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.

Secretion of chemokines and cytokines by human tubular epithelial cells in response to proteins.

BACKGROUND: Chronic interstitial scarring contributes to the progression of renal failure in glomerular disease but its cause is unknown. The development of proteinuria could stimulate tubular cells to release cytokines, chemoattractants and matrix proteins into the interstitium, thus contributing to interstitial disease. METHODS: Polarized human tubular epithelial cells were grown on permeable supports and exposed to serum proteins on their apical surface. The release of tumour necrosis factor alpha(TNFalpha), platelet derived growth factor (PDGF) and monocyte chemoattractant protein-1 (MCP-1) by the cells was measured using immunoassays. RESULTS: Under control conditions there was polarized release of PDGF-AB with predominant basolateral secretion (basolateral to apical ratio 4.7+/-1.6). MCP-1 release was less polarized (ratio 1. 7+/-0.5). TNFalpha was not detected. Exposure of the cells to normal human serum proteins on their apical side increased basolateral release of PDGF-AB (1.7+/-0.4 fold) and MCP-1 (2.4+/-0.2 fold). Fractionation of the serum showed that this effect on human tubular epithelial cells was reproduced by a fraction of molecular weight 40-100 kDa. The predominant proteins in this fraction were albumin and transferrin but these purified proteins alone did not alter secretion of PDGF-AB or MCP-1. CONCLUSION: This data demonstrates that human tubular cells exposed to proteins, which would be filtered in glomerular disease, produce inflammatory mediators with the potential to stimulate inflammation and scarring in the interstitium of the kidney.

Can the use of low-dose dopamine for treatment of acute renal failure be justified?

The use of dopamine for the prevention and treatment of acute renal failure is widespread. Its use is based on physiology suggesting selective renal vasodilation when it is infused at low dose. This article reviews the available data on the clinical use of dopamine. When used to prevent acute renal failure in high-risk treatments there is no evidence of benefit of dopamine but, given the low incidence of significant renal failure, the studies are underpowered. In treatment of acute renal failure, the quality of the data is poor. Only in one small randomised trial of moderate acute renal failure in patients with malaria was a clinically significant benefit of dopamine shown. The rest of the data, in the form of case series, showed either no benefit of dopamine or small benefits of little clinical significance. Again, these studies are of insufficient power for conclusions to be drawn as to the overall benefits and risks. We conclude that benefits of dopamine use cannot be ruled out by currently available data but its use cannot be advised until trials examining clinically important endpoints in large numbers of patients have been performed.

The care of patients with diabetic nephropathy: audit, feedback, and improvement.

Diabetic nephropathy is the commonest cause of end-stage renal failure in the developed world. The quality of care of 152 patients with diabetic nephropathy was assessed at the time of referral to a single nephrologist. The type II diabetics (62%) were older than the type I diabetics (38%) (mean 65 years vs. 48 years). The mean duration of diabetes was 17 years. Significant cardiovascular disease was present in 52%. There was diabetic retinopathy in 84% of the type I diabetics and 53% of the type II diabetics. Overall, 63% had hypertension at referral (St Vincent Declaration criteria), untreated in 25%. ACE inhibitors were not prescribed in 48% when no contraindications to their use were present. Glycosylated haemoglobin was > 9.1% in 29%. Twenty were prescribed medications inappropriate to their renal function. Of patients with ischaemic heart disease and serum cholesterol > 5.5 mmol/l, 82% were untreated; 82% of patients with secondary hyperparathyroidism were also untreated. At initial referral, many patients' care was sub-optimal. Referral was too late for adequate preparation for renal replacement therapy in 33%. Following a process of education and feedback of the results to referring practitioners, the timing of referral improved. We emphasize the need for closer co-operation between those managing diabetic patients with nephropathy to optimize their care.

Can intensive treatment alter the progress of established diabetic nephropathy to end-stage renal failure?

Diabetic nephropathy is now the leading cause of end-stage renal disease in the Western world, and is associated with a higher patient morbidity and mortality than other causes of renal failure, largely because of associated cardiovascular disease. Numerous studies have elucidated the factors which influence its onset and progression. The St Vincent Declaration in 1994 proposed standards for the appropriate management of patients with diabetic nephropathy. We assessed whether referral to a nephrology clinic attempting to apply these standards influenced the progression of diabetic nephropathy. The results show a significant improvement in blood pressure, glycosylated haemoglobin and serum cholesterol following referral. There was a significant reduction in the rate of decline of renal function following referral in 39% of patients. With the possible exception of diabetic control there were no significant differences in the management of those that did and did not show improvement. The results show that with intensive out-patient clinic monitoring it is possible to improve the quality of patient care, and that even in established diabetic nephropathy it is possible to slow the rate of progression to end-stage renal failure.

Hypoxia induces intercellular adhesion molecule-1 on cultured human tubular cells.

The adverse effects of acute renal ischemia are partly mediated through an infiltration of inflammatory cells into the tubulointerstitium. The expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) by resident renal cells (endothelial cells and tubular cells) may facilitate this process. We investigated whether hypoxia stimulates the expression of ICAM-1 by cultured human proximal tubular cells (HPTC). Hypoxic culture conditions (PO2 < 4 kPa) stimulated the expression of ICAM-1 by HPTC in a time-dependent manner (P < 0.0001) as demonstrated by quantitative flow cytometry analysis. Quantitative PCR demonstrated an increase in ICAM-1 transcription. Re-oxygenation of tubular cells did not increase ICAM-1 expression further. TNF alpha concentration in culture supernatants increased with hypoxia, but blocking experiments demonstrated that TNF alpha was not implicated in hypoxia-induced expression of ICAM-1. Furthermore, the cytokines IL-6 and IL-1 beta were not involved, but the effect of hypoxia was blocked by PDTC, an antioxidant that may inhibit the activation of the transcription factor NF-kappa B. These data demonstrate that hypoxia is a stimulus that induces the synthesis and expression of the adhesion molecule ICAM-1, presumably via the activation of NF-kappa B.

Fibronectin production by human tubular cells: the effect of apical protein.

In progressive renal disease the degree of renal failure correlates with interstitial scarring and the rate of progression correlates with the degree of proteinuria. This has led to the hypothesis that proteinuria may cause interstitial scarring. Human tubular cells (HTC) grown on permeable membrane supports were characterized to be predominantly of proximal tubular origin. HTC produce the matrix protein fibronectin in a polarised fashion the ratio of basolateral to apical secretion being 2.9 +/- 0.2 at 48 hours. The addition of serum proteins (1.0 mg/ml) to the apical medium resulted in increased basolateral secretion of fibronectin, 2.62 +/- 0.23-fold after 24 hours and 2.40 +/- 0.16-fold after 48 hours. Serum fractionation revealed that the stimulant to fibronectin production had a molecular weight 40 to 100 kDa. Platelet derived growth factor secretion was also stimulated to apical exposure to serum but transforming growth factor beta secretion was not detected. Addition of neutralizing anti-PDGF antibodies did not decrease fibronectin secretion. The activity of serum was not reproduced by albumin or by transferrin. Exposure of HTC to serum resulted in increased release of lactate dehydrogenase, suggesting a degree of cytotoxicity. This evidence could provide a mechanism for the link between proteinuria and interstitial scarring.

Growth of proximal tubular cells in the presence of albumin and proteinuric urine.

The degree of interstitial scarring and proteinuria both correlate with renal function in progressive renal disease. Cellular hypertrophy and hyperplasia have been shown to occur under different experimental conditions. This study investigated the effect of protein on the growth of OK cells. Bovine serum albumin (BSA)- and fatty-acid-free BSA (FFBSA)-stimulated proliferation of OK cells and hypertrophy occurred when the cells were incubated with 10 mg/ml of BSA or FFBSA. Incubation with proteinuric urine from nephrotic rats resulted in much greater proliferation. Hence protein can alter proximal tubular cell growth in culture and the mixture of proteins in proteinuric urine has a greater effect than can be explained by albumin alone. These findings may be of significance in the progression of renal disease and indicate the potential importance of urinary proteins other than albumin in modulating tubular cell growth.