- Email: [email protected]
Regeneration of peritoneal effluent by Madin–Darby canine kidney cells-lined hollow fibers
Materials Science and Engineering C 6 Ž1998. 221–226
Regeneration of peritoneal effluent by Madin–Darby canine kidney cells-lined hollow fibers Akira Saito a
a,)
, Hideaki Suzuki b, Karol Bomsztyk b, Suhail Ahmad
b
DiÕision of Nephrology and Metabolism, Department of Medicine, Tokai UniÕersity School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan b DiÕision of Nephrology, Department of Medicine, UniÕersity of Washington, School of Medicine, Seattle, WA, USA Received 19 June 1998
Abstract Regeneration of plasma ultrafiltrate is a significant step towards the development of wearable artificial kidneys ŽWAK.. Monolayers of Madin–Darby canine kidney ŽMDCK. cells were grown on inner surface of cellulose diacetate hollow fibers contained in mini-filters. Eight such devices were tested as bioartificial tubules for regeneration of peritoneal dialysis effluent ŽPD drainage. from a patient. PD drainage was perfused at a rate of 25 ml miny1, under 50 mm Hg hydraulic pressure and ultrafiltrate was produced from apical to basal direction of the cells. Cell-uncovered membranes Ž n s 8. were also similarly tested as controls. Hydraulic permeability of the cell-uncovered membranes was 0.49 " 0.06 ml miny1 , whereas that of the cell-covered membranes Žexperimental. was 0.28 " 0.04 ml miny1 Ž P - 0.0001.. Concentrations of sodium, chloride and potassium in the filtrate through the cell-covered membranes were 21.3 " 11.6, 17.8 " 13.8 and 0.56 " 0.14 mEq ly1 , respectively, in comparison, across cell-uncovered membranes Žcontrol., 137.9 " 3.3, 104.5 " 1.1 and 2.53 " 0.18 mEq ly1, respectively Ž P - 0.0001, experimental vs. control.. Urea nitrogen, creatinine, uric acid and glucose concentrations were 5.4 " 4.5, 0.81 " 0.66, 0.85 " 0.47 and 137.5 " 134.3 mg dly1, respectively, in the filtrate across the cell-covered membranes, and 49.1 " 4.9, 7.06 " 0.60, 4.26 " 0.47 and 1098.1 " 147.4 mg dly1, respectively, in the filtrate across the cell-uncovered membranes Ž P - 0.0001.. In conclusion, MDCK cell-covered membranes used in this study removed electrolytes, glucose and uremic metabolites significantly from the PD drainage from a patient. Thus, such a device has potential for use in the development of a WAK. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Renal failure; Hemofiltration; Bioartificial kidney; Artificial tubules; MDCK cells
1. Introduction Continuous dialysis therapies are better tolerated than intermittent treatments. However, almost all of long term hemodialysis patients receive intermittent treatment. Continuous hemofiltration as a long term dialysis therapy is desirable since it is most likely technique to be used for wearable artificial kidneys ŽWAK.. However, the requirement for large quantities of replacement fluid is a major hindrance in common utilization of this form of therapy. The WAK has to be small in size and light in weight, thus the problem large bulk of replacement fluid needs to be solved. One of the solutions for these problems is to use plasma ultrafiltrate to generate replacement fluid. This would reduce cost, complexity and bulk associated with the external replacement fluid and would make it possible ) Corresponding author. Tel.: q81-463-931121 Ext. 2350; Fax: q81463-924374; E-mail: [email protected]
to develop a small WAK. Ideally, the regeneration method should separate electrolytes and water from protein metabolites including urea and creatinine. Renal tubular epithelial cells allow the electrolytes to be absorbed Žtranscellular transport. while rejecting the protein metabolites and may be used for this purpose. Two research groups have grown tubular epithelial cells on ultrafiltration devices w1–7x. Aebischer et al. w1x and Ip et al. w2x succeeded in growing LLC-PK 1 and Madin–Darby canine kidney ŽMDCK. cells on the outer surface of hollow fibers. Humes et al. w6x and Nikolovski et al. w7x succeeded in growing a single layer of MDCK cells and LLC-PK 1 cells on the inner surface of single fiber. However, the efficacy of these devices in terms of transport characteristics of uremic substances and electrolytes have not been investigated. We have developed a MDCK epithelial cell-covered ultrafiltration device and transport characteristics of this device is reported in this manuscript.
0928-4931r98r$ - see front matter q 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 9 8 . 0 0 0 5 4 - X
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A. Saito et al.r Materials Science and Engineering C 6 (1998) 221–226
2. Materials and methods 2.1. Cell culture MDCK cells, derived from the distal tubule of dog kidney, were obtained from American Tissue Type Culture Collection ŽATCC, Rockville, MD. and were grown in RPMI 1640 supplemented with 5% fetal bovine serum ŽGibco Laboratories, Grand Island, NY., 200 mg mly1 y1 L-glutamine, 100 IU ml penicillin and 100 mg mly1 streptomycin. Cells were maintained at 378C in 5% CO 2 in air. 2.2. Mini-filters for filtrate regeneration Mini-filters which consisted of five fibers Ž10 cm long. were kindly supplied by Althin Medical ŽMiami Lakes, FL. with a total surface area of 2.9 cm2 . The membrane of the hollow fibers was cellulose diacetate which are normally used in Altra Novaw dialyzer 1. 2.3. Preparation of MDCK cell-coÕered membrane For transport studies, MDCK cells were seeded on the inner surfaces of hollow fiber capillaries from Altra Novaw dialyzer. Prior to seeding with MDCK cells, inner surfaces of the membrane were coated with bovine type 1 collagen ŽCollaborative Biomedical Products, Bedford, MA. and incubated at 378C overnight for polymerization. The cells Žin the above mentioned culture medium. were seeded inside the hollow fibers at a density of 6 = 10 6 to 1 = 10 7 MDCK cells mly1 . The cell-seeded capillary membranes were kept in the incubator for 2 weeks and the medium was exchanged at intervals of 48 h, and then placed in a flow chamber for transport studies. Two methods were used to verify the confluency of epithelial cell monolayer. 2.3.1. Phenol red transport study The concentrations of phenol red were measured in perfusate and filtrate obtained from all the mini-filters Žfor description of experiment and terminology, please see Section 2.4.. The same measurements were performed with the cell-uncovered hollow fiber membranes as controls. At start of the studies, RPMI 1640 medium was replaced by hormone-supplemented, serum-free medium as described by Taub et al. w8x containing 15 mg ly1 phenol
1
The wall thickness of the membrane is 30 mm, and internal diameter is 195 mm. The Altra Novaw is a conventional dialyzer with ultrafiltration coefficient of 7.1 ml mm Hgy1 hy1 1.4 my2 with bovine blood at 378C. Urea and creatinine clearance of 161 and 148 ml miny1 , respectively, at Q B s 200 ml miny1 , Q D s 500 ml miny1 and Qf s10 ml miny1 .
red as perfusate, and the flow rate of perfusate was kept at 25 ml miny1 . The hormone-supplemented, serum-free medium was made of an 1:1 mixture of Ham’s F-12 nutrient mixture ŽGibco. and Dubelcco’s modified Eagle’s medium ŽGibco., to which selenium, transferrin, insulin, hydrocortisone, triiodothyronine and prostaglandin E 1 were added. The mini-filters were kept in a water bath at 378C and hydraulic pressure was applied to maintain perfusate under 50 mm Hg during the studies. Samples were alkalinized with NaOH and 800 ml of each sample was applied to Lambda Bio UVrVIS Spectrometer ŽPerkin-Elmer, Ueberlingen, Germany. to determine phenol red concentration by measuring UV absorbance at 560 nm. Leak rate of phenol red of each filter was calculated as concentration ratio of phenol red filtraterperfusate in terms of UV absorption at 560 nm. 2.3.2. Scanning electron microscopy MDCK cell monolayers on hollow fiber capillaries after the studies were perfused with PBS and then fixed by perfusing the interior of hollow fiber capillary with 1.5% gluteraldehyde in PBS at 48C for 60 min. The fibers were then dehydrated with 35, 50, 75, 90, 95 and 100% of absolute alcohol for 20 min each, dried with critical point dryer, Samdri w -PVT-3B ŽTousimis Research, Rockville, MD., sputter coated with gold–palladium using SEM Coating Unit E 5100 ŽPolaron Instruments, Hatfield, PA. and examined under a scanning electron microscope JSM 35C ŽJEOL, Tokyo, Japan.. 2.4. ConÕectiÕe transport study Convective transport was investigated using eight mini-filters, each containing five hollow fibers, in which MDCK cells were grown on the inner surfaces. For control experiments, eight mini-filters without MDCK cells were used. During the experiments the mini-filters were kept in a water bath at 378C, but the fluid and tubes were exposed to ambient temperature ŽFig. 1.. In order to conduct experiments, peritoneal outflow from a PD patient was used. Patient had been free of peritonitis for a year and used 2.5% glucose Dianiel w solution ŽBaxter, MacGaw Park, IL. and donated PD drainage for the experiments. The drainage was used as perfusate for all the experiments. Medical grade silicon tubing ŽDow Corning, Midland, MI. was used for connecting the circuit. Hydraulic pressure was applied by gravity by adjusting the height of the perfusate container to maintain the pressure of 50 mm Hg inside the hollow fiber capillaries ŽFig. 1.. Flow rate of the perfusate through the mini-filter was kept at 25 ml miny1 using a mini-pump. The samples of PD drainage affluent, effluent and the filtrate through the cell-covered membranes were collected for chemical measurements. The same samples were also collected during the control experiments using cell-uncovered mini-filters.
A. Saito et al.r Materials Science and Engineering C 6 (1998) 221–226
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tion of phenol red crossed the MDCK cell-covered membrane. In contrast, 0.98 " 0.04 phenol red leaked through the cell-uncovered membrane Ž P - 0.0001, experimental vs. control.. Thus, the epithelial cells layered the inside of fibers without space in between preventing any significant leaks. Concentrations of several plasma constituents in perfusate affluent, effluent and filtrate obtained across MDCK cell-covered Žexperimental. and -uncovered membranes Žcontrol. are shown in the Table 1. Concentrations of nitrogen metabolites, electrolytes and glucose in controls were not significantly different from those of perfusate affluent and effluent. In contrast, concentrations of these plasma constituents were significantly lower in the ultrafiltrate than in perfusate affluent or effluent Ž P - 0.0001.. Fig. 1. Schematic drawing of experiment for transport study. Peritoneal dialysis ŽPD. drainage was perfused at a rate of 25 ml miny1 under hydraulic pressure of 50 mm Hg, from inner to outer direction of hollow fiber capillaries, applied by gravity by adjusting the height of perfusate. During the experiments the mini-filters were kept in a water bath at 378C.
3.3. Hydraulic permeability Hydraulic permeabilities of control devices were 0.49 " 0.06 ml miny1 50 mm Hgy1 of hydraulic pressure.
2.5. Measurements of substances in perfusate and ultrafiltrate Urea nitrogen, creatinine, uric acid, sodium, potassium, chloride, and glucose concentrations in PD drainage affluent, effluent and the filtrate were measured by applying 0.6 ml each sample to the Automated Chemistry Analyzer Paramax 720ZX ŽBaxter.. 2.6. Statistics Results are expressed as means " S.D. Statistical significance is determined by the Student’s t-test.
3. Results 3.1. Cell morphology Cross-sectional scanning electron micrograph showed that MDCK cells formed a monolayer to confluence on inner surface of cellulose diacetate hollow fiber 2 weeks after seeding ŽFig. 2A.. The cells exhibited polarized morphologic characteristics including apical microvilli, tight junctions and desmosomes ŽFig. 2B.. The electron microscopic cross-sectional findings of the hollow fiber at 5 weeks after the cell seeding still showed a monolayer of the cells attached on the inner surface of membrane. 3.2. Transport characteristics Two kinds of transport studies were carried out with MDCK cell-covered and -uncovered filters, the former was phenol red and the latter, plasma constituents in PD effluent. In phenol red transport study, only 0.10 " 0.03 frac-
Fig. 2. ŽA. Cross-sectional scanning electron micrograph of MDCK cells grown on inner surface of cellulose diacetate hollow fiber 2 weeks after seeding Ž=480.. ŽB. Scanning electron micrograph of the same section of the fiber by higher magnification Ž=2600.. Short and long apical microvilli, tight junctions of MDCK cells were shown.
224
CAPD drainage Filtrate Žcontrol. Filtrate Žexperimental. Effluent Žexperimental.
Urea nitrogen Žmg dly1 .
Creatinine Žmg dly1 .
Uric acid Žmg dly1 .
Glucose Žmg dly1 .
Sodium ŽmEq ly1 .
Chloride ŽmEq ly1 .
Potassium ŽmEq ly1 .
50.3"5.1 49.1"4.9 5.4"4.5) a 50.5"6.1
7.05"0.62 7.06"0.60 0.81"0.66) a 7.06"0.68
4.33"0.48 4.26"0.47 0.85"0.47) a 4.33"0.50
1020.7"150.1 1098.1"147.4 137.5"134.3) a 1073.3"149.3
137.3"3.1 137.9"3.3 21.3"11.6) a 138.1"4.0
104.2"1.2 104.5"1.1 17.8"13.8) a 104.6"1.4
2.50"0.26 2.53"0.18 0.56"0.14) a 2.53"0.30
)Comparison with CAPD drainage P - 0.0001. a Comparison with filtrate Žcontrol. P - 0.0001.
A. Saito et al.r Materials Science and Engineering C 6 (1998) 221–226
Table 1 Concentrations of nitrogen metabolites, glucose and electrolytes in CAPD drainage, control filtrate, experimental filtrate and experimental effluent
A. Saito et al.r Materials Science and Engineering C 6 (1998) 221–226
Hydraulic permeabilities of the experimental devices Žapical to basal permeability. were 0.28 " 0.04 ml miny1 50 mm Hgy1 . Thus hydraulic permeabilities of cell-covered membranes were significantly lower than those of the cell-uncovered membranes Ž P - 0.001..
4. Discussion This is the first time that regeneration of biological fluid across renal epithelial cell-covered membranes has been reported. Our results demonstrate marked reductions of solute concentrations in the regenerated filtrate through MDCK cell-covered membranes. Nitrogen metabolites such as urea, creatinine and uric acid, were significantly removed from ultrafiltrate. Thus, the regenerated filtrate can be used as a replacement fluid without risk of returning back the protein metabolites. However, electrolytes were also removed from the perfusate and if this method is to be used, electrolytes will have to be supplemented. Since the concentrations of nitrogen metabolites, electrolytes and glucose in perfusate effluent were not significantly different from that of affluent, the transport across the cells appears to be mostly convective. It appears that substances were rejected by MDCK cells, were not absorbed across the apical cell membrane and would not have accumulated in the cells. Small amount of these substances, however, may have been transported either from apical to basal direction intercellularly or transcellularly. The fraction of phenol red transported through the cell-covered membranes was only 0.10 " 0.03, which was similar to the fractions of urea Ž0.11 " 0.05. and creatinine Ž0.11 " 0.04. calculated from filtrate and CAPD drainage levels. However, the fractions of electrolytes transported across the device such as sodium Ž0.15 " 0.03. and chloride Ž0.17 " 0.05. were higher under the same conditions. This extra electrolytes transport has to be confirmed whether or not transcellular active transport contributes to the results. High Naq, Kq ATPase activity in MDCK cells has been reported w9,10x. Active transports of electrolytes has also been reported in distal convoluted tubule and collecting duct in animal kidneys w11–16x. However, electrolyte transport across the MDCK and LLC-PK 1 cellcovered membrane has not previously been reported. In order to use the devices for clinical purpose, some electrolytes will have to be supplemented. Alternatively, a combination of MDCK and LLC-PK 1 cells might be effective for better electrolyte transport. Naq, Kq ATPase activity w10x, gene expressions of electrolyte transporter such as NaqrHq exchanger w17x, Naqrglucose cotransporter w18x and Naqramino acid cotransporter w19x have been reported in LLC-PK 1 cells. LLC-PK 1 cell monolayer, however, was reported not to allow filtration in the apical to basal direction when positive transmembrane pressure of 5 to 30 mm Hg was applied on the apical side w5x.
225
Measurable filtration in the basal to apical direction through the cell-covered membrane were confirmed w5x, however, the filtrate obtained in the basal to apical direction should not be returned to patient since the filtrate might contain heterogeneous proteins from damaged cells. Our study was done under 50 mm Hg of hydraulic pressure in the apical to basal direction, and MDCK cell monolayers were able to maintain function during and after the study. Histological assessment demonstrate confluent monolayers of the MDCK cells along the inner surfaces of hollow fiber membranes. Monolayers of MDCK cells on the fibers were able to keep their functional confluency during and after studies of convective transport. It took 2 weeks to achieve functional confluency. Three of the MDCK cell-covered membrane filters were confirmed to keep almost the same permeability performance 3 weeks after the first permeability studies Ždata not shown.. We have previously reported that an ultrafiltration rate of 7 ml miny1 with continuous hemofiltration kept plasma levels of small molecules at negative 30 to negative 60% of pre-dialysis values of intermittent conventional dialysis w20x. Allowing for daily intake of fluid the net replacement fluid would be required at the rate of 4 ml miny1 . Our device with a surface area of 2.9 cm2 produced 0.28 ml miny1 of regenerated ultrafiltrate. Thus, a device with 1 m2 surface area should produce at least 0.97 ml miny1 of ultrafiltrate. Since this rate is not yet practical for clinical use, further investigation should be done to increase the ultrafiltration rate across cell-covered membranes. PD drainage was used as perfusate in this study for several reasons. Main reasons for this include, the PD drainage is usually sterile and contains important proteins and other substances necessary for the cell growth such as albumin, transferrin, growth factors, glucose, etc. w21x. In addition, it also contains considerable amounts of uremic toxins, which allowed us to study the kinetics of these metabolites. However, the unused fresh PD solution does not seem to be good for cell culture, when the cells were cultured with fresh 1.5% glucose Dianiel w solution, 80% of the cells died within 48 h due to low pH of the solution Ždata not shown.. In previous reports w3,6x, high-flux Polysulfon membrane filters were used, through which plasma proteins can pass. Cellulose diacetate membrane used in this study are generally impermeable to proteins. It is important for this system to protect against the penetrance of immunologically competent cells and heterogenic proteins from damaged cells. Newer high flux membranes may be less permeable to proteins and could be used since ultrafiltration rate might be higher without the risk of cross-contamination. References w1x P. Aebischer, T.K. Ip, L. Miracoli, P.M. Galletti, Trans. Am. Soc. Artif. Intern. Organs 33 Ž1987. 96–102.
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w2x T.K. Ip, P. Aebischer, P.M. Galletti, Trans. Am. Soc. Artif. Intern. Organs 34 Ž1988. 351–355. w3x H. Uludag, G. Panol, P. Aebischer, Trans. Am. Soc. Artif. Intern. Organs 35 Ž1989. 523–527. w4x P. Aebischer, T.K. Ip, G. Panol, P.M. Galletti, Life Support Systems 5 Ž1987. 159–168. w5x T.K. Ip, P. Aebischer, Artif. Organs 13 Ž1989. 58–65. w6x H.D. Humes, D.A. Cieslinski, A.J. Funke, J. Am. Soc. Nephrol. 6 Ž1995. 465. w7x J. Nikolovski, S. Poirier, A.J. Funke, D.A. Cieslinski, H.D. Humes, J. Am. Soc. Nephrol. 7 Ž1996. 1376–1377. w8x M. Taub, L. Chuman, M.H. Saier, Proc. Natl. Acad. Sci. 76 Ž1979. 3338–3342. w9x M.J. Rinder, L.M. Chuman, L. Shaffer, M.H. Saier Jr., J. Cell Biol. 81 Ž1979. 35–648. w10x G. Gstraunthaler, W. Pfaller, P. Kotanko, Am. J. Physiol. 248 Ž1985. F536–F544, ŽRenal Fluid Electrolyte Physiol. 17.. w11x K. Hierholzer, M. Wiederholz, Kidney Int. 9 Ž1976. 198–203.
w12x R.N. Khuri, M. Wiederholz, N. Streider, G. Giebisch, Am. J. Physiol. 228 Ž1975. 1262–1268. w13x E. Slatopolsky, Plenum, New York, 1983, pp. 269–330. w14x G.R. Shareghi, L.C. Stoner, Am. J. Physiol. 235 Ž1978. F367–F375. w15x E. Pastoriza-Munoz, R. Colindres, W. Lassiter, C. Lechene, Am. J. Physiol. 235 Ž1978. F321–F330. w16x G.R. Shareghi, Z.S. Agus, Am. J. Physiol. 242 Ž1982. F379–F384. w17x R.F. Reilly, F. Hildebrandt, D. Biemesderfer, C. Sardet, J. Pouyssegur, P.S. Aronson, C.W. Slayman, P. Igarashi, Am. J. Physiol. 261 Ž1991. F1088–F1094, ŽRenal Fluid Electrolyte Physiol. 30.. w18x T. Shioda, T. Ohta, K.J. Isselbacher, D.B. Rhoads, Proc. Natl. Acad. Sci. USA 91 Ž1994. 11919–11923. w19x C.T. Kong, S.F. Yet, J.E. Lever, J. Biol. Chem. 268 Ž1993. 1509– 1512. w20x A. Saito, T. Takagi, K. Sugiura, M. Ono, K. Minakuchi, S. Teraoka, K. Ota, Nephrol. Dial. Transplant 10 Ž1995. 52–56, Suppl. 3. w21x A. Saito, H. Ogawa, T.G. Chung, K. Maeda, Field, Rich and Associates, New York, 1986, pp. 307–311.
Regeneration of peritoneal effluent by Madin–Darby canine kidney cells-lined hollow fibers Akira Saito a
a,)
, Hideaki Suzuki b, Karol Bomsztyk b, Suhail Ahmad
b
DiÕision of Nephrology and Metabolism, Department of Medicine, Tokai UniÕersity School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan b DiÕision of Nephrology, Department of Medicine, UniÕersity of Washington, School of Medicine, Seattle, WA, USA Received 19 June 1998
Abstract Regeneration of plasma ultrafiltrate is a significant step towards the development of wearable artificial kidneys ŽWAK.. Monolayers of Madin–Darby canine kidney ŽMDCK. cells were grown on inner surface of cellulose diacetate hollow fibers contained in mini-filters. Eight such devices were tested as bioartificial tubules for regeneration of peritoneal dialysis effluent ŽPD drainage. from a patient. PD drainage was perfused at a rate of 25 ml miny1, under 50 mm Hg hydraulic pressure and ultrafiltrate was produced from apical to basal direction of the cells. Cell-uncovered membranes Ž n s 8. were also similarly tested as controls. Hydraulic permeability of the cell-uncovered membranes was 0.49 " 0.06 ml miny1 , whereas that of the cell-covered membranes Žexperimental. was 0.28 " 0.04 ml miny1 Ž P - 0.0001.. Concentrations of sodium, chloride and potassium in the filtrate through the cell-covered membranes were 21.3 " 11.6, 17.8 " 13.8 and 0.56 " 0.14 mEq ly1 , respectively, in comparison, across cell-uncovered membranes Žcontrol., 137.9 " 3.3, 104.5 " 1.1 and 2.53 " 0.18 mEq ly1, respectively Ž P - 0.0001, experimental vs. control.. Urea nitrogen, creatinine, uric acid and glucose concentrations were 5.4 " 4.5, 0.81 " 0.66, 0.85 " 0.47 and 137.5 " 134.3 mg dly1, respectively, in the filtrate across the cell-covered membranes, and 49.1 " 4.9, 7.06 " 0.60, 4.26 " 0.47 and 1098.1 " 147.4 mg dly1, respectively, in the filtrate across the cell-uncovered membranes Ž P - 0.0001.. In conclusion, MDCK cell-covered membranes used in this study removed electrolytes, glucose and uremic metabolites significantly from the PD drainage from a patient. Thus, such a device has potential for use in the development of a WAK. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Renal failure; Hemofiltration; Bioartificial kidney; Artificial tubules; MDCK cells
1. Introduction Continuous dialysis therapies are better tolerated than intermittent treatments. However, almost all of long term hemodialysis patients receive intermittent treatment. Continuous hemofiltration as a long term dialysis therapy is desirable since it is most likely technique to be used for wearable artificial kidneys ŽWAK.. However, the requirement for large quantities of replacement fluid is a major hindrance in common utilization of this form of therapy. The WAK has to be small in size and light in weight, thus the problem large bulk of replacement fluid needs to be solved. One of the solutions for these problems is to use plasma ultrafiltrate to generate replacement fluid. This would reduce cost, complexity and bulk associated with the external replacement fluid and would make it possible ) Corresponding author. Tel.: q81-463-931121 Ext. 2350; Fax: q81463-924374; E-mail: [email protected]
to develop a small WAK. Ideally, the regeneration method should separate electrolytes and water from protein metabolites including urea and creatinine. Renal tubular epithelial cells allow the electrolytes to be absorbed Žtranscellular transport. while rejecting the protein metabolites and may be used for this purpose. Two research groups have grown tubular epithelial cells on ultrafiltration devices w1–7x. Aebischer et al. w1x and Ip et al. w2x succeeded in growing LLC-PK 1 and Madin–Darby canine kidney ŽMDCK. cells on the outer surface of hollow fibers. Humes et al. w6x and Nikolovski et al. w7x succeeded in growing a single layer of MDCK cells and LLC-PK 1 cells on the inner surface of single fiber. However, the efficacy of these devices in terms of transport characteristics of uremic substances and electrolytes have not been investigated. We have developed a MDCK epithelial cell-covered ultrafiltration device and transport characteristics of this device is reported in this manuscript.
0928-4931r98r$ - see front matter q 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 9 8 . 0 0 0 5 4 - X
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2. Materials and methods 2.1. Cell culture MDCK cells, derived from the distal tubule of dog kidney, were obtained from American Tissue Type Culture Collection ŽATCC, Rockville, MD. and were grown in RPMI 1640 supplemented with 5% fetal bovine serum ŽGibco Laboratories, Grand Island, NY., 200 mg mly1 y1 L-glutamine, 100 IU ml penicillin and 100 mg mly1 streptomycin. Cells were maintained at 378C in 5% CO 2 in air. 2.2. Mini-filters for filtrate regeneration Mini-filters which consisted of five fibers Ž10 cm long. were kindly supplied by Althin Medical ŽMiami Lakes, FL. with a total surface area of 2.9 cm2 . The membrane of the hollow fibers was cellulose diacetate which are normally used in Altra Novaw dialyzer 1. 2.3. Preparation of MDCK cell-coÕered membrane For transport studies, MDCK cells were seeded on the inner surfaces of hollow fiber capillaries from Altra Novaw dialyzer. Prior to seeding with MDCK cells, inner surfaces of the membrane were coated with bovine type 1 collagen ŽCollaborative Biomedical Products, Bedford, MA. and incubated at 378C overnight for polymerization. The cells Žin the above mentioned culture medium. were seeded inside the hollow fibers at a density of 6 = 10 6 to 1 = 10 7 MDCK cells mly1 . The cell-seeded capillary membranes were kept in the incubator for 2 weeks and the medium was exchanged at intervals of 48 h, and then placed in a flow chamber for transport studies. Two methods were used to verify the confluency of epithelial cell monolayer. 2.3.1. Phenol red transport study The concentrations of phenol red were measured in perfusate and filtrate obtained from all the mini-filters Žfor description of experiment and terminology, please see Section 2.4.. The same measurements were performed with the cell-uncovered hollow fiber membranes as controls. At start of the studies, RPMI 1640 medium was replaced by hormone-supplemented, serum-free medium as described by Taub et al. w8x containing 15 mg ly1 phenol
1
The wall thickness of the membrane is 30 mm, and internal diameter is 195 mm. The Altra Novaw is a conventional dialyzer with ultrafiltration coefficient of 7.1 ml mm Hgy1 hy1 1.4 my2 with bovine blood at 378C. Urea and creatinine clearance of 161 and 148 ml miny1 , respectively, at Q B s 200 ml miny1 , Q D s 500 ml miny1 and Qf s10 ml miny1 .
red as perfusate, and the flow rate of perfusate was kept at 25 ml miny1 . The hormone-supplemented, serum-free medium was made of an 1:1 mixture of Ham’s F-12 nutrient mixture ŽGibco. and Dubelcco’s modified Eagle’s medium ŽGibco., to which selenium, transferrin, insulin, hydrocortisone, triiodothyronine and prostaglandin E 1 were added. The mini-filters were kept in a water bath at 378C and hydraulic pressure was applied to maintain perfusate under 50 mm Hg during the studies. Samples were alkalinized with NaOH and 800 ml of each sample was applied to Lambda Bio UVrVIS Spectrometer ŽPerkin-Elmer, Ueberlingen, Germany. to determine phenol red concentration by measuring UV absorbance at 560 nm. Leak rate of phenol red of each filter was calculated as concentration ratio of phenol red filtraterperfusate in terms of UV absorption at 560 nm. 2.3.2. Scanning electron microscopy MDCK cell monolayers on hollow fiber capillaries after the studies were perfused with PBS and then fixed by perfusing the interior of hollow fiber capillary with 1.5% gluteraldehyde in PBS at 48C for 60 min. The fibers were then dehydrated with 35, 50, 75, 90, 95 and 100% of absolute alcohol for 20 min each, dried with critical point dryer, Samdri w -PVT-3B ŽTousimis Research, Rockville, MD., sputter coated with gold–palladium using SEM Coating Unit E 5100 ŽPolaron Instruments, Hatfield, PA. and examined under a scanning electron microscope JSM 35C ŽJEOL, Tokyo, Japan.. 2.4. ConÕectiÕe transport study Convective transport was investigated using eight mini-filters, each containing five hollow fibers, in which MDCK cells were grown on the inner surfaces. For control experiments, eight mini-filters without MDCK cells were used. During the experiments the mini-filters were kept in a water bath at 378C, but the fluid and tubes were exposed to ambient temperature ŽFig. 1.. In order to conduct experiments, peritoneal outflow from a PD patient was used. Patient had been free of peritonitis for a year and used 2.5% glucose Dianiel w solution ŽBaxter, MacGaw Park, IL. and donated PD drainage for the experiments. The drainage was used as perfusate for all the experiments. Medical grade silicon tubing ŽDow Corning, Midland, MI. was used for connecting the circuit. Hydraulic pressure was applied by gravity by adjusting the height of the perfusate container to maintain the pressure of 50 mm Hg inside the hollow fiber capillaries ŽFig. 1.. Flow rate of the perfusate through the mini-filter was kept at 25 ml miny1 using a mini-pump. The samples of PD drainage affluent, effluent and the filtrate through the cell-covered membranes were collected for chemical measurements. The same samples were also collected during the control experiments using cell-uncovered mini-filters.
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tion of phenol red crossed the MDCK cell-covered membrane. In contrast, 0.98 " 0.04 phenol red leaked through the cell-uncovered membrane Ž P - 0.0001, experimental vs. control.. Thus, the epithelial cells layered the inside of fibers without space in between preventing any significant leaks. Concentrations of several plasma constituents in perfusate affluent, effluent and filtrate obtained across MDCK cell-covered Žexperimental. and -uncovered membranes Žcontrol. are shown in the Table 1. Concentrations of nitrogen metabolites, electrolytes and glucose in controls were not significantly different from those of perfusate affluent and effluent. In contrast, concentrations of these plasma constituents were significantly lower in the ultrafiltrate than in perfusate affluent or effluent Ž P - 0.0001.. Fig. 1. Schematic drawing of experiment for transport study. Peritoneal dialysis ŽPD. drainage was perfused at a rate of 25 ml miny1 under hydraulic pressure of 50 mm Hg, from inner to outer direction of hollow fiber capillaries, applied by gravity by adjusting the height of perfusate. During the experiments the mini-filters were kept in a water bath at 378C.
3.3. Hydraulic permeability Hydraulic permeabilities of control devices were 0.49 " 0.06 ml miny1 50 mm Hgy1 of hydraulic pressure.
2.5. Measurements of substances in perfusate and ultrafiltrate Urea nitrogen, creatinine, uric acid, sodium, potassium, chloride, and glucose concentrations in PD drainage affluent, effluent and the filtrate were measured by applying 0.6 ml each sample to the Automated Chemistry Analyzer Paramax 720ZX ŽBaxter.. 2.6. Statistics Results are expressed as means " S.D. Statistical significance is determined by the Student’s t-test.
3. Results 3.1. Cell morphology Cross-sectional scanning electron micrograph showed that MDCK cells formed a monolayer to confluence on inner surface of cellulose diacetate hollow fiber 2 weeks after seeding ŽFig. 2A.. The cells exhibited polarized morphologic characteristics including apical microvilli, tight junctions and desmosomes ŽFig. 2B.. The electron microscopic cross-sectional findings of the hollow fiber at 5 weeks after the cell seeding still showed a monolayer of the cells attached on the inner surface of membrane. 3.2. Transport characteristics Two kinds of transport studies were carried out with MDCK cell-covered and -uncovered filters, the former was phenol red and the latter, plasma constituents in PD effluent. In phenol red transport study, only 0.10 " 0.03 frac-
Fig. 2. ŽA. Cross-sectional scanning electron micrograph of MDCK cells grown on inner surface of cellulose diacetate hollow fiber 2 weeks after seeding Ž=480.. ŽB. Scanning electron micrograph of the same section of the fiber by higher magnification Ž=2600.. Short and long apical microvilli, tight junctions of MDCK cells were shown.
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CAPD drainage Filtrate Žcontrol. Filtrate Žexperimental. Effluent Žexperimental.
Urea nitrogen Žmg dly1 .
Creatinine Žmg dly1 .
Uric acid Žmg dly1 .
Glucose Žmg dly1 .
Sodium ŽmEq ly1 .
Chloride ŽmEq ly1 .
Potassium ŽmEq ly1 .
50.3"5.1 49.1"4.9 5.4"4.5) a 50.5"6.1
7.05"0.62 7.06"0.60 0.81"0.66) a 7.06"0.68
4.33"0.48 4.26"0.47 0.85"0.47) a 4.33"0.50
1020.7"150.1 1098.1"147.4 137.5"134.3) a 1073.3"149.3
137.3"3.1 137.9"3.3 21.3"11.6) a 138.1"4.0
104.2"1.2 104.5"1.1 17.8"13.8) a 104.6"1.4
2.50"0.26 2.53"0.18 0.56"0.14) a 2.53"0.30
)Comparison with CAPD drainage P - 0.0001. a Comparison with filtrate Žcontrol. P - 0.0001.
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Table 1 Concentrations of nitrogen metabolites, glucose and electrolytes in CAPD drainage, control filtrate, experimental filtrate and experimental effluent
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Hydraulic permeabilities of the experimental devices Žapical to basal permeability. were 0.28 " 0.04 ml miny1 50 mm Hgy1 . Thus hydraulic permeabilities of cell-covered membranes were significantly lower than those of the cell-uncovered membranes Ž P - 0.001..
4. Discussion This is the first time that regeneration of biological fluid across renal epithelial cell-covered membranes has been reported. Our results demonstrate marked reductions of solute concentrations in the regenerated filtrate through MDCK cell-covered membranes. Nitrogen metabolites such as urea, creatinine and uric acid, were significantly removed from ultrafiltrate. Thus, the regenerated filtrate can be used as a replacement fluid without risk of returning back the protein metabolites. However, electrolytes were also removed from the perfusate and if this method is to be used, electrolytes will have to be supplemented. Since the concentrations of nitrogen metabolites, electrolytes and glucose in perfusate effluent were not significantly different from that of affluent, the transport across the cells appears to be mostly convective. It appears that substances were rejected by MDCK cells, were not absorbed across the apical cell membrane and would not have accumulated in the cells. Small amount of these substances, however, may have been transported either from apical to basal direction intercellularly or transcellularly. The fraction of phenol red transported through the cell-covered membranes was only 0.10 " 0.03, which was similar to the fractions of urea Ž0.11 " 0.05. and creatinine Ž0.11 " 0.04. calculated from filtrate and CAPD drainage levels. However, the fractions of electrolytes transported across the device such as sodium Ž0.15 " 0.03. and chloride Ž0.17 " 0.05. were higher under the same conditions. This extra electrolytes transport has to be confirmed whether or not transcellular active transport contributes to the results. High Naq, Kq ATPase activity in MDCK cells has been reported w9,10x. Active transports of electrolytes has also been reported in distal convoluted tubule and collecting duct in animal kidneys w11–16x. However, electrolyte transport across the MDCK and LLC-PK 1 cellcovered membrane has not previously been reported. In order to use the devices for clinical purpose, some electrolytes will have to be supplemented. Alternatively, a combination of MDCK and LLC-PK 1 cells might be effective for better electrolyte transport. Naq, Kq ATPase activity w10x, gene expressions of electrolyte transporter such as NaqrHq exchanger w17x, Naqrglucose cotransporter w18x and Naqramino acid cotransporter w19x have been reported in LLC-PK 1 cells. LLC-PK 1 cell monolayer, however, was reported not to allow filtration in the apical to basal direction when positive transmembrane pressure of 5 to 30 mm Hg was applied on the apical side w5x.
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Measurable filtration in the basal to apical direction through the cell-covered membrane were confirmed w5x, however, the filtrate obtained in the basal to apical direction should not be returned to patient since the filtrate might contain heterogeneous proteins from damaged cells. Our study was done under 50 mm Hg of hydraulic pressure in the apical to basal direction, and MDCK cell monolayers were able to maintain function during and after the study. Histological assessment demonstrate confluent monolayers of the MDCK cells along the inner surfaces of hollow fiber membranes. Monolayers of MDCK cells on the fibers were able to keep their functional confluency during and after studies of convective transport. It took 2 weeks to achieve functional confluency. Three of the MDCK cell-covered membrane filters were confirmed to keep almost the same permeability performance 3 weeks after the first permeability studies Ždata not shown.. We have previously reported that an ultrafiltration rate of 7 ml miny1 with continuous hemofiltration kept plasma levels of small molecules at negative 30 to negative 60% of pre-dialysis values of intermittent conventional dialysis w20x. Allowing for daily intake of fluid the net replacement fluid would be required at the rate of 4 ml miny1 . Our device with a surface area of 2.9 cm2 produced 0.28 ml miny1 of regenerated ultrafiltrate. Thus, a device with 1 m2 surface area should produce at least 0.97 ml miny1 of ultrafiltrate. Since this rate is not yet practical for clinical use, further investigation should be done to increase the ultrafiltration rate across cell-covered membranes. PD drainage was used as perfusate in this study for several reasons. Main reasons for this include, the PD drainage is usually sterile and contains important proteins and other substances necessary for the cell growth such as albumin, transferrin, growth factors, glucose, etc. w21x. In addition, it also contains considerable amounts of uremic toxins, which allowed us to study the kinetics of these metabolites. However, the unused fresh PD solution does not seem to be good for cell culture, when the cells were cultured with fresh 1.5% glucose Dianiel w solution, 80% of the cells died within 48 h due to low pH of the solution Ždata not shown.. In previous reports w3,6x, high-flux Polysulfon membrane filters were used, through which plasma proteins can pass. Cellulose diacetate membrane used in this study are generally impermeable to proteins. It is important for this system to protect against the penetrance of immunologically competent cells and heterogenic proteins from damaged cells. Newer high flux membranes may be less permeable to proteins and could be used since ultrafiltration rate might be higher without the risk of cross-contamination. References w1x P. Aebischer, T.K. Ip, L. Miracoli, P.M. Galletti, Trans. Am. Soc. Artif. Intern. Organs 33 Ž1987. 96–102.
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