Lüder M. Fels

A randomized trial comparing the nephrotoxicity of cisplatin/ifosfamide-based combination chemotherapy with or without amifostine in patients with solid tumors

This study evaluates the degree of kidney damageduring cisplatin/ifosfamide-based combinationchemotherapy and its possible prevention byamifostine. Thirty-one patients with solid tumorsstratified according to pretreatment were randomizedto receive VIP- or TIP-chemotherapy with or withoutamifostine (910 mg/m2) given as a short infusion priorto cisplatin. Chemotherapy consisted of cisplatin(50 mg/m2), ifosfamide (4 g/m2) and either etoposide(500 mg/m2) (= VIP) or paclitaxel (175 mg/m2) (= TIP)repeated at 3 weekly intervals. For all patients theglomerular filtration rate (GFR) measured bycreatinine-clearance, serum creatinine, electrolytesand differential urinary protein/enzyme excretion weredetermined prior to, during and after each cycle. Atotal of 62 cycles of chemotherapy were evaluable. Inthe amifostine-group GFR was fully maintained afterapplication of two cycles of chemotherapy, whereas inthe control group a > 30%-reduction of median GFR(108 to 80 ml/min) was observed (p < 0.001). Patientsreceiving amifostine had a lower degree of highmolecular weight proteins excretion indicating lessglomerular damage. In both groups significantincreases of tubular marker profiles peaking at day 3after chemotherapy were observed with a nearlycomplete reversibility of these changes prior to thenext chemotherapy cycle. The number of patients withlow magnesium serum levels during treatment was 17%after amifostine application versus 69% in controlpatients. The results seem to indicate that treatmentwith amifostine can preserve GFR after application oftwo cisplatin/ifosfamide-based chemotherapy cycles.This may be advantageous if repetitive cycles ofchemotherapy or subsequent administration of high dosechemotherapy is planned.

The use of reduced doses of amifostine to ameliorate nephrotoxicity of cisplatin/ifosfamide-based chemotherapy in patients with solid tumors.

This study evaluates the degree of kidney damage during cisplatin/ifosfamide-based combination chemotherapy and its possible prevention by amifostine. Thirty-one patients with solid tumors stratified according to pretreatment were randomized to receive cisplatin/ifosfamide-based chemotherapy with or without amifostine (1000 mg absolute) given as a short infusion prior to cisplatin. Chemotherapy consisted of cisplatin (50 mg/m2), ifosfamide (4 g/m2) and either etoposide (500 mg/m2) (VIP regimen) or paclitaxel (175 mg/m2) (TIP regimen) repeated at 3 weekly intervals. For all patients the glomerular filtration rate (GFR) measured by creatinine clearance, serum creatinine, electrolytes and differential urinary protein excretion were determined prior to, during and after each treatment cycle. A total of 62 cycles of chemotherapy were evaluable. In the amifostine arm the GFR was almost completely maintained after application of two cycles of chemotherapy (121 to 108 ml/min), whereas in the control group a 30% reduction of the GFR (105 to 80 ml/min) was observed. In both groups marked increases of glomerular and tubular marker profiles peaking at day 3 after chemotherapy were found with a nearly complete reversibility of these changes prior to the next chemotherapy cycle. Patients receiving amifostine had a lower degree of hypomagnesemia, as well as a lower urinary excretion of N-acetyl-glucosaminidase and albumin, indicating less tubular damage compared to the control patients. Treatment with 1000 mg amifostine resulted in an almost complete preservation of GFR. This corresponded to a slightly reduced excretion of tubular marker proteins and a lower incidence of hypomagnesemia during chemotherapy in amifostine patients compared to controls. This dose of amifostine may be sufficient for nephroprotection in patients without pre-existing risk factors for renal damage who undergo a restricted number of chemotherapy cycles.

Risk Assessment of Nephrotoxicity of Cadmium

Cadmium as an environmental or occupational toxin has been well studied. Exposure can fairly easily be detected by analysis of cadmium in biological material. Different routes of cadmium uptake, such as via airborne particles, cigarette smoke or contaminated food, have been identified. Urinary concentrations associated with renal effects vary, depending on urinary marker of effect, and are as low as 2 micrograms cadmium/g creatinine. Different segments of the nephron are affected by cadmium. Urinary enzymes and serum derived proteins, mainly low-molecular weight proteins, as well as eicosanoids or glycosaminoglycans are among the tubular or glomerular markers of effect. Predisposing factors, such as smoking, dietary habits and low body iron stores have been described. It is concluded that even for low level cadmium exposure effects can be detected with sensitive diagnostic tools.

Evaluation of late nephrotoxicity in long-term survivors of Hodgkin's disease

Chemotherapy (Ctx) and/or radiotherapy (Rtx) are effective in the treatment of Hodgkins disease (HD) but potentially involve late toxicities, including nephrotoxic side effects. Therefore a follow-up study has been performed to screen patients for late signs of an impaired tubular or glomerular function and to correlate data of renal function with type of therapy and cumulative doses of cytotoxic agents applied. 81 patients in complete remission for at least 2 years and a median follow-up of 96 (39-304) months, and 53 controls were examined. Clinical routine parameters such as creatinine and electrolytes were determined. A differentiation of proteinuria into the albumin, high molecular weight (HMW) and low molecular weight (LMW) fractions made it possible to assess glomerular and tubular function based on the LMW/HMW ratio. The structural protein fibronectin served as an additional, sensitive marker of glomerular integrity. Routine parameters of kidney function did not show any signs of late nephrotoxicity. However, patients treated for HD had a higher ratio of LMW/HMW in comparison to the group of healthy volunteers (p < 0.01), indicating subclinical tubular renal damage. When the cutoff for tubular damage was defined as LMW/HMW > 1.5, 50% of the patients treated with combined modality, and 42 and 37% of the patients with Ctx or Rtx alone had subclinical tubular alterations, respectively. A tendency towards a higher prevalence of subclinical tubular changes was observed in patients with higher cumulative doses of methotrexate or ifosfamide and in patients with combined Ctx and Rtx with radiation fields involving the renal area. Changes in glomerular function were not observed. It is concluded that treatment of HD is not associated with clinically apparent long-term impairment of renal function but can lead to subclinical alterations. Further clinical implications of these subclinical tubular alterations cannot be assessed at present. A differentiation of proteinuria does not have to be performed routinely but might be useful in the follow-up of selected patients with an increased risk.

Filtration Characteristics of the Single Isolated Perfused Glomerulus of Myxine glutinosa

Using an in vitro microperfusion technique, the filtration characteristics of single isolated glomeruli of the Atlantic hagfish (Myxine glutinosa) were investigated. The suitability of the method as a model to study glomerular function was evaluated. Experiments with a protein-free perfusate led to a filtration coefficient Kf (0.189 +/- 0.181 nl x s-1 x mm Hg-1) that was in the same order of magnitude as determined in vitro for other vertebrates. Morphometric analysis on serial thin sections (1 microns) revealed a glomerular capillary surface (A) of 1.84 +/- 0.72 mm2. A linear relationship between glomerular diameter and A allowed the calculation of the hydraulic conductivity Lp (0.618 +/- 0.384 microliter x s-1 x mmHg-1 x cm-2). The data indicate that the isolated perfused glomerulus of M. glutinosa has filtration characteristics similar to higher vertebrates. The glomeruli of M. glutinosa, which in contrast to glomeruli of other animals are easy to dissect, are a suitable model for the study of glomerular filtration and permeability characteristics of vertebrates. An example of a possible application of the model is a study into the effects of adrenaline on glomerular filtration characteristics. Adrenaline led to increases in ultrafiltration pressure (PUF), single nephron glomerular filtration rate (SNGFR) and filtration fraction (FF); Kf remained unaffected.

Cathepsin B and L in Isolated Proximal Tubular Segments during Acute and Chronic Proteinuria

Acute and chronic proteinuria were studied in rats, using lysosomal cathepsin B and L as marker enzymes for tubular protein degradation. The activity of cathepsin B and L has been determined in microdissected segments S1, S2 and S3 of the proximal tubule by an ultramicroassay. Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as a substrate. In normoproteinuric Sprague-Dawley rats, induction of acute unselective glomerular proteinuria with Adriamycin (5 mg/kg body weight) revealed a moderate activity increase of cathepsin B and L in the S2 segment, reaching 12.6 +/- 5.6 versus 8.6 +/- 4.2 pmol.mm-1.min-1 in controls. In contrast, Munich Wistar Frömter (MWF) rats, that are characterized by a genetically determined, chronically elevated glomerular protein excretion, showed a very high activity of cathepsin selectively in S2 of 25.0 +/- 12.1 pmol.mm-1.min-1. Acute proteinuria induced by Adriamycin in chronic proteinuric MWF rats could increase cathepsin activity in the S3 segment only, showing 12.0 +/- 8.3 versus 6.8 +/- 4.0 pmol.mm-1.min-1 in MWF control rats. In conclusion, chronically increased protein filtration changes the functional reserve capacity of the proximal tubule. While acutely induced glomerular proteinuria in normoproteinuric rats stimulates lysosomal proteolytic activity mainly in S2 segment, chronic proteinuric MWF rats may display already a maximally stimulated cathepsin activity in this segment probably due to long-term increased tubular protein load. In case of acute elevation of chronic proteinuria, the consecutive S3 segment shows increased lysosomal function for protein conservation.

The Hagfish Kidney as a Model to Study Renal Physiology and Toxicology

The kidney of the adult hagfish is a comparatively simple organ consisting of large, segmentally arranged glomeruli and two tubules (archinephric ducts, AND) which drain into the cloaca. The biochemical composition, morphology, and function in vitro (permeability of the glomerular barrier to water and proteins) of the glomeruli is similar to that of higher vertebrates. The AND is morphologically characterized by an epithelium with a brush border. The AND reab-sorbs molecules such as glucose or amino acids, expresses enzymes, such as cathepsins or acid phosphatase, but seems to be less active than the corresponding segments (proximal tubules) in higher vertebrates.

THE “FIXED” CHARGE OF GLOMERULAR CAPILLARY WALL AS DETERMINANT OF PERMSELECTIVITY

The determinants of glomerular capillary wall (GCW) permeability to proteins have been subject of controversial discussion. To study this question we have developed a modified isolated perfused rat kidney model in which tubular transport processes are completely blocked by perfusion fixation with glutaraldehyde. This model allows to directly titrate the charge density of the GCW using albumin solutions buffered over a wide pH-range, a manipulation that cannot be performed in the intact kidney. Analyzing the results of these experiments helped to determine a fixed charge density of the GCW of 43mEq/L. In the present work, we used the isolated perfused fixed rat kidney model to study the influence of this fixed charge on the transglomerular passage of proteins. To do this, the fixed kidney was perfused with albumin solutions containing different isoforms of horseradish peroxidase. The lowest sieving coefficient was obtained with the acidic isoform (0.035±0.008, n=7), while the isoforms at pI 6.85 and 8.45 showed higher sieving coefficients (0.059±0.008, n=7 and 0.090±0.008, n=4, respectively). The highest sieving coefficient (0.59±0.031, n=6) was observed in perfusion experiments of the fixed kidney with cationic HRP (pI≥9.30). However, when comparing the sieving coefficients, the highly cationic isoform was excluded because it has a lower molecular weight than the other isoforms. The sieving coefficients of the other isoforms were significantly different (p < 0.05, ANOVA, Scheffé test). In conclusion, the presence of a discrete (even if lower than previously thought) “fixed” charge on the GCW of 43 mEq/L restricts the transglomerular passage of isoforms of horseradish peroxidase by a factor 2–3. These results imply that the influence of charge selectivity has been overstated in the literature.

Isolated glomeruli of the Atlantic hagfish Myxine glutinosa as an alternative in vitro model to study glomerular protein metabolism in pharmaco-toxicology of anticancer drugs

This study was designed to validate an alternative in vitro system with isolated glomeruli of the Atlantic hagfish Myxine glutinosa as a model to study alterations in glomerular protein metabolisms in pharmaco-toxicology of anticancer drugs. A morphometric characterization of the glomeruli of Myxine glutinosa reveals a calculated glomerular volume of 180 nl/glomerulus. The glomerular extracellular volume, measured as inulin space, is 38.5 nl/glomerulus. Total glomerular protein content of Myxine glutinosa amounts to 3.56 micrograms/glomerulus and total DNA content to 0.44 microgram/glomerulus. Metabolic properties, estimated as glomerular protein synthesis, are comparable with mammalian glomeruli. The glomeruli of Myxine glutinosa are viable in a tissue culture for up to 12 hr. The incorporation rate of radiolabeled amino acids into glomerular, acid-precipitable proteins is almost identical to that of rats (e.g. Myxine glutinosa 1091 +/- 98 DPM/micrograms DNA vs. rat 1340 +/- 84 DPM/micrograms DNA after 4 hr incubation). To evaluate how nephrotoxic substances affect glomerular metabolism in this model, the anticancer drug Adriamycin (ADR) was used to experimentally induce a glomerular lesion. ADR caused an increase in glomerular protein synthesis in isolated glomeruli of Myxine glutinosa, which is in accordance with data found in rats. Cisplatin, in contrast, known to mainly interfere with tubular integrity, had no effect on glomerular protein synthesis, confirming the specificity of the model. The isolated glomeruli of Myxine glutinosa are suggested as a valid alternative multicellular in vitro system for studying alterations in glomerular metabolism under pharmaco-toxicological conditions and for the evaluation of specific target-cell toxicity of selected nephrotoxins.

The Effects of Cadmium and Adriamycin on the Isolated Perfused Glomerulus of Myxine Glutinosa (Cyclostomata)

The severity and mechanisms of action of environmental and drug nephrotoxicity needs to be evaluated. While impaired renal function has often been based on chronic exposure studies, little attention has been paid to acute effects, especially renal dysfunction that occurs shortly after exposure to heavy metals or drugs. It may be difficult to define such changes in the whole animal, but in vitro techniques offer a simpler solution to assessing such changes.