Turnover of human tubular cells exposed to proteins in vivo and in vitro

Kidney International, Vol. 59 (2001), pp. 507–514

Turnover of human tubular cells exposed to proteins in vivo and in vitro CHRISTOPHER J. BURTON, STEVEN J. HARPER, ELAINE BAILEY, JOHN FEEHALLY, KEVIN P.G. HARRIS, and JOHN WALLS Department of Nephrology, Leicester General Hospital, Leicester, England, United Kingdom

Turnover of human tubular cells exposed to proteins in vivo and in vitro. Background. The cause of tubulointerstitial pathology in glomerular disease is uncertain. Proteinuria may be a causative factor and has been shown to increase the turnover of tubular cells in a rat model of proteinuria. We investigated whether increased tubular cell proliferation occurs in human proteinuric renal disease. A human cell culture model was used to investigate the effects of proteins on tubular cell turnover further. Methods. Tubular proliferation in renal biopsies from patients with membranous nephropathy (MN) and minimal change nephropathy (MCN) was assessed by in situ hybridization for histone mRNA. Proliferation was measured in polarized human tubular cell cultures using tritiated thymidine, following addition of proteins to the apical medium. Toxicity was assessed by lactate dehydrogenase (LDH) release and monolayer permeability to inulin. Results. Increased tubular cell histone mRNA expression occurred in biopsies in MN (3.0-fold increase, P ⬍ 0.002) and MCN (3.6-fold increase, P ⬍ 0.02). Serum proteins added to the medium on human tubular cell cultures increased thymidine uptake (1.3-fold, P ⬍ 0.005), LDH release (1.5-fold, P ⬍ 0.001), and monolayer permeability (1.7-fold, P ⬍ 0.005). The effects were reproduced by a fraction of molecular weight 40 to 100 kD, but not by pure albumin or transferrin. Conclusions. Proliferation of tubular cells is associated with proteinuria in vivo and is caused by proteins in cell culture. Toxicity of proteins to tubular cells and increased cell turnover may contribute to tubulointerstitial pathology in glomerular disease.

alters the environment within the tubulointerstitium [2, 3], and could provoke interstitial pathology and tubular atrophy. In the presence of glomerular proteinuria, tubular cells are exposed to an increased total quantity of protein and also to types of protein from which they are normally protected by the glomerular barrier. Proximal tubular cells, which are required to reabsorb filtered proteins, would be particularly vulnerable to any adverse effects of proteinuria. We have previously shown that apical exposure of human proximal tubular cells, in culture, to normal serum proteins of a size that would be filtered in glomerular disease results in an increase in basolateral release of matrix proteins [4]. We have also demonstrated that opossum kidney cells, a proximal tubular cell line, proliferate in the presence of albumin and in the presence of low concentrations of proteins derived from the urine of nephrotic rats [5]. Increased proliferation and apoptosis of tubular cells have recently been shown in vivo in a rat model of heavy proteinuria [6]. This study was designed to investigate whether proliferation of tubular cells occurs in human proteinuric renal disease. In situ hybridization for histone mRNA was used as the marker of cell proliferation as high level expression of histone mRNA is specific to S-phase of the cell cycle [7, 8], and identification of histone mRNA positive cells has been shown to correlate well with the incorporation of 5-bromo-2⬘-deoxyuridine [9], a measure of DNA synthesis. Membranous nephropathy (MN), a disease characterized by nonselective proteinuria, and minimal change nephropathy (MCN), characterized by selective proteinuria, were compared. Using a polarized cell culture model, the components of human serum proteins, which have effects on proliferation and toxicity to human tubular cell monolayers, were investigated.

The mechanisms by which glomerular disease secondarily causes interstitial inflammation and scarring are unknown. The degree of proteinuria is correlated with progression of renal failure [1], and the development of proteinuria is one way by which injury to a glomerulus Key words: kidney tubules, proteinuria, cell proliferation, toxicity, tubulointerstitial disease, glomerular disease, membranous nephropathy, minimal change nephropathy.

METHODS Human renal tissue Archival renal biopsy material from 10 cases of MN and 10 cases of MCN were studied. Individual control

Received for publication June 17, 1999 and in revised form August 7, 2000 Accepted for publication August 10, 2000

 2001 by the International Society of Nephrology

507

508

Burton et al: Proteinuria and human tubular cell turnover Table 1. Baseline characteristics of patient and control biopsies Minimal change nephropathy

Age ⫾ SD years % Male Storage time months % Microwave fixed Fixation timea hours a

Membranous nephropathy

Patients

Controls

Patients

Controls

45.3 ⫾ 13.0 30 19.8 ⫾ 11.1 40 19.0 ⫾ 3.0

46.0 ⫾ 12.1 30 20.1 ⫾ 11.5 40 18.5 ⫾ 2.7

52.6 ⫾ 18.6 50 19.0 ⫾ 14.9 60 18.5 ⫾ 2.6

51.6 ⫾ 16.0 50 21.4 ⫾ 16.7 60 21.3 ⫾ 2.4

Excludes microwave fixed samples

biopsies that had been performed for investigation of microscopic hematuria in the absence of significant proteinuria were matched to each patient sample for age, sex, race, fixation, and storage time. These characteristics of patients and controls are shown in Table 1. All of the patients with MCN and their controls were Caucasian. All of the MN patients and their controls were Caucasian, except one patient of Chinese origin who was matched to a Chinese control. The control biopsies were normal by light microscopy, immunofluorescence, and transmission electron microscopy. All biopsies were fixed in neutral pH 10% formol-saline. Probe and probe labeling Deoxyoligonucleotides (unlabeled sequences were kindly donated by Pathway Services Ltd., Leicester, UK) complementary to histone genes H2b, H3, and H4 were 3⬘ labeled with digoxigenin-11-dUTP using a labeling kit (Boehringer Mannheim, Mannheim, Germany). Labeled probe was purified through Sephadex G50-spun columns, and labeling was confirmed by test filters. All of the deoxyoligonucleotides were 30 bases long, this length having been established as a practical compromise between hybrid stability, hybrid specificity, cost of synthesis and purification, and the efficiency of tissue penetration [10]. Nonisotopic in situ hybridization for histone mRNA Nonisotopic in situ hybridization was adapted from our previously published protocols [11, 12]: RNase-free reagents and glassware [diethylpyrocarbonate (DEPC)treated; Sigma, Poole, UK] were used throughout. Four micrometer sections were cut onto silane-coated slides. Sections were dewaxed in xylene and taken through graded alcohols. Pretreatments included 0.2 mol/L HCl acid for eight minutes and proteinase K at 37⬚C for one hour. Slides were postfixed with 0.4% paraformaldehyde in phosphate-buffered saline (PBS) at 4⬚C for 10 minutes and acetylated with 0.25% acetic anhydride/xylene. Sections were then covered for 10 minutes in prehybridization buffer [600 mmol/L NaCl, 1 ⫻ PE, 10% dextran sulfate, 30% formamide; PE ⫽ 50 mmol/L Tris (pH 7.5), 0.2% bovine serum albumin (BSA), 1% sodium dodecyl sulfate (SDS), 1% polyvinylpyrrolidone (40 kD), and 1% Ficoll (400 kD)]. A 15 ␮L labeled probe cocktail

(250 ng probe/mL prehybridization buffer) was pipetted onto a coverslip. The section was drained of excess fluid, inverted, and allowed to pick up the coverslip. A twohour hybridization at 37⬚C was performed. Post-hybridization washes were as follows: 2 ⫻ 10 minute standard saline citrate solution (SSC)/30% formamide 37⬚C, 2 ⫻ 5 minute SSC at room temperature, and blocking solution (3% BSA/0.1% Triton X100 in PBS) for 15 minutes. Sections were then incubated with alkaline phosphataselabeled sheep polyclonal antidigoxigenin antibody (Boehringer Mannheim) 1:600 in blocking solution for 30 minutes. They were washed with blocking solution for five minutes and buffer 3 (0.1 mol/L Tris-HCl, pH 9.5, 0.1 mol/L NaCl, 0.05 mol/L MgCl2) for five minutes. Sections were left overnight in developing substrate: 44 ␮L nitro blue tetrazoleum (NBT; 75 mg/mL 70% dimethylformamide; Sigma), 33 ␮L 5-bromo-4-chloro-3indoyl phosphate (BCIP; 50 mg/mL dimethylformamide; Sigma) in 10 mL Buffer 3. They were washed under running tap water, counterstained with hematoxylin, and mounted with an aqueous mountant before examination. Negative controls were as previously listed [12]. These included omission of probe, omission of ␣-digoxigenin antibody, random oligonucleotide similarly labeled, RNAase pretreatment control, and a nonhomologous probe cocktail containing nine oligonucleotides with the same G-C content as the histone probe also similarly labeled. Specimens from diseased kidneys and controls were processed in parallel to ensure identical experimental conditions. A single observer, unaware of whether the samples were from diseased kidneys or controls, examined all specimens using a Nikon Optiphot microscope. The number of histone-positive cells was counted in sequential high-powered fields (hpf) in the whole length of the human biopsies. Cell culture Human tubular cells were isolated by a modification of the method of Detrisac et al as reported previously [4, 13]. In brief, the outer cortex was dissected from the normal pole of kidneys, which had been removed for treatment of carcinoma, or from donor kidneys deemed unsuitable for transplantation. Fragmented cortex was suspended in type II collagenase (1.0 mg/mL) at 37⬚C

Burton et al: Proteinuria and human tubular cell turnover

for 30 minutes. The digest was passed through a series of sieves of diminishing mesh size, and the glomeruli were removed on the top of the 90 ␮m mesh. The remaining tubular fragments were seeded into 75 cm2 flasks, which had been coated with bovine collagen type I and adsorbed fetal calf serum (FCS) proteins. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM):F12 with the addition of 25 mmol/L HEPES buffer, insulin (5 ␮g/mL), transferrin (5 ␮g/mL), selenium (5 ng/mL), tri-iodo-thyronine (4 pg/mL), hydrocortisone (36 ng/mL), benzyl penicillin (100 IU/mL), and streptomycin (50 ␮g/mL). After 10 to 14 days, the cells reached confluence and were subcultured. At the second passage, the cells were transferred into six-well plates containing polycarbonate semipermeable membrane supports of 3.0 ␮m pore size, coated with bovine collagen type I. They were grown in the medium described previously with the addition of 5% FCS. We have previously demonstrated that cells isolated and grown in this way stain positive for cytokeratin but not factor VIII-related antigen, and produce cAMP in response to parathyroid hormone, confirming the cells to be of predominantly proximal tubular origin [4]. In addition, the cells form a polarized uniform monolayer with a very low permeability to albumin [4]. Serum preparation When cells from a nephrectomy specimen became available, venous blood was collected from normal volunteers and allowed to coagulate. The serum was separated and exhaustively dialyzed against PBS. The protein concentration in the serum was measured using a Bio-Rad DC protein assay, and the serum samples were diluted to a standard protein concentration of 10 mg/mL in PBS and stored at ⫺20⬚C for a maximum of one week. The dialyzed serum proteins were fractionated by molecular weight using a Superdex 200 gel filtration column (Pharmacia, Uppsala, Sweden). One milliliter of serum containing 50 mg of total protein was added to the column and eluted using a 0.15 mol/L NaCl/0.05 mol/L Na2HPO4 pH 7.2 buffer. Four molecular weight fractions were collected: (1) ⬎440 kD, (2) 440 to 100 kD, (3) 100 to 40 kD, and (4) ⬍40 kD. The greatest concentration of proteins occurred in fraction C, which was adjusted to a final protein concentration of 10 mg/mL. The other fractions were diluted identically to fraction C to ensure that the protein concentrations were in the same ratio to fraction C as they were in unseparated serum. Effects of serum proteins on growth and toxicity to tubular cells Once the cells on the permeable supports formed confluent monolayers, the medium was changed to DMEM with addition of 25 mmol/L HEPES, hydrocortisone (36 ng/mL), benzyl penicillin (100 IU/mL), and streptomycin

509

(50 ␮g/mL). This medium contained a physiological concentration of glucose (5.5 mmol/L) and was used because the high level of glucose found in DMEM:F12 had previously been shown to alter tubular cells behavior in cultures [14]. After 24 hours in this medium, the media bathing both the apical and basolateral surfaces of the cells were changed. The stored serum proteins were added to the apical medium at a further dilution of 1:10, yielding the following final concentrations: unfractionated serum, 1.0 mg/mL; fraction A, 0.075 ⫾ 0.09 mg/mL; fraction B, 0.457 ⫾ 0.030 mg/mL; fraction C, 1.0 mg/mL; and fraction D, 0.007 ⫾ 0.005 mg/mL. An equivalent volume of PBS was added to the controls to ensure equal dilution of culture medium ingredients. Fresh serumfree medium was placed on the basolateral side of the monolayer. After 48-hour tritiated thymidine incorporation, monolayer permeability and release of lactate dehydrogenase (LDH) were assessed. Tritiated thymidine incorporation DNA synthesis was quantitated by tritiated thymidine incorporation using a method adapted from Golchini et al [15] and Greenberg et al [16]. Five hundred microliters of DMEM containing 2 ␮Ci/mL of 3H-thymidine was added to both the apical and basolateral sides of the cell monolayer. After two hours of incubation at 37⬚C, the cells were washed with normal saline and exposed to 0.1 mmol/L unlabeled thymidine in DMEM for 20 minutes to remove nonspecific binding. The cells were washed four times on both sides with normal saline. One-percent nonidet P40 in PBS was added to the apical side of the cells, and the plates were incubated at ⫹4⬚C for four hours. The cells were scraped from the surface of the membrane and disrupted by sonnication. Cell solution was added to Ecoscint A and scintillation counted on an LKB 1219 liquid scintillation counter. Background disintegrations per minute (dpm) were subtracted from the dpm of the samples. Total cell protein was measured in the cell solutions by Bio-Rad DC assay kit. Lactate dehydrogenase release by human tubular epithelial cells Following exposure to the experimental conditions, LDH was measured in the culture media using a commercially available method based on the reduction of pyruvate to lactate producing a change in optical density at 340 nm. Apical and basolateral LDH was added to give the total LDH released. Total cellular LDH was determined in cell lysates prepared by scraping the cells from the supports into a solution of 1% Triton X-100 in PBS followed by disruption of the cells by sonnication. Permeability of monolayers to 14C-inulin The apical medium was replaced with medium containing 0.02 MBq/mL of 14C-inulin carboxylic acid, and

510

Burton et al: Proteinuria and human tubular cell turnover

Table 2. Clinical data in patients with minimal change nephropathy

Patient 1 2 3 4 5 6 7 8 9 10

Proteinuria g/24 h at time of biopsy

Serum creatinine lmol/L at time of biopsy

Tubular histone-positive cells per hpf

1.9 5.0 13.1 1.96 8.8 1.15 NA 7.1 2.9 0.6

96 117 69 94 103 108 64 80 116 91

0.57 0.30 2.20 1.16 0.70 0.54 0.98 0.2 0.41 0.24

Abbreviations are: NA, not available; hpf, high-powered field.

Table 3. Clinical data in patients with membranous nephropathy

Patient 1 2 3 4 5 6 7 8 9 10

Proteinuria g/24 h at time of biopsy

Serum creatinine lmol/L at time of biopsy

Tubular histone-positive cells per hpf

4.5 1.4 4.0 4.7 5.0 NA 18.7 4.0 2.7 2.6

83 70 156 97 NA 835 114 378 66 70

0.71 0.63 0.63 1.14 0.48 3.57 0.86 4.05 0.97 1.63

Abbreviations are: NA, not available; hpf, high-powered field.

fresh medium was added to the basolateral side, ensuring that the levels of fluid on the apical and basolateral sides were equal to avoid a hydrostatic pressure gradient. After a two-hour incubation, the apical and basolateral media were added to Ecoscint A, and the activity was counted on a liquid scintillation counter. The permeability of the monolayer to inulin was calculated as the flux of inulin per unit time relative to the concentration gradient for inulin and the surface area of the monolayer as previously described [17]. Cytokine assays Platelet-derived growth factor (PDGF)-AB and transforming growth factor-␤ (TGF-␤) were assayed using kits supplied by R&D Systems (Abingdon, UK). Statistics The results are expressed as mean ⫾ SEM. Statistics were performed using Student t tests or analysis of variance where there were more than two groups for comparison. The Bonferroni correction for multiple comparisons was used where appropriate, and P ⬍ 0.05 was taken as significant. RESULTS Proliferation of renal tubular cells in human glomerular disease Clinical data relating to the patients with MCN and MN are shown in Tables 2 and 3. In the 10 patients with MN, the level of proteinuria ranged between 1.4 and 18 g/24 h (mean 5.3 ⫾ 1.7 g/24 hours). In the 10 patients with MCN, proteinuria ranged between 1.2 and 13.1 g/24 hours (mean 5.3 ⫾ 1.4 g/24 hours). Control biopsies were from patients with microscopic hematuria but no proteinuria. As expected level of histone mRNA expression in tubules of control kidneys was very low, with approximately one histone positive cell in every third high-powered field (0.35 ⫾ 0.05 positive tubular cells/

Fig. 1. Quantitation of histone positive cells in (A) membranous nephropathy (MN) and (B) minimal change nephropathy (MCN). Biopsies from 10 patients with MN and 10 patients with MCN were compared with control biopsies from patients with no proteinuria. Tubular cells per high-powered field (hpf) expressing histone mRNA were counted. **P ⬍ 0.002; *P ⬍ 0.02.

high-powered field). However, the number of tubular cells expressing histone mRNA significantly increased in both proteinuric renal diseases [MN by 3.0-fold (P ⬍ 0.002) and MCN by 3.6-fold (P ⬍ 0.02)] as shown in Figure 1. In the patients with MN, the two patients with a markedly increased creatinine level also had the highest level of tubular expression of histones, suggesting that this may be associated with progressive disease in this condition, but there were insufficient numbers of patients to allow valid comparison between those with progressive disease with those with stable renal function. Proliferation of human tubular cells exposed to apical proteins To investigate whether proteinuria per se is the stimulus to tubular cell proliferation, human renal tubular cells in polarized culture were exposed to serum proteins on their apical surface and proliferation measured by tritiated thymidine incorporation. Tubular cells exposed to normal human serum proteins had a modest increase in tritiated thymidine uptake (1.32-fold, P ⬍ 0.005), as

511

Burton et al: Proteinuria and human tubular cell turnover

Table 4. Effects of pure albumin and partially iron-saturated transferrin on tritiated thymidine incorporation, lactate dehydrogenase (LDH) release, and monolayer permeability to inulin of human tubular cell cultures

3

H-thymidine dpm LDH mIU/mg cell protein Permeability ⫻ 10⫺3 cm/h

Control

Albumin 1.0 mg/mL

Transferrin 0.05 mg/mL

4320 ⫾ 230 401 ⫾ 92 1.4 ⫾ 0.2

4980 ⫾ 410 426 ⫾ 80 1.2 ⫾ 0.2

4539 ⫾ 50 458 ⫾ 47 1.4 ⫾ 0.4

Data are from three kidney preparations, each in triplicate.

Fig. 2. Tritiated thymidine incorporation into human tubular cells exposed to apical proteins. Tubular cells were exposed to normal human serum (S), control conditions (0), or molecular weight fractions A–D, as defined in the text. After 48 hours, tritiated thymidine uptake in disintegrations per minute (dpm) per mg of cell protein was determined. The experiments shown used serum samples from five normal volunteers and cells produced from three different kidney preparations. *P ⬍ 0.005.

shown in Figure 2. There was no significant change in total protein content of the cultures on exposure to apical serum. To investigate which proteins within serum were responsible for the proliferative effect, serum fractions were applied to the culture system. Figure 2 shows that apical exposure to a fraction of molecular weight 40 to 100 kD (fraction C) completely reproduced the effect of unfractionated serum, increasing mean tritiated thymidine uptake by 1.35-fold (P ⬍ 0.005). There was no significant increase after exposure to fractions A, B, or D. Most cytokines and growth factors that might be released into serum and cause cell proliferation are of low molecular weight and would be expected to be found predominantly in fraction D. This was confirmed by the assay of PDGF-AB, which was present at a concentration of 0.17 ng/mL in fraction D and 0.01 ng/mL in fraction C. TGF-␤, which can also cause cell proliferation, was found predominantly in fraction A (concentration 0.34 ng/mL) because it is bound to ␣2-macroglobulin in serum [18]. No TGF-␤ was detectable in fraction C. The predominant proteins in fraction C were albumin and transferrin. Purchased (Sigma) pure human albumin (1.0 mg/mL) and pure partially iron saturated transferrin (0.05 mg/mL) were added to the medium bathing the apical surface of human tubular cells. The concentration of transferrin used was equivalent to its concentration assayed in fraction C. Neither of these proteins significantly increased thymidine incorporation by the tubular cells (Table 4). Tubular toxicity of serum proteins Release of LDH by cells in culture is evidence of loss of cell membrane integrity and hence toxicity [19]. Figure 3 demonstrates that exposure to unfractionated serum proteins increased the release of LDH from the cell

Fig. 3. Lactate dehydrogenase (LDH) release by human tubular cell cultures following exposure to proteins. After 48 hours of exposure to human serum (S), control conditions (0), or molecular weight fractions A–D, release of LDH into the medium was measured as described in the text. Total LDH release into apical and basolateral media is shown in mIU per mg of cell protein. The experiments shown used serum samples from five normal volunteers and cells produced from three different kidney preparations. *P ⬍ 0.001.

monolayers (1.5-fold, P ⬍ 0.001). The increased release of LDH was reproduced by exposure to serum protein fraction C (1.5-fold, P ⬍ 0.001), but not by exposure to any other serum fraction. There was no significant difference in total cellular LDH activity between cells exposed to control conditions and those exposed to serum or serum fractions. Permeability to small molecules such as inulin is an indicator of the integrity of the monolayer as a whole. Under basal conditions, the permeability of the monolayers (1.3 ⫾ 0.2 ⫻ 10⫺3 cm/hour) was similar to that previously reported for mouse tubular cell cultures (1.2 ⫻ 10⫺3 cm/hour) [17]. As shown in Figure 4, permeability to inulin was increased by exposure to unfractionated serum proteins (1.7-fold, P ⬍ 0.005). Again, this effect was reproduced by exposure to serum protein fraction C (2.0-fold, P ⬍ 0.02). Neither pure albumin nor pure partially iron saturated transferrin at the concentrations used were able to reproduce the effects of serum on LDH release or on permeability (Table 4).

512

Burton et al: Proteinuria and human tubular cell turnover

Fig. 4. Permeability of tubular cell monolayers to inulin following exposure to proteins. After 48 hours of exposure to human serum (S), control conditions (0), or molecular weight fractions A–D, permeability of the monolayers to 14C-inulin was measured as described in the text. The experiments shown used serum samples from five normal volunteers and cells produced from three different kidney preparations. *P ⬍ 0.02.

DISCUSSION Tubulointerstitial inflammation and scarring are features of many glomerular diseases [20–22], but the cause is unknown. One hypothesis is that the presence of proteins in the glomerular ultrafiltrate alters the behavior of tubular cells, provoking inflammation and scarring within the interstitium. In support of this, we and others have shown that polarized tubular cells in culture release matrix proteins, cytokines, and chemokines into the basolateral medium when exposed to proteins that would be filtered in glomerular disease [4, 23, 24]. However, evidence to link these in vitro effects to the behavior of tubular cells exposed to proteinuria in vivo is lacking. Proliferation of cells can readily be assessed in both pathology specimens and in cell cultures. Increased proliferation and apoptosis of tubular cells has recently been shown in protein overload nephropathy, a rat model of proteinuria [6]. In these experiments, we have investigated proliferation of tubular cells in vivo in human proteinuric renal disease and compared with the behavior of human tubular cells in vitro when exposed to serum proteins. Up-regulation of histone mRNA expression is an established marker of cell proliferation. Although histone mRNA is constitutively expressed at low levels, the mRNA levels increase 20- to 100-fold during S-phase and rapidly decline in G2 [7, 8]. Low levels of histone mRNA expression were found in the cortical tubules from the control human kidney biopsies, confirming previous work [25] and showing that normal human renal tubular epithelial cells undergo a slow rate of turnover. However, proliferation of tubular cells is a normal response to injury, for instance, in the recovery from acute

tubular necrosis [25]. In our study, significantly higher expression of histone mRNA was seen in renal cortical tubules from patients with proteinuria caused by both MN and MCN compared with controls. Differences in the two control groups may be explained by their different baseline characteristics brought about by the matching to the different patient groups. There were differences in age and sex distribution of the two groups, and more of the membranous controls were fixed by microwave. Tubulointerstitial changes are known to occur with age [26]. The effects of sex and fixation method are not known. Because of these differences, it is not possible to compare directly the results from membranous and minimal change kidneys. Even though MCN is not associated with progressive interstitial scarring, increased urinary N-acetyl glucosaminidase has been found, indicating tubular injury in this condition [27]. There was a suggestion that in MN, the patients with most severe renal damage were those with the highest level of tubular histone expression. However, the presence of tubular proliferation in MCN, which does not progress, suggests that this mechanism alone is not the cause of progressive disease, which must require additional pathological stimuli. We used our previously established polarized human tubular cell culture model to investigate the effects of proteins on tubular cell proliferation. Since proliferation may be a response to injury, the cell culture model was also used to investigate toxicity of proteins for tubular cells. In a human system, it is not possible to reproduce perfectly the mixture of proteins that are present within proximal tubular fluid. Proteins excreted in urine have been altered by their passage through the urinary tract and additional proteins are added by the urothelium so that proteinuric urine would not be a satisfactory model. The use of plasma proteins is not ideal, as it would require the addition of anticoagulants to the system, and we have therefore used diluted normal human serum as our model. To address the concern that cytokines released during the process of blood coagulation were responsible for the effects of proteins in our system, we used fractionation of the serum proteins and demonstrated that the active fraction was of a molecular weight that does not contain low-molecular-weight cytokines, and we have demonstrated that PDGF is contained almost exclusively in fraction D, which was inactive. The human tubular cells in polarized culture increased tritiated thymidine incorporation when exposed to serum proteins on their apical surface. The increased proliferation was modest in comparison to the increase in histone expression in vivo possibly because of the higher baseline level of proliferation in the culture system, but additional, as yet unidentified, factors may contribute to proliferation in vivo. The same conditions that resulted in proliferation of tubular cells also resulted in release

Burton et al: Proteinuria and human tubular cell turnover

of LDH and increased monolayer permeability. LDH release is a commonly used indicator of loss of cell integrity, although it does not necessarily always correlate with toxicity, but the combination of increased LDH release and increased permeability of the membrane strongly suggests toxicity of the proteins for the tubular cells. The observed tubular proliferation could therefore be a response to cellular injury. A combination of injury and proliferation could explain the lack of change in total protein content of the cultures when exposed to serum or serum fractions; however, the relative insensitivity of the protein measurement requires caution in making this interpretation. The protein fraction that reproduced the effect of whole serum contained proteins of molecular weight 40 to 100 kD. These proteins would be expected to be filtered in glomerular disease, including in the selective proteinuria of MCN. This may explain the lack of difference in tubular proliferation in vivo between MN and MCN. Although the predominant proteins in the active fraction were albumin and transferrin, pure human albumin and partially iron saturated transferrin at concentrations found in fraction C did not reproduce its effects. Effects of pure albumin and transferrin have been reported in tubular cell culture systems, but, for the most part, at much higher protein concentrations than were used in our system [23, 24]. The biological significance of effects of proteins at these very high concentrations must be questioned as tubular cells are unlikely to be exposed to more than 1.0 mg/mL of protein even in severe nephrosis [28]. Since albumin at 1.0 mg/mL was ineffective, the effect of serum could not simply be due to the quantity of protein to which the cells had been exposed. The precise nature of the agent in the active fraction that caused proliferation of tubular cells is yet to be determined. Possibilities are another protein at low concentration within this molecular weight range, a molecule carried by albumin but removed during the manufacturing process or perhaps the requirement for a combination of proteins. The potential importance of molecules carried by proteins is shown by the differing effects of albumin on opossum kidney cell culture according to the type of fatty acid being carried. Albumin conjugated with oleate increased cell proliferation, while albumin palmitate inhibited proliferation [29]. The experiments described here have shown that proteinuric renal disease in humans is associated with a proliferative response in tubules. Exposure of human tubular cells in culture to proteins that are likely to be filtered in glomerular disease results in proliferation and also evidence of toxicity, which is in keeping with the observed increased tubular cell turnover in rat protein overload nephropathy. The mechanism by which proteins cause this increased turnover of tubular cells is yet to be elucidated.

513

ACKNOWLEDGMENTS This work was supported by National Kidney Research Fund project Grants 93/2/6, R40/2/94, and R35/1/98. Dr. Harper is supported by the Wellcome Trust Grant 057936/99. Reprint requests to Christopher J. Burton, Ph.D., The Richard Bright Renal Unit, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, England, United Kingdom. E-mail: [email protected]

APPENDIX Abbreviations used in this study are: BSA, bovine serum albumin; DEPC, diethylpyrocarbonate; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; LDH, lactate dehydrogenase; MCN, minimal change nephropathy; MN membranous nephropathy; NBT, nitroblue tetrazoleum; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; TGF-␤, transforming growth factor-␤.

REFERENCES 1. Benigni A, Remuzzi G: Glomerular protein trafficking and progression of renal disease to terminal uremia. Semin Nephrol 16:151– 159, 1996 2. Schreiner G: Renal toxicity of albumin and other lipoproteins. Curr Opin Nephrol Hypertens 4:369–373, 1995 3. Burton CJ, Harris KPG: The role of proteinuria in the progression of chronic renal failure. Am J Kidney Dis 27:765–775, 1996 4. Burton CJ, Combe C, Walls J, et al: Fibronectin production by human tubular cells: The effect of apical protein. Kidney Int 50:760– 767, 1996 5. Burton CJ, Harris KPG, Bevington A, et al: The growth of proximal tubular cells in the presence of albumin and proteinuric urine. Exp Nephrol 2:345–350, 1994 6. Thomas ME, Brunskill NJ, Harris KPG, et al: Proteinuria induces tubular cell turnover: A potential mechanism for tubular atrophy. Kidney Int 55:890–898, 1999 7. Zhong R, Roeder RG, Heintz N: The primary structure and expression of four cloned human histone genes. Nucleic Acids Res 11:7409–7425, 1983 8. Harris ME, Bohni R, Schneiderman MH, et al: Regulation of histone mRNA in the unperturbed cell cycle: Evidence suggesting control at two posttranscriptional steps. Mol Cell Biol 11:2416– 2424, 1991 9. Alison M, Chaudry Z, Baker J, et al: Liver regeneration: A comparison of in situ hybridisation for histone mRNA with bromodeoxyuridine labelling for the detection of S-phase cells. J Histoch Cytochem 42:1603–1608, 1994 10. Pringle JH, Ruprai AK, Primrose L, et al: In situ hybridisation of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes. J Pathol 162:197–207, 1990 11. Harper SJ, Allen AC, Pringle JH, et al: Increased dimeric producing B cells in the bone marrow in IgA nephropathy determined by in situ hybridisation for J chain mRNA. J Clin Pathol 49:38–42, 1996 12. Jones PH, Harper SJ, Watts F: Stem cell patterning and fate in human epidermis. Cell 80:83–93, 1995 13. Detrisac CJ, Sens MA, Garvin AJ, et al: Tissue culture of kidney epithelial cells of proximal tubule origin. Kidney Int 25:383–390, 1984 14. Rocco MV, Chen Y, Goldfarb S, et al: Elevated glucose stimulates TGF-beta gene expression and bioactivity in proximal tubule. Kidney Int 41:107–114, 1992 15. Golchini K, Norman J, Bohman R, et al: Induction of hypertrophy in cultured proximal tubule cells by extracellular NH4Cl. J Clin Invest 84:1767–1779, 1989 16. Greenberg DB, Barsh GS, Tsung-Shang HO, et al: Serum stimulated phosphate uptake and initiation of fibroblast proliferation. J Cell Physiol 90:193–210, 1977 17. Kroshian VM, Sheridan AM, Lieberthal W: Functional and

514

18. 19. 20. 21.

22. 23.

Burton et al: Proteinuria and human tubular cell turnover

cytoskeletal changes induced by sublethal injury in proximal tubular epithelial cells. Am J Physiol 266:F21–F30, 1994 O’Connor-McCourt MD, Wakefield LM: Latent transforming growth factor-␤ in serum. A specific complex with ␣2-macroglobulin. J Biol Chem 262:14091–14099, 1987 Wheeler DC, Persaud JW, Fernando R, et al: Effects of lowdensity lipoproteins on mesangial cell growth and viability in vitro. Nephrol Dial Transplant 5:185–191, 1990 Schainuck LI, Striker GE, Cutler RE, et al: Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1:631–641, 1970 Wehrmann M, Bo¨hle A, Bogenschutz O, et al: Long-term prognosis of chronic idiopathic membranous glomerulonephritis: An analysis of 334 cases with particular regard to tubulointerstitial changes. Clin Nephrol 31:67–76, 1989 Abe S, Amagasaki Y, Iyon S, et al: Significance of tubulointerstitial lesions in biopsy specimens of glomerulonephritic patients. Am J Nephrol 9:30–37, 1989 Wang Y, Chen J, Chen L, et al: Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein. J Am Soc Nephrol 8:1537–1545, 1997

24. Zoja C, Donadelli R, Colleoni S, et al: Protein overload stimulates RANTES production by proximal tubular cells depending on NF-␬B activation. Kidney Int 53:1608–1615, 1998 25. Nadasdy T, Laszik Z, Blick KE, et al: Human acute tubular necrosis: A lectin and immunohistochemical study. Hum Pathol 26:230–239, 1995 26. Thomas SE, Anderson S, Gordon KL, et al: Tubulointerstitial disease in aging: Evidence for underlying peritubular capillary damage, a potential role for renal ischemia. J Am Soc Nephrol 9:231–242, 1998 27. Kunin CM, Chesney RW, Craig WA, et al: Enzymuria as a marker of renal injury and disease: Studies of N-acetyl-␤-glucosaminidase in the general population and in patients with renal disease. Pediatrics 62:751–760, 1978 28. Landwehr DM, Carvalho JS, Oken DE: Micropuncture studies of the filtration and absorption of albumin by nephrotic rats. Kidney Int 11:7–9, 1977 29. Thomas ME, Schreiner GF: Contribution of proteinuria to progressive renal injury: Consequences of tubular uptake of fatty acid bearing albumin. Am J Nephrol 13:385–398, 1993