Martin K. Kuhlmann

Reduction of cisplatin toxicity in cultured renal tubular cells by the bioflavonoid quercetin.

Abstract Quercetin (QC), a polyphenolic compound widely distributed in fruits and vegetables has recently gained interest due to its cisplatin (CP) sensitizing properties in cancer cells. It is currently unknown, whether quercetin also increases the susceptibility of the kidneys to cisplatin toxicity. We studied the effects of various bioflavonoids on CP toxicity in an in vitro model of cultured tubular epithelial cells (LLC-PK1). Viability of LLC-PK1 cells, as assessed by lactate dehydrogenase (LDH) release and MTT-test, was affected by CP (100–400 μM) in a time and dose dependent fashion. Pretreatment of cells with QC for 3 h significantly reduced the extent of cell damage. The protective activity of QC was concentration dependent, starting at 10–25 μM and reaching a plateau between 50 and 100 μM. Other bioflavonoids (catechin, silibinin, rutin) did not diminish cellular injury, even at higher concentrations (100–500 μM). Quercetin itself showed some intrinsic cytotoxicity at concentrations exceeding 75 μM. Our data indicate that quercetin reduces cisplatin toxicity in cultured tubular epithelial cells. The exact mechanism of protection is unclear, though scavenging of free oxygen radicals may play an important role.

In vivo effects of dialysate flow rate on Kt/V in maintenance hemodialysis patients

It is generally assumed that hemodialysis adequacy is only minimally affected by increasing the dialysate flow rate (Qd). Recent in vitro studies showed that dialyzer urea clearance (Kd(urea)) may increase substantially more than expected in response to an increase in Qd. Because these studies implied that dialysis efficacy may benefit from greater Qds, we studied in vivo the effects of various Qds on the delivered dose of dialysis in 23 maintenance hemodialysis (MHD) patients. Hemodialysis was performed at Qds of 300, 500, and 800 mL/min for at least 3 weeks each, whereas specific dialysis prescriptions (treatment time, blood flow rate [Qb], ultrafiltration volume, and type and size of dialyzer) were kept constant. Delivered dose of dialysis, assessed by single-pool Kt/V (Kt/V(sp)) and double-pool Kt/V (Kt/ V(dp)), was measured at least three times for each Qd (218 measurements). Mean +/- SEM Kt/V(sp) was 1.19 +/- 0.03 at Qd of 300 mL/min, 1.32 +/- 0.04 at 500 mL/min, and 1.45 +/- 0.04 at 800 mL/min. The relative gains in Kt/V(sp) for increasing Qd from 300 to 500 mL/min and 500 to 800 mL/min were 11.7% +/- 8.7% and 9.9% +/- 5.1%, respectively. Kt/V(dp) increased at a similar percentage (11.2% +/- 8.9% and 10.3% +/- 5.1%, respectively). The observed gain in urea clearance by increasing Qd from 500 to 800 mL/min was significantly greater than the increase in Kd(urea) predicted from mathematical modeling (5.7% +/- 0.4%; P = 0.0008). Removal ratios for creatinine and the high-molecular-weight marker, beta(2)-microglobulin, were not affected by increasing Qd from 500 to 800 mL/min. The proportion of patients not achieving adequacy (Kt/V(sp) >/= 1.2) was reduced from 56% at Qd of 300 mL/min to 30% at 500 mL/min and further to 13% at 800 mL/min. It is concluded that increasing Qd from 500 to 800 mL/min is associated with a significant increase in Kt/V. Hemodialysis with Qd of 800 mL/min should be considered in selected patients not achieving adequacy despite extended treatment times and optimized Qbs.

High Protein/Energy vs. Standard Protein/Energy Nutritional Regimen in the Treatment of Malnourished Hemodialysis Patients

Although malnutrition is frequently encountered in maintenance hemodialysis (MHD) patients, a clear method of treating this complication is still lacking. Failure of nutritional support regimens may be due to inadequate support of dietary needs. Therefore, a high vs. standard or low protein/energy dietary regimen was studied in malnourished MHD patients. A total of 18 malnourished MHD patients selected according to subjective global assessment (SGA)-scores and biochemical indicators of malnutrition (serum albumin <40 g/l, cholesterol <200 mg/dl, prealbumin <30 mg/dl; two out of three) were assigned to three treatment groups: (A: 45 kcal/kg/d and 1.5 g protein/kg/d; B: 35 kcal/kg/d and 1.2 g protein/kg/d; C: spontaneous intake supplemented with 10% of mean protein and energy intake). A and B received food supplements at appropriate dosing to reach the targeted nutritional intake. During 3-month follow-up nutrient intake was assessed by repeated 4-day dietary diaries. Compliance and tolerance was good in each group. Weight gain (1.2±0.4 kg) was observed in group A, but not in B and C. Serum albumin levels increased by 1.0±0.5 g/l in group A, but not in B and C. Prealbumin and cholesterol levels were unaffected. Weight change correlated with mean dietary energy intake, but not with mean dietary protein intake. We conclude that prescription of 45 kcal/kg/d and 1.5 g protein/kg/d may be necessary to achieve weight gain and improvement of nutritional indices in malnourished MHD pts. Oral food supplements can be used safely and effectively to increase nutrient intake to high levels in these patients.

Improved cold preservation of kidney tubular cells by means of adding bioflavonoids to organ preservation solutions

Background. Cold ischemia and reperfusion during renal transplantation result in release of reactive oxygen species. The aim of this study is to examine whether cold storage induced cell injury can be ameliorated by adding flavonoids directly to preservation solutions. Methods. Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin (UW) or Euro-Collins (EC) solution at 4°C for 20 hours. Preservation solutions were supplemented with various flavonoids. After rewarming, structural and metabolic cell integrity was measured by lactate dehydrogenase (LDH) release and MTT-test, and lipid peroxidation was assessed from generation of thiobarbituric acid-reactive substances (TBARS). Results. Twenty hours of cold storage resulted in a substantial loss of cell viability in both preservation solutions (in EC: LDH release 92.4±2.7%; MTT-test 0.5±0.7%). Addition of luteolin, quercetin, kempferol, fisetin, myricetin, morin, catechin, and silibinin significantly reduced cell injury (for luteolin in EC: LDH release 2.4±1.6%; MTT-test 110.3±10.4%, P<0.01; TBARS-production (related to cold stored control cells) 8.9±2.6%). No cytoprotection was found for apigenin, naringenin, and rutin. Protective potency of flavonoids depends on number of hydroxyl-substituents and lipophilicity of the diphenylpyran compounds. Conclusion. Cold storage induced injury of renal tubular cells was substantially ameliorated by adding selected flavonoids directly to preservation solutions.

Cellular uptake of vitamin B12 in patients with chronic renal failure.

Background/Aims: Elevated concentration of plasma homocysteine (tHcy) is common in renal patients, however, the reason behind the resistance to vitamin B12 and folate therapy are poorly understood. Methods: We investigated vitamin B12 uptake by mononuclear cells (MC) from predialysis patients (n = 19) as compared to healthy controls (n = 15). Serum levels of tHcy, methylmalonic acid and cystathionine, holotranscobalamin (holoTC), total vitamin B12 and folate were also measured. Results: The uptake of vitamin B12 by MC from renal patients was lower than that by MC from controls (9.3 vs. 12.5 pg/3 × 106 cells; p = 0.001). Nonetheless, the receptor-binding capacity was comparable between patients and controls (6.1 vs. 6.5 pg/3 × 106 cells; p = 0.627). Average reduction of vitamin B12 uptake in patients as compared to the controls was 18.1%. Conclusions: Our results show that vitamin B12 uptake is impaired in MC from renal patients, with no evidence that the surface receptor is down-regulated. High serum concentrations of holoTC are common in renal patients and might be related to a generalized resistance to this vitamin. Serum concentrations of vitamin B12 within the reference range are not likely to ensure vitamin delivery into the cells. Supraphysiological doses of vitamin B12 may be necessary to deliver a sufficient amount of the vitamins to the cells via mechanisms largely independent of holoTC receptor.

Iohexol plasma clearance measurement in older adults with chronic kidney disease—sampling time matters

BACKGROUND Accurate and precise measurement of GFR is important for patients with chronic kidney disease (CKD). Sampling time of exogenous filtration markers may have great impact on measured GFR (mGFR) results, but there is still uncertainty about optimal timing of plasma clearance measurement in patients with advanced CKD, for whom 24-h measurement is recommended. This satellite project of the Berlin Initiative Study evaluates whether 24-h iohexol plasma clearance reveals a clinically relevant difference compared with 5-h measurement in older adults. METHODS In 104 participants with a mean age of 79 years and diagnosed CKD, we performed standard GFR measurement over 5 h (mGFR300) using iohexol plasma concentrations at 120, 180, 240 and 300 min after injection. With an additional sample at 1440 min, we assessed 24-h GFR measurement (mGFR1440). Study design was cross-sectional. Calculation of mGFR was conducted with a one compartment model using the Brochner-Mortensen equation to calculate the fast component. mGFR values were compared with estimated GFR values (MDRD, CKD-EPI, BIS1, Revised Lund-Malmö and Cockcroft-Gault). RESULTS In all 104 subjects, mGFR1440 was lower than mGFR300 (23 ± 8 versus 29 ± 9 mL/min/1.73 m(2), mean ± SD; P < 0.001). mGFR1440 was highly correlated with mGFR300 (r = 0.9). The mean absolute difference mGFR300 - mGFR1440 was 5.9 mL/min/1.73 m(2) corresponding to a mean percentage difference of 29%. In individuals with eGFRCKD-EPI ≤ 30 mL/min/1.73 m(2), percentage difference of mGFR300 and mGFR1440 was even higher (35%). To predict mGFR1440 from mGFR300, we developed the correction formula: mGFR1440 = -2.175 + 0.871 × mGFR300 (1-fold standard error of estimate: ±2.3 mL/min/1.73 m(2)). The GFR estimating equation with the best accuracy and precision compared with mGFR300 and mGFR1440 was the Revised Lund Malmö. CONCLUSIONS In elderly CKD patients, measurement of iohexol clearance up to 5 h leads to a clinically relevant overestimation of GFR compared with 24-h measurement. In clinical care, this effect should be bore in mind especially for patients with considerably reduced GFR levels. A new correction formula has been developed to predict mGFR1440 from mGFR300. For accurate GFR estimates in elderly CKD patients, we recommend the Revised Lund Malmö equation.

Effect of quercetin on hypoxic injury in freshly isolated rat proximal tubules

The bioflavonoid quercetin, which has antioxidant properties, protects renal tubular epithelial cells from oxidant-induced injury by inhibiting lipid peroxidation. We examined the effect of quercetin on hypoxia-induced injury in freshly isolated rat renal proximal tubules. Hypoxia induced rapid loss of cellular ATP, followed by functional and structural alterations measured as a decrease in tubular potassium content and sequentially by an increase in lactate dehydrogenase release. Furthermore, hypoxia increased lipid peroxidation, measured as thiobarbituric acid-reactive substances. Quercetin significantly inhibited hypoxia-induced functional and structural tubular injury in addition to lipid peroxidation but did not alter hypoxia-induced ATP depletion. These results demonstrate the potency of the bioflavonoid quercetin in protecting proximal tubules from hypoxic injury, which is independent of tubular energy metabolism and may be related to the inhibition of lipid peroxidation.

Calf Bioimpedance Spectroscopy for Determination of Dry Weight in Hemodialysis Patients: Effects on Hypertension and Left Ventricular Hypertrophy

Background/Aims: Dry weight estimation in hemodialysis patients is still a substantial problem. Despite meticulous clinical assessment, fluid overload is common, leading to hypertension and left ventricular hypertrophy (LVH). Segmental calf bioimpedance spectroscopy (cBIS) is a novel tool for dry weight assessment. Here we tested the hypothesis, that its clinical routine use reduces arterial hypertension and left ventricular mass. Methods: Left ventricular mass (determined by magnetic resonance imaging), blood pressure and antihypertensive medication (defined daily doses, ddd) were assessed at baseline (BL). Thereafter post-dialytic target weight was reduced until cBIS-defined dry weight was reached (DW). During a 6-month follow up, DW was re-evaluated monthly by cBIS and end-dialytic weight was adjusted correspondingly. At the end, left ventricular mass, blood pressure and antihypertensive medication were determined a 3rd time (follow-up, FU). Results: Eleven out of 15 patients were available for analysis after 6 months. Left ventricular mass showed a declining trend during the study period (Mean±SD; BL 145±54g; DW 142±55g; FU 137±52g; p=0.61, linear mixed model). Comparable results were obtained for systolic blood pressure (BL 158±18mmHg; DW 144±19mmHg; FU 149±21mmHg; p=0.07), and antihypertensive medication (BL 3.28±2.82ddd; DW 2.86±2.81ddd; FU 3.36±3.05ddd; p=0.37). Conclusions: We conclude that attainment of dry weight assessed by cBIS tends to reduce left ventricular mass and blood pressure while antihypertensive medication remains unchanged. While the study was underpowered, its results provide an important hypothesis generating data basis for the design of larger studies.

Slope analysis of blood volume and calf bioimpedance monitoring in hemodialysis patients

BACKGROUND Continuous intradialytic bioelectrical impedance spectroscopy of the calf (cBIS) monitors changes in calf extracellular fluid volume (cECV), thus allowing estimation of hydration in end-stage renal disease patients. Blood volume monitoring (BVM) during hemodialysis (HD) provides information about relative changes in intravascular volume, which indirectly reflects plasma refilling. We hypothesize that the rate of plasma refilling changes when cBIS-determined dry weight (BIS-DW) is reached. METHODS Post-HD weight was reduced from baseline (BL) in 15 patients until dry weight was reached according to cBIS criteria (BIS-DW). The slopes of cBIS and BVM curves were analysed during the first 30 and last 20 min in 31 BL treatments, which were compared to the slopes during 31 treatments when BIS-DW was reached. RESULTS During BL treatments, BVM slopes did not differ between the first 30 and last 20 min (-0.112 ± 0.157%/min versus -0.089 ± 0.036, P = n.s.), while cBIS slopes were generally steeper at the beginning than at the end of HD (-0.184 ± 0.139%/min versus 0.10 ± 0.127, P < 0.01). During BIS-DW treatments, BVM and cBIS slopes were steeper at the beginning than at the end (BVM: -0.131 ± 0.122 versus -0.064 ± 0.051, P < 0.01; cBIS: -0.192 ± 0.129 versus -0.035 ± 0.012, P < 0.001) and the cBIS slopes were steeper than BVM slopes at the beginning of HD. This relationship is inverted at the end of HD, when BIS-DW is reached (beginning: -0.192 ± 0.129 versus -0.131 ± 0.122, P < 0.05, end: -0.035 ± 0.012 versus -0.064 ± 0.051, P = 0.05). CONCLUSIONS This study demonstrates that cECV changes faster at the beginning than at the end of HD. A reversal steepness of the cBIS slope in relation to BVM slope is observed at the time when BIS-DW is reached. Therefore, combined analysis of cBIS and BVM aiming at clinical end points may be useful to assess the relationship between plasma refilling and tissue hydration during dialysis.

Differentiation of Monocyte Derived Dendritic Cells in End Stage Renal Disease is Skewed Towards Accelerated Maturation

BACKGROUND Dendritic cells (DC) play an important role in the induction of immune responses. Patients with end stage renal disease (ESRD) suffer from chronic inflammation, leading to a secondary, uremic immunodeficiency associated with alterations in monocyte subpopulations with increased proinflammatory capacities. OBJECTIVES The aim of this study was to examine, under isolated conditions, whether alterations in monocyte subpopulations may affect in vitro maturation of dendritic cells (DC) in patients with ESRD, thus allowing us to draw conclusions for the situation in vivo. MATERIAL AND METHODS Monocytes from 30 patients undergoing hemodialysis (HD) and 15 healthy volunteers were enriched from peripheral blood leukocytes, differentiated into immature DC (iDC) in medium containing IL-4 and GM-CSF, and were induced with LPS to differentiate into mature DC (mDC). Monocyte subpopulations and DC maturation stages were phenotypically characterized using flow-cytometry. RESULTS Although phenotypically indistinguishable, the number of both iDC and mDC that were generated from uremic monocytes was significantly higher compared to those from healthy controls (p=0.02 and p=0.03, respectively). This was associated with an increased number of CD14+ CD16+ monocytes (p=0.02) and by a higher maturation efficiency of mDC in patients (p=0.04). CONCLUSIONS A high percentage of CD14+ CD16+ monocytes in patients with ESRD is associated with an increased propensity to differentiate into DC. This indicates that chronic inflammation may substantiate the biased consistence of monocyte subpopulations leading to profound alteration in DC generation and maturation in ESRD.