and 15.7 % vasculitis. Elevated levels of κ FLCs were found for 84 % of patients and elevated λ for 52.7 %. For the patients with rheumatoid arthritis, FLCs correlated with C-reactive protein (κ, r = 0.368, p

Background: Angiotensin II (ANG II) and advanced glycation end products (AGEs) exert genotoxic effects in vitro which were prevented by the ANG II type 1 (AT1) receptor blocker, candesartan. In end-stage renal disease (ESRD) the incidence of genomic damage is increased. A stimulation of the renin-angiotensin system and accumulation of AGEs could be involved. Methods: We tested whether oral co-administration of candesartan modulates enhanced DNA damage in ESRD patients. Fifteen maintenance hemodialysis (MHD) patients with mild hypertension were treated with candesartan for 4.5 months. Fourteen MHD patients served as conventionally treated uremic controls. DNA damage was measured as micronucleus frequency (MNF) in peripheral blood lymphocytes and evaluated three times before candesartan therapy and afterwards every 6 weeks. Results: Compared to 14 healthy controls, MNF at baseline was significantly elevated in MHD patients. While in the conventionally treated MHD patients the enhanced DNA damage persisted, the co-administration of candesartan ameliorated the genomic damage significantly and independently of blood pressure changes. Conclusion: Blockade of AT1 receptors with candesartan can reduce DNA damage in MHD patients. Long-term studies in larger patient groups are needed to investigate whether the improved genomic damage lowers atherosclerotic complications and cancer development.

We retrospectively analyzed 10 906 lipid apheresis sessions (heparin-induced lipoprotein precipitation, direct adsorption of lipoproteins, double filtration plasmapheresis, dextran sulfate adsorption, and immunoadsorption) in 38 patients who were consecutively treated in our department during the last 20 years. The incidences of major cardiovascular events (MACE) (death, cerebrovascular accident, myocardial infarction, limb amputation, and renal vascular involvement) were taken separately as primary end-points or as a combined end-point. The time-course of secondary end-points (coronary and extracranial status of arteries, left ventricular function, occlusive artery disease, and calculated glomerular filtration rate [cGFR]) were also evaluated, as well as the extent of the reduction in plasma lipids and lipoproteins and the incidence of therapy associated side-effects. MACE decreased from 7.02% events per patient per year at the start of lipid apheresis to 1.17% during lipid apheresis and the rate of myocardial revascularization decreased from 22.8% to 3.8% per patient per year. Classical (diabetes mellitus, arterial hypertension, and smoking history), as well as novel risk factors (cGFR < 60 mL/min, statin withdrawal, mixed hyperlipoproteinemia, and elevated lipoprotein (a)) were associated with an elevated risk for MACE. All applied methods had comparable effects. All lipid apheresis methods proved to be safe and suitable for long-term treatment. The present data demonstrate that treatment with lipid apheresis is very effective and leads to long-term reduction in cardiovascular mortality and morbidity.

The occurrence of microalbuminuria or albuminuria indicates a disturbance of the barrier function of endothelial cells, basement membrane or of a structural-renal disease (including diseased podocytes). The prevalence of microalbuminuria in the general population is about 8 %, however, in high risk groups, prevalence rates of 50 % and more have been observed. Its incidence is strongly associated with increased cardiovascular morbidity and mortality. Blood pressure control and the blockade of the renin-angiotensin-aldosteron-system (RAAS), respectively, is the central mechanism to reduce cardio-vascular-renal end points as well as mortality.

ZusammenfassungDie renale Osteopathie stellt eine schwierige Herausforderung bei der Behandlung niereninsuffizienter Patienten dar. Sie bildet keine einheitliche Entität. Man unterscheidet High- und Low-Turnover-Osteopathien sowie gemischte Formen. Die Prophylaxe und Therapie dieser Erkrankung ist auf diese pathogenetisch unterschiedlichen Formen abzustimmen. Darüber hinaus ist ein erhöhtes Serum-Kalzium-Phosphat-Produkt als Risikofaktor für eine verstärkte Kalzifikation von Gefäßen zu berücksichtigen. Wesentliche La-borparameter im Rahmen der Steuerung der Therapie sind Serum-Phosphat und -Kalzium sowie der Parathormon-Spiegel. Für deren Zielwerte in Abhängigkeit der Nierenfunktion geben die so genannten K-DOQI Richtlinien einen Anhalt. Das therapeutische Spektrum wird durch neue Präparate wie neue Vitamin-D-Analoga, Kalzimimetika und Phosphatbinder wie Lanthankarbonat erweitert.

Background Little is known about sorbitol metabolism in renal papillary interstitial cells. For characterization we studied regulation of sorbitol synthesis by aldose reductase (AR) and degradation by sorbitol dehydrogenase (SDH) in papillary interstitial cells. Methods Interstitial cells were isolated from rat renal inner medulla to a pure cell fraction. mRNA was isolated from cultivated cells and sorbitol, AR and SDH activity were determined enzymatically in homogenates. Results Sorbitol concentration in these cells at 300 mosmol/l was 4.4+/-0.3 vs 78+/-3.6 micro mol/g protein at 600 mosmol/l. At steady-state conditions at 300 mosmol/l, AR activity was nearly the same as SDH activity (15.1+/-1.6 vs 16.6+/-2.0 U/g protein). At 600 mosmol/l, AR activity increased to 82.5+/-11.4 U/g protein and SDH activity to 31.5+/-6.0 U/g protein. Studying the time course of enzyme activity after changing osmolarity from 300 to 600 mosmol/l, we found half maximal stimulation after 2-3 (AR) or 3 (SDH) days. The amount of AR-mRNA preceded the rise of enzyme activity, whereas SDH-mRNA was not significantly influenced. Lowering osmolarity from 600 to 300 mosmol/l, enzyme activity decreased to less than half within 2 (AR) or 1 (SDH) day(s). Conclusions The results suggest that sorbitol metabolism contributes to handling of osmotic stress in rat renal papillary interstitial cells.

A novel model to study renal myofibroblast formation in vitro. Background In chronic renal disease, the decrease of excretory renal function closely correlates with the extent of interstitial fibrosis. A common feature of interstitial fibrosis is the occurrence of myofibroblasts, which are regarded as the predominant cells in matrix synthesis. We studied the transformation of renal fibroblasts into myofibroblasts in vitro as a model to elucidate the mechanisms underlying this process. Methods Primary cultures of freshly isolated rat inner medullary fibroblasts were established as reported previously. mRNA expression of myofibroblast markers and interstitial collagens was examined by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Phenotypic expression was investigated by immunolabeling. Endogenous transforming growth factor-β (TGF-β) production was inhibited by addition of a neutralizing antibody or by TGF-β1 antisense oligodeoxynucleotides (ODNs). Results Initially and during the first 36 hours, primary culture cells expressed neither α-smooth muscle actin nor desmin mRNA. From day 2 of primary culture, we observed a strong increase in these mRNAs, as evaluated by RT-PCR, followed by the phenotypic expression of these myofibroblast markers. Collagen type I mRNA was first detectable from day 4 of primary culture and showed a strong increase in its expression level during the following days, with phenotypic expression predominantly in myofibroblasts. The transformation rate of fibroblasts to myofibroblasts largely decreased in cocultures with collecting duct cells. This effect could be reversed by reducing the seeding density. We examined in this system the effect of TGF-β1 to define further its putative fibrogenic activity. However, neither the addition of exogenous TGF-β1 nor the inhibition of endogenous TGF-β production showed any significant effect on fibroblast transformation, suggesting that this cytokine exerts its effects at other levels or requires a cofactor. Conclusions We present a novel model to examine the de novo expression of myofibroblast markers and collagen type I in freshly isolated fibroblasts under defined conditions in primary culture. This model could provide a strategy for the molecular characterization of myofibroblast formation and the phenomena associated with this process.

Microscopic examination of urinary sediment is an integral component in the evaluation of nephropathies. However, identification and differentiation of the nucleated nonsquamous cells in urine is often difficult using such conventional techniques as phase contrast or bright field microscopy, even after Papanicolaou staining, and requires a lot of experience. We now report a method to differentiate urinary cell types using lectin staining. Twenty-five lectins were examined with respect to their binding pattern on cryosections of the human kidney and urinary tract, as well as binding to blood cells. The specificity of lectin binding to a cell type both in situ and in urine was confirmed by double labeling with specific antibodies directed against various sections of the nephron or nucleated blood cells. For urine cytologic examinations, acetone-fixed cytopreparations of urinary sediments were incubated with a combination of a fluorescein isothiocyanate (FITC)-coupled and a rhodamine-coupled lectin, followed by staining of the nuclei with 4',6-diamidino-2-phenylindole. Specimens were examined in triple immunofluorescence (FITC/rhodamine/UV). Cell types could be identified by their characteristic lectin-binding pattern. For example, the lectin combination of Sophora japonica agglutinin (aggl; SJA) and Erythrina cristagalli aggl (ECA) permitted a differentiation between cells of the proximal tubules (SJA positive [SJA+], ECA+), distal tubules (SJA negative [SJA-], ECA+), collecting ducts (SJA+, ECA-), and lymphocytes (SJA-, ECA-). In preliminary studies, examination of urinary sediment in various chronic nephropathies by this technique showed differences in their cellular excretion pattern. In summary, staining urinary sediments with combinations of lectins provides a rapid and relatively inexpensive method for a facilitated and reliable differentiation of the various nucleated cell types in urine.

To better characterize loop diuretic-sensitive ion fluxes in the inner medullary collecting duct (IMCD) we examined them in IMCD cells grown as a primary culture on permeable supports. A polarization of the cells with their basolateral side to the support was confirmed morphologically by electron microscopy and functionally by flux studies with ouabain. Within 7 days cells developed a transepithelial resistance of 974+/-52 omega per cm2 and a low transepithelial potential difference (-0.7+/-0.8 mV). Measurements of intracellular ion content by electron probe microanalysis in IMCD depleted of intracellular ions by preincubation in a Na(+)-K(+)-Cl(-)-free medium revealed, compared to the control receiving solvent, significant reductions in intracellular Na+ content (-17.6% within 10 min) and intracellular Cl- content (-43.8% within 30 min) by the addition of bumetanide (10(-4) mol/l) to the apical but not basolateral incubation medium. In 22Na+ and 86Rb+ isotope uptake studies, fluxes from the apical side were significantly inhibited at bumetanide concentrations of 100 micromol/l by 0.27+/-0.10 and 0.21+/-0.04 nmol/cm2 in 10 min, respectively, whereas basolateral fluxes of 86Rb+ but not 22Na+ were significantly reduced by this substance. Removal of Cl- had a similar but not additional effect. mRNA encoding the apical isoform of the Na+2Cl(-)K+ cotransporter could be specifically amplified by reverse transcriptase polymerase chain reaction from the inner medulla and highly purified IMCD cells. Northern blot of mRNA isolated from the inner medulla with a riboprobe of the apical isoform revealed a transcript of approximately 4.9 kb. This probe localized under "low-stringency" conditions to the IMCD in in situ hybridization studies. These results suggest the presence of an apically localized isoform of bumetanide-sensitive Na+2Cl(-)K+ cotransport in at least a subfraction of IMCD cells. This transport may be involved in the ultimate adjustment of urinary electrolyte concentration by this final segment of the tubular system.

Conditions for the highly specific selection of a cell type by the use of lectin-coated magnetic beads are reported for the isolation of inner medullary collecting duct (IMCD) cells from a heterogeneous inner medullary cell suspension, containing both single cells and tubular fragments of variable size. The lectin Dolichos Biflorus Agglutinin (DBA), which binds in rat inner medulla exclusively to IMCD cells, was coupled via the avidin-biotin system to beads. By isolating DBA-bead-IMCD cells in a magnetic field (positive selection) from a suspension containing about 50% IMCD, a fraction of 98 ± 1% purity was obtained; recovery of cells was up to 90%. Suspensions negative on reverse-transcriptase polymerase chain reaction for vimentin as a marker of contaminating interstitial and vascular cells could be received by repeating this procedure and additional trypsinization. On the other hand, it was possible to reduce the portion of IMCD cells in the suspension by one isolation step to 1.5 ± 0.9% (negative selection). Performing this step twice resulted in virtually pure suspensions. No significant effects of this isolation technique on cell viability, growth characteristics, and biochemical parameters were observed. Therefore, this method appears to be a powerful tool for the highly specific separation of heterogeneous cell populations.

To further characterize cells of the lower portion of the thin limb of Henle (TLHlp) under defined conditions in vitro, we developed a technique to enrich this cell population in suspension. TLHlpcells were isolated by enzymatic digestion of rat inner medulla, elimination of collecting ducts by lectin-coated beads, and differential centrifugation. Immunohistochemical staining of primary cultures of TLHlpcells with various markers revealed the preparations to be >90% pure. The hormonal stimulation pattern of PGE2and cAMP production by arginine vasopressin, angiotensin II, and dopamine in the isolated cells also argued against significant contamination by other cell types. Staining with an antibody against the aquaporin-1 water channel showed the distribution of cells from the ascending and descending limbs to be approximately equal in the isolated population. This technique allows the enrichment of cells from the lower portion of the thin limb of Henle in suspension to a very high degree of purity with the option to start primary cultures. Because these segments of the tubular system in particular are relatively inaccessible for microdissection, the presented method renders the possibility of addressing new questions regarding these tubular segments under defined conditions in vitro.

Normal rat and human kidney was mapped immunohistochemically with human monoclonal cold agglutinins against the biochemically related erythrocyte glycoconjugate antigens I and i, which represent branched and linear polylactosamines, respectively. The antibodies worked well with both cryostat and paraffin-embedded material. Anti-i gave specific staining of collecting duct cells (mostly principal cells) as evidenced by double labelling with antibodies to band 3 and a 23 kD protein of intercalated cells. In contrast, anti-I turned out to be the first exclusive marker for the ascending and descending thin limb of Henle's loop. In addition, in rat kidney, the binding of anti-I/i was preserved in primary cultures both of principal cells of the papillary collecting duct and cells of the thin limb of Henle's loop. Our results suggest that both antibodies might be interesting tools for in vitro studies of renal cell physiology and the investigation of kidney development.

Background. Urine cytology, although considered a valuable diagnostic tool in the monitoring of kidney graft function, is hampered by difficulty in differentiating the nucleated non-squamous cells in urine using conventional techniques. We have now developed a method for the simple identification of urinary cell types by lectin staining. Methods. Acetone-fixed cytopreparations of urinary sediments were incubated with the lectin combination Sophora Japonica agglutinin (SJA; rhodaminelabelled) and Erythrina cristagalli agglutinin (ECA; fluorescein isothiocyanate (FITC)-labelled) for 15 min, followed by staining of the nuclei with 49,6-diamidino2-phenylindole (DAPI). The courses of 38 patients were serially monitored after kidney transplantation during the period in hospital. Results. Nucleated urinary cell types could be easily identified from one specimen by their characteristic lectin-binding pattern using triple-immunofluorescence microscopy (FITCurhodamineuultra violet), permitting a differentiation between proximal (SJAquECAq) and distal tubules (SJAuECAq), collecting ducts (SJAquECA) and lymphocytes (SJAuECA). Stable graft function was characterized by low numbers of lymphocytes, tubular cells and urothelia. During rejection episodes, but not graft dysfunction unrelated to rejection, urinary excretion of lymphocytes as well as of distal tubular cells (from 1.0 to 6.0 and from 1.4 to 4.0 per 10 high-power fields, respectively) increased significantly up to 3 days prior to clinical diagnosis. Conclusions. Lectin staining facilitates unambiguous differentiation of the urinary cell types, in particular the various tubular epithelial cells, which are otherwise difficult to identify. This technique provides a rapid and easily applicable tool to evaluate the significance of the respective cell types in the monitoring of kidney graft function.

Soluble guanylyl cyclase (sGC) catalyzes the biosynthesis of cGMP in response to binding of L-arginine-derived nitric oxide (NO). Functionally, the NO-sGC-cGMP signaling pathway in kidney and liver has been associated with regional hemodynamics and the regulation of glomerular parameters. The distribution of the ubiquitous sGC isoform alpha 1 beta 1 sGC was studied with a novel, highly specific antibody against the beta 1 subunit. In parallel, the presence of mRNA encoding both subunits was investigated by using in situ hybridization and reverse transcription-PCR assays. The NO-induced, sGC-dependent accumulation of cGMP in cytosolic extracts of tissues and cells was measured in vitro. Renal glomerular arterioles, including the renin-producing granular cells, mesangium, and descending vasa recta, as well as cortical and medullary interstitial fibroblasts, expressed sGC. Stimulation of isolated mesangial cells, renal fibroblasts, and hepatic Ito cells with a NO donor resulted in markedly increased cytosolic cGMP levels. This assessment of sGC expression and activity in vascular and interstitial cells of kidney and liver may have implications for understanding the role of local cGMP signaling cascades.

The interstitial cells in the kidney are not a homogeneous cell population but consist of different cell types like fibroblasts, dendritic cells or lymphocyte-like cells. Fibroblasts are the most abundant interstitial cell type. They are regarded as the most important cells for the production and degradation of extracellular matrix and are assumed to play a pivotal role in renal interstitial fibrosis, which correlates directly with the decrease in excretory renal function. Renal fibroblasts also have endocrine activity: cortical fibroblasts are supposed to synthesize erythropoetin, and inner medullary fibroblasts are involved in the regulation of water and electrolyte homeostasis. A powerful tool for the further elucidation of fibroblast function are studies on cultured cells. Different techniques for the isolation of fibroblasts have been reported, including the cultivation of fibroblasts from outgrowths of minced tissue and the selective removal of contaminating epithelial cells by various methods. Several aspects have to be considered while culturing fibroblasts. Fibroblasts in culture exhibit distinct morphologic and biochemical features depending on their site of origin, state of differentiation and culture conditions. Their identification in culture exclusively by morphological criteria is therefore critical especially in mixed cultures with other cell types. Unfortunately, a constitutively expressed, specific marker for all fibroblasts is still not available. Since myofibroblast formation is considered as a key event in renal interstitial fibrosis, the transformation of fibroblasts to myofibroblasts is of special interest. Studies on cultured fibroblasts provide an effective tool to examine factors that affect this transformation and regulate the production and degradation of extracellular matrix. In addition, this technique can be used for further characterization of the endocrine activity of cultured fibroblasts. A better understanding of the biology of fibroblasts is essential to develop therapeutic strategies for the treatment of renal tubulointerstitial fibrosis, the pathologic equivalent of progressive renal failure.

A method was developed to measure the element content of freshly isolated papillary collecting duct (PCD) cells by electron probe microanalysis in a scanning electron microscope. After isolation, the cells were transferred onto a Thermanox support by centrifugation and the extracellular medium was removed by brief exposure to buffered ammonium acetate; cryofixation, freeze-drying, and coating with carbon followed. Under visual control in the scanning electron microscope the Na, Cl, K and P content of cell clusters (about 30 cells/cluster) was then measured by X-ray microanalysis. Cells incubated in control medium showed potassium:sodium ratios identical to those determined previously in cryosections of the same cells. In ouabain-treated cells sodium influx and potassium efflux was demonstrated. Potassium left the cells with a t1/2 of 21.7 min. The t1/2 of Na influx was 12.6 min for the first 15 min of incubation, whereafter further influx was markedly slower. Ouabain-induced sodium influx was inhibited 40% by amiloride. These results indicate that X-ray microanalysis can be applied to analyze the ion content of isolated cell clusters derived from the papillary collecting duct. Using ouabain and amiloride as inhibitors the suitability of the method to identify transport systems is demonstrated.

Transformation of rat inner medullary fibroblasts to myofibroblasts in vitro. Renal fibroblasts play a major role in the pathogenesis of renal interstitial fibrosis. This process is associated at least in some forms of interstitial fibrosis with a differentiation of fibroblasts into myofibroblasts, characterized by the de novo expression of α-smooth muscle (α-sm) actin and/or desmin. Both the mechanisms underlying this differentiation and their effects on cellular function are poorly understood. In vitro studies are difficult since the phenotypes of fibroblasts in culture have as yet not been well defined. We have, therefore, examined the phenotype of inner medullary fibroblasts (IMF) during the transition from in vivo to in vitro in various cell fractions derived from the inner medulla of healthy rats. IMF were positive for the lectin BSL-1 and negative for markers of endothelial cells. IMF first lost their prominent lipid droplets in vitro. Subsequently they developed cytoplasmic processes accompanied by a decrease in their reactivity for the lectin BSL-1 from strong to weak. From day 3 in primary culture, exclusively these weakly positive BSL-1 cells showed a de novo expression of α-sm actin (day 4 of primary culture, 75 ± 4% day 20, 94 ± 2%) and desmin (day 4, 43 ± 8% day 20, 66 ± 6%), classifying them as myofibroblasts. This transformation depended on culture conditions. In a mixed coculture with inner medullary collecting duct (IMCD) cells the transformation of IMF was largely absent: a significantly greater number of strong BSL-1 positive cells contained prominent lipid droplets (39 ± 4 vs. 19 ± 4%, P < 0.05) on day 4 of primary culture, and the transition of strongly to weakly positive BSL-1 IMF was almost completely blocked. By reducing the seeding density of IMCD cells the effect of this condition on IMF transformation could be largely abolished. This first detailed pheno-typic characterization of rat fibroblasts during the transition from in vivo to in vitro demonstrates that these cells-depending on culture conditions-differentiate to myofibroblasts within a few days of primary culture and that subcultured IMF exhibit predominantly this phenotype. The presented model may serve as a useful tool for the in vitro study of myofibroblast formation and the consequences of such a differentiation for the physiological functions of IMF.