Abstract

Background. Haemodialysis therapy does not provide renal tubule function, such as active fluid and solute transport, nor metabolic or endocrine action. Moreover, this treatment is usually associated with serious complications and high mortality. We constructed a bioartificial renal tubule device by using renal tubule epithelial cells in an artificial membrane, and evaluated transport properties of the device for 2 weeks.

Methods. A renal epithelial cell line, LLC-PK1 (Lewis-lung cancer porcine kidney), was seeded on polysulfone hollow fibres in small and large modules. We studied perfusion and leakage of urea nitrogen (UN) and creatinine (Cr), as well as reabsorption of water, glucose and sodium for a period of 2 weeks.

Results. Cell-lined hollow fibre membranes significantly reduced the leakage of UN and Cr throughout the 2 week period. Reabsorption of water, glucose and sodium were adequate from days 3 to 10 and gradually decreased thereafter. LLC-PK1 cells actively transported these substances. Scanning electron microscopy revealed that cells in the hollow fibres on day 8 became completely confluent. However, they became multi-layered and almost obstructed the hollow fibres on day 13.

Conclusions. This bioartificial renal tubule device functioned to reabsorb water, glucose and sodium for ∼10 days. This is the first report of successful long-term evaluation of a bioartificial renal tubule device. This device, in combination with continuous haemofiltration, may provide treatment to prevent complications of dialysis and raise the quality of life in chronic renal failure patients.

Introduction

Haemodialysis therapy, also known as artificial kidney treatment, saves the lives of end-stage renal failure patients. However, associated with this are serious complications and high mortality rates. Increases in complications due to repeated and long-term intermittent dialysis are especially prevalent among patients with chronic renal failure. Although conventional haemodialysis therapy may replace haemofiltration function, it does not perform the reabsorption, excretion, metabolic and endocrine functions of renal tubules. Failure to reabsorb filtered extracellular sodium during dialysis leads to impairment of intravascular fluid refilling and reduced blood volume, resulting in hypotension [1]. Reduced vitamin D activation may cause secondary hyperparathyroidism and subsequent decrements in bone density, leading to renal osteodystrophy [2]. An inability to metabolise and remove the filtered low molecular-weight proteins, such as β2-microglobulin, produces amyloidosis in long-term haemodialysis patients [3] that is clinically manifested as carpal tunnel syndrome and destructive arthropathy associated with cystic bone lesions [4]. In addition, uraemic patients have high serum levels of advanced glycation end products (AGE) formed from non-enzymatic glycation and oxidation of proteins [5]. AGE levels are highly correlated with the levels of carbonyl compounds [6], which are responsible for carbonyl stress that causes amyloidosis [7] and atherosclerosis [8].

Saito et al. [9] reported that continuous haemofiltration therapy of 10 l/day in chronic renal failure patients maintained a lower level of blood urea nitrogen (UN), creatinine (Cr) and β2-microglobulin, compared with intermittent conventional haemodialysis therapy given three times a week. To prevent the complications of dialysis, we have been developing a portable bioartificial kidney using a continuous haemofilter and a bioartificial renal tubule device, which is composed of renal tubule epithelial cells and an artificial membrane that purifies ∼10 l of blood per day.

Humes et al. [10] reported the use of a bioartificial renal tubule assist device that uses a hollow fibre module and porcine renal tubule progenitor cells. Their short-term study demonstrated that the device created satisfactory fluid, bicarbonate, sodium, glucose and other standard transport properties, as well as adequate metabolic and endocrine activities, such as ammonia production and vitamin D activation in cells. In the current study, we evaluated long-term transport abilities of a bioartificial renal tubule device that used improved hollow fibres and LLC-PK1 porcine renal proximal tubule epithelial cell lines in order to reproduce the functions of in vivo renal tubules. We first evaluated methods for uniformly seeding the cells, for the transport of fluid and solutes, and for the maintenance of a confluent monolayer in small modules. Scaled-up experiments were then performed with large modules for 2 weeks for studying potential future clinical use.

Subjects and methods

Cell culture

We used the renal epithelial cell line, LLC-PK1, that originated from American Type Culture Collection (ATCC, Rockville, MD). The cells were purchased from the Health Science Research Resources Bank (HSRRB, Osaka, Japan). They were cultured at 37°C in Dulbecco's modified Eagle medium (glucose concentration 400 mg/dl; Gibco, Invitrogen Corporation, Grand Island, NY), containing 10% fetal bovine serum (FBS; Biosource International, Camarillo, CA), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). The same medium was used as a basal medium in all of the transport studies described below. The cells were cultured in 225 cm2 tissue culture flasks (Iwaki, Asahi Techno Glass Corporation, Chiba, Japan) or 1700 cm2 expanded surface roller bottles (Corning Incorporated, Corning, NY) at a density of 1–3 × 104 cells/cm2. Culture medium was refreshed three times a week. The cells were removed by rinsing the bottle once with phosphate-buffered solution without magnesium and calcium (PBS; Takara Bio Inc., Shiga, Japan) before incubation with 0.1% trypsin-ethylenediaminetetraacetic acid (Cosmo Bio Co., Ltd, Tokyo, Japan). After incubation for 10 min at 37°C, the reaction was stopped by adding the medium containing 10% FBS. Third-passage cells were used for module studies.

Preparation of modules

The small and large polysulfone hollow fibre modules were kindly supplied by Nipro, Osaka, Japan. The hollow fibre had an inner diameter of 300 µm. Its membrane was 100 µm thick and had a molecular weight cut-off of 6000 Da. A small module contained 40 hollow fibres of 17 cm in length, with an effective intraluminal membrane surface area of 56 cm2 and inner compartment volume of 0.5 cm3. The large one contained 1600 hollow fibres of 34 cm in length with effective intraluminal membrane surface area of 4000 cm2 and inner compartment volume of 35 cm3. The inner surfaces of the hollow fibres were coated with 40 µg/ml synthetic extra cellular matrix (ECM) pronectin-F (Sanyo Chemical Industries, Kyoto, Japan) before seeding with 1–2 × 107 LLC-PK1 cells/ml. To achieve a uniform distribution of cells on the entire intraluminal surface of the hollow fibres, cell-seeding was performed four times at intervals of 1 h each, during which the module was rotated 90°. Culture medium was circulated at a rate of 0.25 ml/min for the small module and 20 ml/min for the large module on the outer compartment of the module during this period to supply oxygen and nutrients to the cells. At approximately 1 h after final seeding, media was circulated in the inner compartment at a rate of 0.25 ml/min for the small module and 20 ml/min for the large module. For small modules, a single-pass perfusion was performed throughout the experiment. For the large modules, a closed-circuit circulation perfusion was performed except during transport studies when a single-pass perfusion was performed. Medium was fed with 5% CO2 in air at a rate of 1 l/min through a hollow fibre oxygenator (UOXY 10, membrane surface area 0.6 m2; Unisyn Technologies, Hopkinton, MA). Oxygen content and pH of the medium was monitored with a pH, DO controller (Mettler-Toledo Process Analytical Inc., Wilmington, MA), and 100% oxygen was fed when oxygen levels fell below 5.5 p.p.m. The culture medium was replaced when pH fell below 7.2 (Figure 1).

Fig. 1.

Schematic presentation of the large hollow fibre module experiment. Dotted line represents the closed-circuit circulation perfusion when the single-pass perfusion study for fluid and substances transport was not performed.

Fig. 1.

Schematic presentation of the large hollow fibre module experiment. Dotted line represents the closed-circuit circulation perfusion when the single-pass perfusion study for fluid and substances transport was not performed.

Transport studies

Small hollow fibre modules

At 5–6 h after the first seeding with the cells, the inner compartment of the module was perfused with the basal medium containing 50 mg/dl UN and 5.0 mg/dl Cr, at a flow rate of 0.25 ml/min. The outer compartment was perfused with the basal medium without UN and Cr at the same flow rate. At 2 days after cell seeding, inlet bottles from both the compartments were replaced with new medium, and leakage of UN and Cr, and reabsorption of water, glucose and sodium were evaluated for 24 h. Synthetic ECM pronectin-F-coated hollow fibres without cells were used as a control. In another series of experiments, 2.5 g/dl of bovine serum albumin (Wako, Osaka, Japan) was added to the basal medium perfusing the outer compartment in order to study the influence of oncotic pressure on the movement of substances. To study the effect of aquaporin inhibition, cells were fixed for 5 minutes in ice-cold 0.15 mol/l cacodylate buffer (pH 7.4) containing 1% glutaraldehyde. To this, 1 mmol/l HgCl2 was then added to the medium in the inner compartment and it was perfused through the inner compartment for 5 min before the transport studies [11]. To study the influence of active transport inhibition, 0.1 mmol/l ouabain, an inhibitor of Na+, K+-ATPase, was added to the outer compartment medium. For all of these transport studies, samples were taken before and after perfusion from the medium bottles of inner and outer compartments, and the media were also weighed. We measured concentrations of UN, Cr, glucose and sodium, and calculated movement of substances across the hollow fibre membrane.

Large hollow fibre modules

Evaluations of leakage of UN and Cr, and reabsorption of water, glucose and sodium through the hollow fibres were performed by single-pass perfusion, at 24 h after cell seeding, and at every 2–3 days thereafter up to 2 weeks. The basal medium perfusing the inner compartment was added with UN and Cr at the same concentration as in the study using small hollow fibre modules, and the medium perfusing the outer compartment was without UN or Cr. The flow rate was set at 20 ml/min for both the inner and outer compartments. Inlet bottles of both compartments were replaced with new medium and single-pass perfusion was performed for 90 min, and movement of substances across the hollow fibres was evaluated. We used synthetic ECM pronectin-F-coated hollow fibres without cells as a control. To study the influence of oncotic pressure on the movement of substances, 2.5 g/dl albumin was added to the basal medium perfusing the outer compartment, as was done with the small hollow fibre modules. In this experiment, single-pass perfusion was performed for 45 min. Inhibition of sodium-dependent glucose transport was studied by adding 1 mmol/l phlorizin to the basal medium perfusing the inner compartment. In all experiments, samples were taken from the medium bottles, before and after perfusion and the media were also weighed (Figure 1). We measured concentrations of glucose, sodium, UN and Cr using standard laboratory assays and calculated movement of substances across the hollow fibres.

Determination of leakage and reabsorption

The net movement of substances from inner to outer compartment was calculated as follows:

Net movement of the substance = amount collected in the outlet bottle from the outer compartment – amount that passed to the outer compartment – amount in the dead space

Each amount of the substance was calculated by multiplying the concentration of the substance by the respective volume:

Net movement of the substance = CO × VOCI × (VIVR − VD) − CD × VD

CO, concentration of the substance in the outlet bottle of the outer compartment; VO, volume in the outlet bottle of the outer compartment; CI, concentration of the substance in the inlet bottle of the outer compartment; VI, volume in the inlet bottle of the outer compartment at the start of the experiment; VR, volume remaining in the inlet bottle of the outer compartment at the end of the experiment; CD, concentration of the substance in the dead space at the start of the experiment; VD, volume of the dead space.

The volume of the dead space in the small module studies was so small compared with the 24 h perfusate (360 ml) that it was not included in the calculations.

Therefore, the net movement of substances from inner to outer compartment in small modules was calculated as follows:

Net movement of the substance = CO × VOCI×(VIVR)

The net movement of UN or Cr from the inner to outer compartment in the direction of the concentration and pressure gradients was designated as the leakage. It was also presented as a percentage of the total amount that had passed from the inner compartment. Leakage of UN and Cr served as an indicator of the confluency of LLC-PK1 cells on the hollow fibre membranes. A smaller leakage indicated a greater confluency of the cells.

The net movement of glucose or sodium from the inner to outer compartment was designated as reabsorption by LLC-PK1 cells, since their concentrations at the inlets of the inner and outer compartments were almost the same.

Scanning electron microscopy

After 4, 8 and 13 days of incubation, the modules were disconnected from the incubation system for examination by scanning electron microscopy. The cells were first rapidly washed with 0.1 mol/l PBS once and prefixed with 2.5% glutaraldehyde in 0.05 mol/l PBS for 2 h at 4°C. After being washed once with 0.1 mol/l PBS, the fibres were recovered from the module and sectioned into pieces. Then, the samples were washed twice in 0.1 mol/l PBS and fixed with 1% osmium tetroxide in 0.05 mol/l PBS for 1 h at 4°C. After that, the samples were dehydrated in a graded series of ethanol solutions, passed through three changes of 100% t-butyl alcohol, and finally dried in a freeze drying device (JFC-310; JOEL, Tokyo, Japan). After being coated with gold in an ion sputter coater (JFC-1100; JEOL), cell-attached hollow fibres were examined with a scanning electron microscope (JSM-840A; JOEL).

Statistical analysis

Data are shown as means±SEM. Comparisons of data were done using two-tailed Student's t-tests, either paired or nonpaired, by employing Microsoft Excel software. A P-value of ≤0.05 was considered statistically significant.

Results

Leakage of UN and Cr and transport studies in small hollow fibre modules

To evaluate the confluency of LLC-PK1 cells in the small hollow fibre module, UN and Cr were added to the basal medium perfusing the inner compartment, and their leakages to the outer compartment were measured. Leakages of UN and Cr in small modules without LLC-PK1 cells were 40±3 and 36±2%, respectively, and were 8±3 and 7±2%, respectively, in modules with cells on day 2 of cell-seeding (P<0.05) (Table 1). The reabsorption of water, glucose and sodium by LLC-PK1 cells in small modules at 2 days after seeding were 14±2 ml/day, 27±2 mg/day and 1.6±0.3 mEq/day, respectively (Table 1). When 2.5 g/dl albumin was added to the basal medium perfusing the outer compartment to study the influence of oncotic pressure, reabsorption of water significantly increased from 0.5±0.1 to 1.0±0.2 ml/hr (Table 2). In contrast, HgCl2, an inhibitor of aquaporin, significantly decreased the reabsorption of water from 0.5±0.1 to 0.2±0.1 ml/hr. Similarly, ouabain, an inhibitor of Na+,K+-ATPase, significantly decreased the reabsorption of sodium from 0.06 ± 0.02 to 0.03 ± 0.02 mEq/h (Table 2), indicating that transport of water and electrolytes was facilitated by aquaporin and Na+, K+-ATPase in the LLC-PK1 cells.

Table 1.

Leakage and reabsorption of substances in small modules

 Leakage
 
  Reabsorption
 
 
 UN (%) Cr (%) H2O (ml/day) Glucose (mg/day) Na+ (mEq/day) 
Control group (without cell) 40±3 36±2 NA NA NA 
Study group (with cell) 8±3* 7±2* 14±2 27±2 1.6±3 
 Leakage
 
  Reabsorption
 
 
 UN (%) Cr (%) H2O (ml/day) Glucose (mg/day) Na+ (mEq/day) 
Control group (without cell) 40±3 36±2 NA NA NA 
Study group (with cell) 8±3* 7±2* 14±2 27±2 1.6±3 

Experiments were performed on day 2. n = 6 each. Values are expressed as means±SEM.

*P<0.05 vs control group (without cell). NA, negligible amount.

Table 2.

Reabsorption of water and sodium in small modules under the influence of enhancer or inhibitors

 H2O (ml/h) H2O (ml/h) Na+ (mEq/h) 
Control group (base line) 0.5±0.1 0.5±0.1 0.06±0.02 
Study group (enhancer/inhibitor) 1.0±0.2* (albumin) 0.2±0.1* (HgCl20.03±0.02* (ouabain) 
 H2O (ml/h) H2O (ml/h) Na+ (mEq/h) 
Control group (base line) 0.5±0.1 0.5±0.1 0.06±0.02 
Study group (enhancer/inhibitor) 1.0±0.2* (albumin) 0.2±0.1* (HgCl20.03±0.02* (ouabain) 

Albumin enhances the osmotic pressure, HgCl2 inhibits the aquaporin and ouabain inhibits the Na+,K+-ATPase. Experiments were performed on day 2. n = 6 each. Values are expressed as means±SEM.

*P<0.05 vs control group.

Leakage of UN and Cr as well as transport studies in large hollow fibre modules

UN and Cr leakage. To monitor the confluency of LLC-PK1 cells in the hollow fibres of large modules, UN and Cr were added to the basal medium perfusing the inner compartment, and leakage of UN and Cr through the hollow fibres was measured for 90 min every 2–3 days. Leakages of UN and Cr in large modules without LLC-PK1 cells were 44 ± 1.3 and 40 ± 2.4%, respectively, but were ∼10% in modules with cells up to 13 days after cell-seeding (Figure 2). A similar tendency was observed in the concentration ratio of exiting tubular fluid to the presenting ultrafiltrate (TF/UF) of the inner compartment and for the absolute leak rate (Table 3).

Fig. 2.

Leakage of UN and Cr in large modules with or without cells. Mean ± SEM (n = 4 for without cells; n = 6 for with cells up to day 8; n = 2 on days 10 and 13). *P<0.05 vs without cell.

Fig. 2.

Leakage of UN and Cr in large modules with or without cells. Mean ± SEM (n = 4 for without cells; n = 6 for with cells up to day 8; n = 2 on days 10 and 13). *P<0.05 vs without cell.

Table 3.

TF/UF ratios and absolute leak rates of UN and Cr in large modules

  Without Cell With Cell (day)
 
     
   10 13 
 Alb− 0.7 ± 0.03 0.84 ± 0.04 0.85 ± 0.04 0.82 ± 0.03 0.87 ± 0.03 0.80 ± 0.1 0.76 ± 0.08 
  (327 ± 6.5) (143 ± 36)* (161 ± 10)* (168 ± 5)* (131 ± 20)* (190 ± 46) (125 ± 45)* 
UN         
 Alb+ 0.66 ± 0.01 0.80 ± 0.03* 0.82 ± 0.02* 0.82 ± 0.01* 0.83 ± 0.02* 0.86 ± 0.14  
  (353 ± 3.0) (205 ± 44) (211 ± 29) (216 ± 55) (196 ± 19) (242 ± 56)  
 Alb− 0.74 ± 0.02 0.86 ± 0.05 0.87 ± 0.04 0.86 ± 0.02 0.91 ± 0.03 0.83 ± 0.09 0.80 ± 0.08 
  (25.5 ± 0) (11 ± 4.0) (12 ± 2.6) (13 ± 0.4) (11 ± 1.3) (16 ± 4.1) (11 ± 5.3) 
Cr         
 Alb+ 0.71 ± 0.01 0.82 ± 0.03 0.85 ± 0.01* 0.84 ± 0.02* 0.85 ± 0.02* 0.90 ± 0.2  
  (29.7 ± 0.4) (20 ± 4.1) (18 ± 2.6) (18 ± 4.7) (17 ± 0.9) (19 ± 7.1)  
  Without Cell With Cell (day)
 
     
   10 13 
 Alb− 0.7 ± 0.03 0.84 ± 0.04 0.85 ± 0.04 0.82 ± 0.03 0.87 ± 0.03 0.80 ± 0.1 0.76 ± 0.08 
  (327 ± 6.5) (143 ± 36)* (161 ± 10)* (168 ± 5)* (131 ± 20)* (190 ± 46) (125 ± 45)* 
UN         
 Alb+ 0.66 ± 0.01 0.80 ± 0.03* 0.82 ± 0.02* 0.82 ± 0.01* 0.83 ± 0.02* 0.86 ± 0.14  
  (353 ± 3.0) (205 ± 44) (211 ± 29) (216 ± 55) (196 ± 19) (242 ± 56)  
 Alb− 0.74 ± 0.02 0.86 ± 0.05 0.87 ± 0.04 0.86 ± 0.02 0.91 ± 0.03 0.83 ± 0.09 0.80 ± 0.08 
  (25.5 ± 0) (11 ± 4.0) (12 ± 2.6) (13 ± 0.4) (11 ± 1.3) (16 ± 4.1) (11 ± 5.3) 
Cr         
 Alb+ 0.71 ± 0.01 0.82 ± 0.03 0.85 ± 0.01* 0.84 ± 0.02* 0.85 ± 0.02* 0.90 ± 0.2  
  (29.7 ± 0.4) (20 ± 4.1) (18 ± 2.6) (18 ± 4.7) (17 ± 0.9) (19 ± 7.1)  

n = 2 each for the group without cells, and for day 13 with cell group. n = 3 each for the remaining groups with cells. Alb+ and Alb− imply with or without albumin addition. Values are expressed as means ± SEM. Data in the parentheses are absolute leak rates in mg/dl.

*P<0.05 vs without cell group.

Reabsorption of water

The reabsorption of water from the inner to the outer compartment of large modules increased gradually and reached a plateau (100 ml/90 min) on day 6 after cell seeding. Reabsorption then decreased gradually to 80 ml/90 min on day 13 (Figure 3). When albumin was added to the basal medium perfusing the outer compartment, reabsorption of water significantly increased on day 1 and day 3 (160 ml/90 min and 170 ml/90 min, respectively) compared with the group not given albumin (Figure 3). Reabsorption then decreased gradually to ∼120 ml/90 min on day 10. Although reabsorption tended to be higher than in the albumin group from day 6 onward too, this difference was not statistically significant (Figure 3).

Fig. 3.

Reabsorption of water in large modules with or without albumin addition. Mean ± SEM (for albumin not added, n = 6 up to day 8, and n = 2 on days 10 and 13; for albumin added, n = 3 up to day 8, and n = 2 on day 10). *P<0.05 vs albumin not added.

Fig. 3.

Reabsorption of water in large modules with or without albumin addition. Mean ± SEM (for albumin not added, n = 6 up to day 8, and n = 2 on days 10 and 13; for albumin added, n = 3 up to day 8, and n = 2 on day 10). *P<0.05 vs albumin not added.

Reabsorption of glucose

The reabsorption of glucose from the inner to the outer compartment of the large module was ∼300 mg/90 min or more during days 3–10 (Figure 4). When albumin was added on day 1 to the basal medium perfusing the outer compartment, glucose reabsorption significantly increased to twice or more above the group not given albumin (Figure 4). This difference narrowed and lost statistical significance on the following days. When sodium-dependent glucose reabsorption was inhibited by the addition of phlorizin to the basal medium perfusing the inner compartment, glucose reabsorption decreased significantly from 260 to 125 mg/90 min (P<0.02) (Figure 5), indicating that glucose reabsorption was facilitated by the sodium-dependent glucose transporter (SGLT).

Fig. 4.

Reabsorption of glucose in large modules with or without albumin addition. Mean ± SEM (for albumin not added, n = 6 up to day 8, and n = 2 on days 10 and 13; for albumin added, n = 3 up to day 8, and n = 2 on day 10). *P<0.05 vs albumin not added.

Fig. 4.

Reabsorption of glucose in large modules with or without albumin addition. Mean ± SEM (for albumin not added, n = 6 up to day 8, and n = 2 on days 10 and 13; for albumin added, n = 3 up to day 8, and n = 2 on day 10). *P<0.05 vs albumin not added.

Fig. 5.

Reabsorption of glucose in large modules with or without phlorizin. Mean ± SEM (n = 3 each). *P<0.02 vs without phlorizin.

Fig. 5.

Reabsorption of glucose in large modules with or without phlorizin. Mean ± SEM (n = 3 each). *P<0.02 vs without phlorizin.

Reabsorption of sodium

The reabsorption of sodium was similar to that of water and glucose, and was at 10 mEq/90 min or more during days 3–13, with the peak of 19 mEq/90 min on day 8 (Figure 6). When albumin was added to the basal medium perfusing the outer compartment, sodium reabsorption was significantly greater on days 1 and 3 (25.5 mEq/90 min and 26.2 mEq/90 min, respectively, P<0.05 vs albumin not added). It decreased thereafter on days 8 and 10 to levels even lower in the group not given albumin. On day 10, the albumin group had reabsorption that was significantly lower than on day 1.

Fig. 6.

Reabsorption of sodium in large modules with or without albumin addition. Mean ± SEM (for albumin not added, n = 6 up to day 8, and n = 2 on days 10 and 13; for albumin added, n = 3 up to day 8, and n = 2 on day 10). *P<0.05 vs albumin not added. **P<0.05 vs albumin added day 1.

Fig. 6.

Reabsorption of sodium in large modules with or without albumin addition. Mean ± SEM (for albumin not added, n = 6 up to day 8, and n = 2 on days 10 and 13; for albumin added, n = 3 up to day 8, and n = 2 on day 10). *P<0.05 vs albumin not added. **P<0.05 vs albumin added day 1.

Scanning electron microscopy

Hollow fibres were recovered from the large modules on days 4, 8 and 13, and were observed under a scanning electron microscope (Figure 7). The hollow fibre membrane was covered with a confluent monolayer of cells on day 4 (Figure 7A). On day 8, the cells became completely confluent and revealed a large number of microvilli, although there were a few areas of multi-layered cells (Figure 7B). However, the cells on day 13 became multi-layered inside the hollow fibre, and the microvilli were shorter and in smaller number than those observed on day 8 (Figure 7C).

Fig. 7.

Scanning electron micrographs of hollow fibres retrieved from the large modules on day 4 (A), day 8 (B) and day 13 (C). Arrows indicate the hollow fibres. Cracks and fissures (arrowheads) seen on the surface of the cell layer are artefacts that appear during preparation of the samples. Scale bars = 20 µm.

Fig. 7.

Scanning electron micrographs of hollow fibres retrieved from the large modules on day 4 (A), day 8 (B) and day 13 (C). Arrows indicate the hollow fibres. Cracks and fissures (arrowheads) seen on the surface of the cell layer are artefacts that appear during preparation of the samples. Scale bars = 20 µm.

Discussion

The major obstacles in the development of an artificial renal proximal tubule device are the obtaining of a large number of viable renal tubule cells and an adequate and even lining of the intraluminal surface of hollow fibres of the modules with these cells in order to maintain cell function over a longer period of time. To examine the potential of using cells from xenogenic origins, we compared three immortalized cell-lines, including JTC-12 (Japan Tissue Culture-12, monkey renal proximal tubule cell line), LLC-PK1 and MDCK (Madin-Darby canine kidney cell line), to test their ability for expansion, their compatibility with membranes, and their viability during manipulation [12]. Among these, the LLC-PK1 cell line was best characterized as having properties of renal epithelial cells and that showed easy expansion into large numbers in in vitro culture. LLC-PK1 cells have also been reported to provide a reliable model of renal proximal tubule cells in a number of studies [13,14]. They displayed typical characteristics of renal proximal tubule cells, including tight junctions, dome formation, sodium-dependent phlorizin-sensitive glucose transport and sodium-dependent amino acid and phosphate transport. Moreover, LLC-PK1 cells maintained monolayer formation for a long period on membranes coated with an ECM [15].

Humes et al. [10] constructed a bioartificial renal tubule assist device by seeding porcine renal proximal tubule cells into the intraluminal spaces of the hollow fibres and then studied the short-term metabolic and endocrine functions of these tubule cells. They found that the cell-seeded module required at least 14 days of incubation to obtain the confluent layer of primary cells before starting their study. In our study, LLC-PK1 cells reached confluency within 24 h after seeding, which saved time, labour and expense. This decreased time to confluency was probably due to an improved polysulfone hollow fibre surface. Our surface was hydrophobic since it was not coated with polyvinyl pyrrolidone (PVP), and as a result the cells showed easy and firm adhesion. Our hollow fibres had a molecular weight cut-off of 6000 Da and wall thicknesses of 100 µm, and those by Humes et al. [10] were 45 000–50 000 Da and 40–70 µm. The smaller molecular weight cut-off with thicker-walled hollow fibres can more efficiently shut out non-self proteins and other unwanted particles from entering the body of the patient, thus ensuring better safety in future clinical uses. The inner diameter of 300 µm was wide enough for the culture medium to circulate uniformly in the hollow fibres and to supply sufficient nutrients to the cells.

Substances may move across a membrane covered with a cell layer according to three pathways: by transport through the cell membranes (transcellular route), by transport through the junctional spaces between the cells (paracellular route), and by leakage through areas where cells fail to cover. For an ideal bioartificial renal tubule, cells should completely cover the intraluminal surface of the hollow fibres with a confluent monolayer. This suppresses leakage to a minimum and sustains this condition and function for a long period. We assessed the confluency of the cell layer through the movement of UN and Cr across the hollow fibre membrane, as both were an indicator of leakage. Urea is partly reabsorbed by renal tubules although Cr is not reabsorbed under physiological conditions [16]. In a previous study, we found that tight junctions between the LLC-PK1 cells of a confluent monolayer were well preserved when cultured on a membrane filter (Transwell, Corning), and that there was significantly less movement of UN and Cr from the apical to the basolateral side of the cells under 15–20 mmHg hydrostatic pressure plus 100 mOsm osmotic pressure for 2 h compared with the control group without cells [12]. Inulin, which is not physiologically excreted or reabsorbed by renal tubule cells, may be a more appropriate indicator of leakage. However, the molecular weight of inulin (∼5000) was as large as the molecular weight cut-off of our hollow fibre membrane. Moreover, inulin leakage was as low as 10% through the hollow fibre membrane without cells in our preliminary study. Therefore, leakage of relatively low molecular weight UN and Cr was designated to represent the confluency status of cells on the intraluminal surfaces of hollow fibres in the current study.

Leakage and reabsorption through the hollow fibre membrane were first studied using the small modules (effective surface area 56 cm2), which were made of the same materials as the large modules (effective surface area 4000 cm2). The leakage of UN and Cr, as well as the reabsorption of water, glucose and sodium by LLC-PK1 cells in the small module indicated that cell confluency was achieved and the cells actively transported fluid and substances (Table 1). Moreover, after treatment with HgCl2, an aquaporin inhibitor, reabsorption of water decreased significantly to ∼40% of that without HgCl2 treatment. To avoid toxic effects of HgCl2 on living cells, the cells were pre-treated with glutaraldehyde, which preserves water permeability characteristics of urinary bladders and renal descending vasa recta [11]. In addition, in the presence of ouabain, a Na+,K+-ATPase inhibitor, reabsorption of sodium significantly decreased to ∼50% of that without ouabain (Table 2). These results reconfirmed that aquaporin-facilitated water reabsorption and sodium-dependent active transport by LLC-PK1 cells occurred inside the small module [13,14]. After achieving the confluent monolayer of LLC-PK1 cells in the small module by the method of cell seeding and the materials employed, we scaled-up the large module studies while examining possibilities of future clinical use.

The leakage of UN and Cr in large modules with LLC-PK1 cells was significantly lower than in modules without cells throughout the 2 week period (Figure 2), indicating that cell confluency inside the large modules was achieved. There was, however, some UN and Cr leakage (Figure 2 and Table 3). Tight junctions between proximal tubule cells are weaker than those between distal tubule cells, and it was likely that UN and Cr diffused through the intercellular pathways followed by the osmotic movement of water. Moreover, ∼50% of urea that is filtered into renal tubules is passively reabsorbed in kidneys under physiological conditions [16].

The reabsorption of water and glucose gradually increased from day 3, reached a plateau on days 6–10, and tended to decrease thereafter (Figures 3 and 4). It has been previously shown that LLC-PK1 cells reabsorb glucose by a SGLT as they became confluent, and that phlorizin inhibited the SGLT [17,18]. In agreement, we found that addition of phlorizin to the medium perfusing the inner compartment caused a significant decrease in glucose reabsorption from 260 to 125 mg/90 min (P<0.02) (Figure 5), indicating that glucose reabsorption by the LLC-PK1 cells lining the hollow fibres was facilitated by SGLT. Sodium reabsorption gradually increased and reached a plateau on day 8 (Figure 6). These findings suggested that the expression of Na+, K+-ATPase increased in parallel with the formation of tight junctions between the cells after they became confluent. They further indicated that after day 10, overgrowth of the cells appeared to form multiple layers, resulting in the blockage of substance transport. At that time, even the addition of albumin failed to cause increases in sodium reabsorption. Although immortalized cell lines such as LLC-PK1 tend to exhibit overgrowth after confluency, they have the advantage of higher viability and expandability in in vitro cultures, compared with primary cells. Ip et al. [15] reported that LLC-PK1 cells cultured on membranes coated with ECM containing the RGD (Arg-Gly-Asp) amino acid sequence maintained their monolayer and retained their ability for active glucose transport over a long term. Here, we also coated the intraluminal surfaces of the hollow fibres with pronectin-F, an ECM containing the RGD sequence. However, the cells became over-confluent and formed multiple layers over time (Figure 7C). We believe that this over-confluency produced the untoward effects on the transport of water, glucose and sodium. These adverse responses were more evident under the influence of increased osmotic pressure. When albumin was added to the medium perfusing the outer compartment, the reabsorptions of water, glucose and sodium were significantly higher than that in the group not given albumin on day 1 and/or day 3 (Figures 3, 4 and 6). However, this difference narrowed with time, which was probably due to the dense overgrowth of cells that partly blocked the movement of fluid and substances, even under the influence of osmotic pressure by day 10. The use of tissue-engineered cells that have contact inhibition between cells may prevent overgrowth and thus prolong the efficiency of this module.

We calculated that this module with a surface area of 0.4 m2 could reabsorb ∼2.4 l of water/day when albumin was added. The module with a surface area of 1 m2 could reabsorb ∼6 l of water out of 10 l of continuous haemofiltration/day, an amount that would satisfy our preliminary requirements [9]. The insufficient reabsorption of 4 l could be covered by oral intake of meals and drinks. Normal blood contains a greater amount of proteins than was present in the albumin-added medium used in this study. Therefore, normal blood would cause more fluid reabsorption due to high colloid osmotic pressure in clinical use. Recently, we demonstrated that aquaporin 1-transfected LLC-PK1 cells had approximately two times higher transcellular osmotic water permeability than untransfected cells [19]. Employment of these highly efficient transfected cells may lead to availability of smaller portable bioartificial renal tubule devices.

A number of clinical trials recently reported the use of human renal proximal tubule cells in renal tubule devices [20–22]. These devices, used for treating patients with acute renal failure (ARF), and patients with multiorgan failure and sepsis for a maximum of 24 h, displayed satisfactory outcomes. These reports have encouraged the development and improvement of bioartificial renal tubule device for clinical uses. In contrast with their limited critical period for management of ARF, we intend to develop bioartificial renal tubule devices that function for longer periods in chronic renal failure patients. To our knowledge, this is the first in vitro 2 week continuous evaluation of a bioartificial renal tubule device. We found that this device functioned to reabsorb water, glucose and sodium for ∼10 days. In a previous study [23] we demonstrated that a renal proximal tubule cell line played a key role in the disposal of AGE, indicating that it may prevent long-term complications in uraemic patients, such as β2-microglobulin amyloidosis and atherosclerosis. Although not evaluated in this study, these findings indicate that this bioartificial renal tubule device may provide other metabolic functions of renal tubules which are absent in haemodialysis therapy. In combination with haemofiltration, this device may perform most renal functions. With the addition of necessary peripheral devices, such as motors, batteries, pressure gauges and safety alarms, this module may be used in portable settings. Although some improvements will be necessary, this artificial renal tubule device has potential for patients with chronic renal failure to prevent the complications of dialysis. The ability to perform long-term therapy at their residence would also raise quality of life.

The authors thank Mr Masayoshi Tokunaga for excellent technical support on scanning electron microscopy. This work was supported in part by a Grant-in-Aid for Scientific Research (B, No. 2-12470213) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest statement. None declared.

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Author notes

1Department of Molecular and Cellular Nephrology, Institute of Medical Sciences, Tokai University, Japan and 2Division of Nephrology, Tokai University School of Medicine, Kanagawa, Japan

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