The role of proteinuria in the progression of chronic renal failure

American Journal of Kidney Diseases IN-DEPTH

TheOfficial Journalof the National

Kidney Foundation

VOL 27, NO 6, JUNE 1996

REVIEW

The Role of Proteinuria in the Progression of Chronic Renal Failure C. Burton, MB, BS, MRCP, and K.P.G. Harris, MB, BS, MRCP • The cause of the relentless progression of chronic renal failure of diverse origins remains unknown and is likely to be mulUfactodal. Numerous studies have now demonstrated a correlation between the degree of proteinuria and the rate progression of renal failure, which has led to the hypothesis that proteinurla may be an independent mediator of progression rather than simply being a marker of glomerular dysfunction. This article reviews the evidence underlying this hypothesis and the mechanisms by which particular proteins may cause renal pathology. The abnormal flotation of proteins across the glomerular basement membrane will bring them into contact with the mesengium and with the tubular cells. There is evidence to support a role of Iipoproteins on mesanglal call function, which ultimately could contribute to glomerular sclerosis. The proximal tubular cells roabsorb proteins from the tubular fluid, which leaves them particularly vulnerable to any adverse effects proteins may have. It has been postulated tifmt the sheer amount of protein to be metabolized by these ceils may overwhelm the lysosomes and result in leakage of cytotoxic enzymes into the ceils, in addition, the increased metabolism of proteins may result in production of ammonia, which can mediate inflammation through activation of complement. Specific proteins that have been shown to be cytotexic are bansfarrin/iron, low-density lipoprotein, and complement components, all of which appear in the urine in proteinuric states. Other specific proteins have been shown to stimulate production of cytokines, chemoatbactents, and mab'ix proteins by tubular cells and thus may stimulate inte,~;;;lal inflammation and scarring. The mechanisms by which the presence of proteins in the tubular fluid alters tubular ceil biology is yet to be determined. © 1996 by the National Kidney Foundation, Inc. INDEX WORDS: Proteinuria; mesanglal cell; proximal tubular cell; progression of renal failure; cytokines; chemoattractants; extracellular matrix proteins.

A

UNIVERSAL FINDING in progressive renal disease is the development of proteinuria. This review evaluates the evidence that proteinuria is of pathogenetic importance to the progression of chronic renal failure, rather than simply being a marker of the severity of the renal disease, and discusses the potential mechanisms whereby proteinuria may be harmful to the kidney. Once a renal injury has produced a certain degree of renal damage, progressive kidney failure is often inexorable.J Observations in animal models of progressive renal disease as well as in human renal disease have led to the hypothesis that after renal damage a maladaptive response occurs in the remaining nephrons, which results in their eventual destruction and chronic renal failure. This hypothesis has been most exten-

sively tested and studied in the subtotal nephrectomy model in the rat, in which surgical ablation of renal tissue to produce a "remnant kidney" results in the development of progressive proteinuria, progressive renal impairment, and glomeruFrom the Department of Nephrology, University of Leicester and Leicester General Hospital, Leicester, United Kingdom. Received December 11, 1995; accepted in revised form January 2, 1996. Supported by a grant from the National Kidney Research Fund, Huntington, United Kingdom. Address reprint requests to K.P.G. Harris, MB, BS, MRCP, Department of Nephrology, Leicester General Hospital, Gwendolen Rd, Leicester, LE5 4PW, United Kingdom. © 1996 by the National Kidney Foundation, Inc. 0272-6386/96/2706-000153.00/0

American Jouma/of Kidney Diseases, Vol 27, No 6 (June), 1996: pp 765-775

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losclerosis. 2 A variety of potential "maladaptive responses" have been identified, including hemodynamic abnormalities, 2'3 lipid abnormalities,4 and renal growth. 5 It is likely that the process is multifactorial. 6 There has also been considerable controversy regarding the relative importance of glomerular pathology and interstitial pathology, because it has been shown that even in primary glomerular disease there is a better correlation of renal function with markers of interstitial disease than with markers of glomerular pathology. 7'8 Under normal circumstances, only low-molecular-weight macromolecules gain access to the urinary space, larger molecules being excluded by an intact glomerular barrier. In glomerular disease, the barrier is disrupted, and much larger serum proteins, including albumin and larger proteins, gain access to both the mesangium and tubular fluid. Hence, the potential exists for one or more of these abnormally filtered proteins to interact adversely with the mesangium or the cells lining the tubular space, promoting further glomerular and interstitial damage. The hypothesis that proteinuria may itself be an independent determinant of progression of renal disease is strengthened by the observation that the degree of proteinuria can be correlated with the rate of progression of renal failure in both human and experimental renal disease. This is an attractive hypothesis because it provides not only a cause of progression but also a means by which glomerular disease ultimately may result in interstitial scarring. CORRELATIONS BETWEEN PROTEINURIA AND PROGRESSION IN HUMAN RENAL DISEASE

A causal role for proteinuria in the development of progressive renal failure is suggested by the strong correlation of rate of progression with quantity of proteinuria in a number of renal diseases. In 40 consecutive patients with biopsyproven diagnosis of focal segmental sclerosis followed for 6 to 16 years, patients presenting with nephrotic syndrome had a worse prognosis than those presenting with less proteinuria. 9 Similar observations have been made in mesangiocapillary glomerulonephritis type I, in which the presence and persistence of nephrotic syndrome predicted renal failure, ~° and in patients with idiopathic membranous nephropathy, in which

BURTON AND HARRIS

41% of patients presenting with nephrotic syndrome (followed for a mean of 54.8 months) developed progressive renal failure, compared with none of those presenting with less than nephrotic range proteinuria.~ ~In addition, a study of nearly 300 patients with immunoglobulin mesangial nephropathy demonstrated that proteinuria of more than 1 g/day was an independent variable associated with poor prognosis.~2 The development of proteinuria correlates with the development of chronic rejection in transplanted kidneys. ~3 The predictive value of the severity of proteinuria on the rate of progression of renal failure has also been recently confirmed in a large prospective study of progressive renal failure in the United States (the MDRD study). ~4 Thus, the rate of progression of a variety of very different primary renal pathologic conditions is predicted by the severity of proteinuria, suggesting a potential pathogenetic link between proteinuria and the development of renal scarring. The pathogenetic significance of proteinuria has recently been further suggested by the observation that a reduction in proteinuria accomplished by the use of an angiotensin-converting enzyme (ACE) inhibitor is associated with a slowing of the rate of progression of renal failure. In this study, the beneficial effect of ACE inhibition on progression was only observed in those patients in whom the drug brought about a reduction in the level of proteinuria. 15 CORRELATIONS BETWEEN PROTEINURIA AND PROGRESSION IN ANIMAL MODELS

Rats with a remnant kidney develop proteinuria and progressive renal impairment. Maneuvers that result in a reduction in the severity of the proteinuria protect the animal against the development of progressive renal impairment. For example, feeding the animals low-protein diets or diets composed of soya rather than casein significantly reduces the amount of proteinuria and abrogates the development of renal failure. 16'17 Similarly, treatment of animals with a remnant kidney with ACE inhibitors reduces the level of proteinuria compared with controls and lessens the degree of structural damage in the glomeruli and the tubulointerstitium. J8Animals treated with an alternative antihypertensive regimen have proteinuria at the same level as controls and evidence of structural damage in glomeruli and in-

PROTEINURIA AND PROGRESSION OF RENAL FAILURE

terstitium similar to that in controls. In this model, therefore, the degree of proteinuria, the degree of structural damage, and the degree of renal impairment are all closely correlated. In the remnant kidney model, the development of proteinuria has been associated with the development of interstitial disease.~8 An infiltration of macrophages and deposition of extracellular matrix proteins are common histological findings in most experimental progressive renal diseases. The evolution of this process has been further investigated in two models characterized by proteinuria and interstitial disease, namely, puromycin animonucleoside (PAN) nephrosis and protein overload nephropathy. In PAN nephrosis, a single dose of PAN results in direct toxicity to glomerular epithelial cells and the development of nephroticrange proteinuria. The proteinuria worsens up to 14 days and then returns to normal. Closely related to the time course for proteinuria, there is an influx into the interstitium of chronic inflammatory cells, including macrophages and T cells. ~9 There is a clear relationship between the degree of the proteinuria and the severity of the interstitial infiltrate, and dietary protein restriction reduces both proteinuria and the interstitial changes. 2° Preliminary data would suggest that as the proteinuria develops there is a significant increase in renal message for a macrophage-specific chemoattractant protein (MCP-1), 21 although it has not been possible to attenuate macrophage recruitment using neutralizing antibodies to MCP-1, suggesting a role for other chemoattractants in this model. This model is also characterized by foci of tubulointerstitial fibrosis and messenger RNA (mRNA) levels for genes encoding extracellular matrix proteins; tissue inhibitors of metalloproteinases and transforming growth factor/31 are all increased in association with the development of proteinuria and the interstitial infiltrate. The institution of dietary protein restriction to reduce proteinuria reverses these effects and affords histological protection. 2° Proteinuria can also be induced in rats by intraperitoneal injection of large amounts (5 g/day) of bovine serum albumin (BSA) (protein overload nephropathy). The animals develop heavy proteinuria with no evidence of immune complex deposition in the glomeruli or the interstitium. Nor is there evidence of circulating anti-BSA antibodies. As the proteinuria increases, there is an influx of chronic inflammatory cells into the

767

interstitium and an accumulation of extracellular matrix proteinsY "23 In addition, there is a significant increase in renal message for the chemotactic substances MCP-1 and osteopontin, 23 although the exact biological significance of these observations to the interstitial infiltrate remains to be determined. In the absence of any other explanation, it has been proposed that in this model the proteinuria is the injurious agent, resulting in recruitment of macrophages and lymphocytes, n with increased matrix protein synthesis and altered matrix degradation and remodeling contributing to the interstitial fibrogenic process. 23 HOW DOES PROTEINURIA LEAD TO PROGRESSIVE RENAL DAMAGE?

Most of the evidence that proteinuria is a determinant of progression of renal failure remains circumstantial and is predominantly based on correlations. In the past few years, several plausible potential mechanisms have been put forward to explain how proteinuria might lead to progressive renal injury. In addition, there is now a large body of evidence, mostly from in vitro studies, supportive of a potential nephrotoxic potential for proteinuria. Such lines of evidence broadly fall into two main groups, namely, mesangial toxicity as a result of mesangial overload with abnormally filtered macromolecules and induction of proximal tubular cell dysfunction as a result of abnormal amounts or types of protein being presented to the proximal tubular brush border. MESANGIAL TOXICITY OF PROTEINURIA

Accumulation of serum proteins in the glomerular mesangium is observed in a variety of animal models of progressive renal failure, including the remnant kidney model 24 and PAN nephrosisY Mesangial accumulation of these macromolecules may produce mesangial cell injury, mesangial cell proliferation, increased production of mesangial matrix and hence glomerulosclerosis. In particular, lipoproteins have been studied in this regard. The apolipoprotein B of low-density lipoprotein (LDL) and very-low-density lipoprotein and apolipoprotein (a) of lipoprotein (a) have been found in glomeruli in proteinuric states. 26'27 In vitro LDL interacts with its receptor on human mesangial cells, stimulating the production of the

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oncogenes, c-fos and c-jun, 28 and causing cell proliferation.29 In addition, LDL promotes the production of the extracellular matrix protein fibronectin by mesangial cells as well as inducing the production of MCP-1 ~° and platelet-derived growth factor,z8 Thus, LDL promotes a series of cellular events in mesangial cells that may propagate glomerulosclerosis, including the recruitment of macrophages, which play a pivotal role in this process. 3~ Once within the mesangium, LDL may undergo oxidation by either macrophages or mesangial cells themselves to form oxidized LDL. LDL modified in this way is known to be more cytotoxic to mesangial cells than LDL itself32 and may therefore further promote glomerular damage. EFFECTS OF PROTEINURIA ON PROXIMAL TUBULAR CELLS

Proteins that are normally filtered by the glomerulus and consequently appear in the tubular fluid are reabsorbed by proximal tubular cells by endocytosis.33 It is known that higher-molecularweight proteins when filtered by a damaged glomerulus are reabsorbed and metabolized by the proximal tubule,34 which then would be vulnerable to any adverse effects that these proteins may produceY Adverse effects of proteinuria (Fig 1) could result either from the quantity of protein in the tubular fluid or from the effects of a particular biologically active protein that is not normally

Fig 1. The mechanisms by which filtered proteins may cause interstl~al inflammaUon and scarring. In glomerular disease, filtered proteins in the tubular lumen alter ~ biology of the tubular epithelium. This results in the release into the interstitium of c h e m o a t t ~ c t a n t s , cytokines, and exlbracelluisr mal~ix proteins. As a result, there is accumulation of macrophages, which also release cytokines to recruit and sUmulate fibroblasts. The resultant increase in extracellular matrix causes scarring.

found in the urine but is present after glomerular damage. DIRECT TUBULAR TOXICITY OF PROTEINURIA

A variety of proteins have been demonstrated to be directly toxic to tubular epithelial cells. 36 Evidence for direct tubular toxicity in proteinuric states comes from the appearance in the urine of so-called tubular proteins. Tubular absorption of lysozyme is a high-capacity, low-affinity process, and the presence of lysozyme in the final urine is a good indicator of failure of tubular reabsorption. 37 The urinary excretion of lysozyme has been shown to closely correlate with degree of albumin excretion in passive Heymann nephritis, and lysozymuria is decreased by treatments that lower albuminuria, such as low-protein diets or ACE inhibitors. 38 There is difficulty interpreting lysozymuria as a marker of tubular damage in the presence of proteinuria because competition for protein reabsorption could result in increased urinary lysozyme in the absence of tubular damage. N-Acetyl-fl-glucosaminidase (NAG) is produced by tubular cells and released into the urine when they are injured. NAG is a large protein (> 125 kd) and is only present in the serum in very low concentrations, so its presence in the urine cannot be accounted for by filtration through the glomerular barrier, making it a useful marker of tubular damage. 39 A strong correlation has been shown between the degree of protein-

PROTEINURIA AND PROGRESSION OF RENAL FAILURE

uria and urinary NAG excretion in children with a variety of glomerular disorders, 4° and an increased NAG excretion has also been demonstrated in patients with nephrotic syndromef There is therefore evidence of injury to tubular cells in the presence of proteinuria, although the mechanism for this is unknown. It is possible that the sheer quantity of protein required to be reabsorbed by proximal tubular cells results in injury to them. It is known that the increased trafficking of protein across the proximal tubular cell as a consequence of proteinuria results in increased lysosomal enzyme activity.42 It has been suggested that excessive proteinuria results in leakage of lysosomal enzymes into the cytoplasm of the tubular cell.43"44The consequent cell injury could stimulate inflammation and scarring. ALTERED TUBULAR CELL BIOLOGY IN RESPONSE TO PROTEINURIA

A number of functions of proximal tubular cells have been discovered that suggest that they can take part in the process of inflammation and scarring, including the production of matrix proteins, pro-inflammatory cytokines, and chemotactic substances (Table 1). This is not surprising, considering that embryologically they are derived from mesenchymal cells, as are fibroblasts and the cells of the immune system.45 Increasing evidence would suggest that proteins may directly modulate tubular cell function, altering both their growth characteristics and their phenotypic expression of cytokines and matrix proteins. Cell growth occurs in many renal diseases associated with proteinuria and may represent a maladaptive response that contributes to the progression of renal failure.46 Urine from nephrotic rats (to a greater extent than albumin alone) induces proliferation of opposum kidney cells (a proximal tubular cell line) 47 at a concentration of protein that micropuncture studies suggest may be present in proximal tubular fluid. Further observations using the same cell line have suggested that the effect of albumin on cellular proliferation is dependent on the fatty acids carried by the albumin, with oleate albumin causing proliferation, but not palmitate albumin.48 Cell culture studies have confirmed that cultured human cortical epithelial cells will produce MCP- 1 on exposure to a variety of cytokines.49'5°

769 Table 1. The Proposed Effects of Proteins on Proximal Tubular Cells Urinary Protein

Effect

Reference

Excess total protein

Lysosomalrupture Ammonia production

43, 44 54, 55

Albumin

Exacerbation of hypoxia Lipid chemoattractant Proliferation

59, 60 64, 65 48

Transferrin/iron HDL LDL Complement (C5b-9)

MCP-1 MCP-1 Cytotoxicity ET-1 Cytotoxicity Cytotoxicity Cytokines Collagen

66 66 66 78 29 56 57 58

NOTE. Proteins present in the urine have been shown to alter tubular cell biology. This may be because of the sheer amount of protein to be metabolized by proximal tubular cells or because of effects of specific proteins, A number of proteins have been shown to interact with tubular cells in ways that may provide interstitial inflammation and scarring either by production of inflammatory mediators or through direct cytotoxicity to the cells. Abbreviations: MCP-1, monocyte chemoattractant protein-1; ET-1, endothelin 1; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

In addition, a role for lipid chemoattractants has been proposed (discussed later). Tubular cells have also been shown to produce a number of potentially pro-inflammatory cytokines. Tubular cells grown from human kidney biopsy specimens express mRNA for interleukin-6, granulocyte macrophage colony-stimulating factor (GMCSF) and platelet-derived growth factor-B (PDGF-B). 5j Greater amounts of mRNA for GMCSF and PDGF-B are found in tubular cells derived from diseased kidneys in comparison with those from normal kidneys. Supernatants from tubular cells in culture are able to stimulate fibroblasts to produce the extracellular matrix protein, fibronectin, 52 and again the effect is greatest using media produced by tubular cells derived from diseased kidneys. Our laboratory has recently demonstrated that human proximal tubular cells could contribute to the fibrogenic process by production of PDGF and the matrix protein fibronectin 53 as well as the chemokine MCP-1. Exposure of the apical surface of these cells to 1.0 mg/mL serum proteins (a concentration that could be found in tubular fluid in nephrotic

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states) results in a significant increase in basolateral release of PDGF, fibronectin, and MCP-1. The active component of serum is localized to a fraction of molecular weight 40 to 100 kd, a size that would readily be filtered by a damaged but not by a normal glomerulus. The nature of the stimulating protein and the mechanisms of its effect are under investigation.

Ammoniagenesis Ammonia generation within the kidney may play a role in the development of progressive interstitial disease. 54 In patients with glomerulonephritis, the level of urinary ammonia correlates well with the level of proteinuria. Increased ammonia production could result from catabolism of the increased amount of reabsorbed proteins.55 Ammonia is known to activate complement by the alternative pathway,54 and once activated complement has a number of proinflammatory effects, including chemoattraction by C5a and cell lysis by C5b-9. 56 C5b-9 in sublytic concentrations has been shown to release cytokiness7 and also to stimulate collagen synthesis58 in glomerular epithelial cells. Thus, an increase in ammonia production secondary to increased tubular catabolism of proteins from proteinuria could result in the activation of complement and consequent inflammation and scarring within the kidney.

Exacerbation of Hypoxia by Proteinuria Proteinuria may have its effect by exacerbating other pathological processes within the tubulointerstitium. It is, for instance, possible that in tubular cells that are already stressed by hypoxia the additional energy required to reabsorb and digest large amounts of proteins could result in damage to those cells. It has been shown that in experimental ischaemic injury the injection of low-molecular-weight proteins exacerbates the development of acute tubular necrosis. Renal ischemia in rats can be produced by clamping of the renal artery for a fixed time or by induction of hemorrhagic shock. In both of these models, injection of myoglobin, ribonuclease, or lysozyme has been shown to worsen the development of acute tubular necrosisfl In the hemorrhagic shock model, it has been shown that there is a greater depletion of adenine nucleotides in the presence of the low-molecular-weight proteins, which

BURTON AND HARRIS

could be the result of the extra work required in reabsorption. 6° There is no reason to suppose that the reabsorption of high-molecular-weight proteins requires any less energy than reabsorption of low-molecular-weight proteins. Oxygen tension in the kidney is known to be less than systemic arterial oxygen tension,6~and hence tubular cells are vulnerable to relatively small changes in oxygen requirement and delivery. In glomerular disease, this vulnerability could be amplified by damage to the postglomerular capillaries induced by the transmission of glomerular hypertension, causing a further decrease in oxygen delivery.62 In support of this, studies of human biopsy specimens have demonstrated loss of postglomerular capillaries. 63 Once scarring has started to develop, the increased distance between tubular cells and peritubular capillaries would further exacerbate any ischemic effect. EFFECTS OF SPECIFIC PROTEINS

Whether the potential adverse effects of proteinuria are attributable to an individual protein or the molecules it carries or caused by the combined effects of the many proteins filtered by a damaged glomerulus remains unknown. Toxicity of a number of proteins has recently been demonstrated (Table 1).

Albumin Recent evidence has suggested a role for albumin as an agent that could stimulate the development of interstitial inflammation through the molecules it carries. It has been shown that rat proximal tubules in short-term culture will produce a powerful chemoattractant for monocytes/ macrophages if exposed to BSA.64Further investigation of this chemoattractant showed that it is a lipid and that it was only produced by tubular cells if the albumin to which they were exposed carded certain fatty acids. Lipid-free bovine serum albumin had no effect. It has further been shown that this lipid chemoattractant is present in the urine of rats with protein overload proteinuria65 and could therefore provide an explanation for the influx of macrophages into the interstitium in this condition. Rat proximal tubular cells exposed to BSA also produce MCP-1, 66 an effect that is not altered by delipidation of the BSA and is also found when the cells are exposed to

PROTEINURIA AND PROGRESSION OF RENAL FAILURE

transferrin, suggesting that it may not be an effect specific to albumin. Two further lines of evidence argue against the hypothesis that albumin per se is the pathogenetic protein in this process. The analbuminemic rat has a genetic abnormality that results in failure to manufacture albumin. The normal functions of albumin are performed by alternative proteins such as transferrin. When these animals develop renal disease such as PAN nephrosis 67 or remnant kidney model, 68 the levels of proteinuria are much lower than in normal animals because of the absence of albumin, but the animals nevertheless go on to progressive renal failure with the same histological changes as control animals. Although the quantity of proteinuria is less than the controls, the animals still have high-molecularweight proteinuria, suggesting that the absolute quantity of proteinuria is not as significant as the types of protein that it contains. Minimal change nephrotic syndrome is characterized by very large amounts of protein, predominantly albumin, leaking through the glomerulus and appearing in the urine. However, renal scarfing and renal failure are rare. This discrepancy may be explained by the limited duration of the proteinuria in minimal change disease, which is usually readily sensitive to steroids, in contrast to progressive renal diseases, in which the proteinuria is prolonged. Increased NAG excretion has been shown in minimal change nephrotic syndrome,4° indicating a degree of tubular damage, and the NAG excretion returns to normal when proteinuria decreases during remission of the disease. Therefore, in minimal change disease, the tubular injury is either of insufficient degree to result in long-term changes or it is of insufficient duration. An alternative explanation to this paradox may be provided by the observation that the effect of albumin on tubular cell function in vitro is dependent on the fatty acid composition of the albumin. This has led to the hypothesis that it is the metabolism of fatty acids carded on albumin rather than the proteins per se that initiates tubulointerstitial injury.69 Significantly, urinary albumin in minimal change disease has a markedly lower fatty acid content compared with that from adult nephrotic patients7° and therefore could have a lower potential for initiating injury.

Lipoproteins Abnormalities of lipid metabolism as a result of proteinuria may play a role in the progression

771

of renal disease. 4'7~ It is known that significant quantities of both high-density lipoprotein (HDL) and LDL appear in the urine of nephrotic patients. 72In addition, albumin, which constitutes the majority of the filtered load, also contains high-affinity fatty acid-binding sites and is also an important lipoprotein. 73 It is clear that tubular cells interact with lipoproteins because lipidladen tubular cells have been found in nephrotic urine, TM and lipid droplets in tubular cells have been demonstrated in renal biopsy specimens from nephrotic patients, 75 as have apoA and apoB. 76 A cell culture study demonstrated uptake of HDL and LDL by human proximal tubular cells. 77 Oxidized LDL or minimally modified LDL both caused cell injury and detachment of cells from the culture plate. Tubular cells themselves may possess LDL oxidizing ability, and it is postulated that transferrinuria accompanying lipoproteinuria could provide a source of iron to catalyze the oxidative process. 77 Human tubular cells exposed to HDL have been shown to increase production of endothelin-1.78 Endothelin1 has effects on the microcirculation79 and fibroblasts 8° and is a monocyte chemoattractant. 8~ Through these effects, HDL could influence tubulointerstitial inflammation and scarring.

Transferrin Transferrin, with a molecular weight only slightly greater than albumin, is also filtered in glomerular proteinuria. Urinary transferrin has also been proposed as a mediator of tubular toxicity. As fluid passes down the tubule, it becomes increasingly acidic. Under these conditions, transferrin releases the iron that it carries. 82 Free Fe ÷+ ions are known to be cytotoxic and could therefore injure tubular cells. It has been demonstrated that exposure of tubular cells in culture to transferrin iron but not to transferrin or albumin increases lactate dehydrogenase release and cytosolic lipid peroxide malondialdehyde. This suggests that reabsorption of transferrin iron results in release of reactive iron in the proximal tubular cell, causing peroxidative injury. It has been shown that exposure of proximal tubular cells to transferrin results in the upregulation of mRNA to MCP-1, 66 although it is yet to be determined whether this is an effect of exposure to any protein or whether it is specific 'for transferrin. A pathogenetic role for iron in progressive re-

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nal injury is provided by the demonstration that, in rats with nephrotoxic serum nephritis, there is a correlation with the quantity of iron excreted in the urine and the degree of tubulointerstitial injury. 8~ A marked increase in the urinary excretion of iron has been noted in patients with diabetic nephropathy and may be of pathogenetic importance in the progressive renal injury seen in this disease, s4 There is, however, some evidence that urinary iron is protective in renal ischemia, 85 so the effects of iron are complex.

Complement As discussed, the activation of complement by ammonia is a mechanism by which proteinuria could influence interstitial inflammation and scarring. In addition, increased glomerular permeability may allow circulating complement components to filter into the tubular fluid. The C5b-9 membrane attack complex has been found in the urine in membranous nephropathy, 86 diabetic nephropathy, 87 and focal segmental glomerular sclerosis. 87 In membranous nephropathy, this is thought to represent leakage of C5b-9 from active glomerular disease. In the other conditions, however, no glomerular deposition of the complex has been detected. It has been shown that the proximal tubular brush border can activate complement by the alternative pathway, s8 and hence urinary C5b-9 may represent activation of filtered complement components by the tubular brush border. Whatever the mechanism by which this complex arrives in the tubular fluid, its presence is a potential cause of cell damage.

Bence-Jones Protein In this situation, excess production of low-molecular-weight light chains that are freely filtered by the glomerulus results in the appearance of protein in the urine in the presence of an otherwise normal kidney. The presence of BenceJones proteinuria is associated with the development of renal failure, s9 although the relationship is not invariable. It is not yet known why some patients with Bence-Jones proteinuria develop renal disease and others do n o t . 36 Theories as to the cause of renal failure in myeloma have included simple obstruction of the tubules by protein casts, although it was noted as long ago as 1968 that the degree of renal failure correlates more closely with tubular atrophy than with pres-

BURTON AND HARRIS

ence of casts. ~° Several alterations in tubular function on exposure to light chains have been investigated. In vivo "tubular type" proteinuria occurs in the presence of urinary light chains, indicating proximal tubular dysfunction. 9t Certain transport functions of rat renal cortex have been shown to be altered by the presence of light chains extracted from the urine. Transport of ammonium and glucose is inhibited, 92 as is the sodium potassium adenosine triphosphatase rat cortical tubules. 93 Sodium-dependent alanine and glucose uptake of brush border membrane vesicles from proximal tubular cells have also been inhibited by urinary light chains. 94 Thus, in the presence of a normal kidney, some types of Bence-Jones proteinuria have significant effects on tubular cell functions and are known to cause renal failure. CONCLUSION

The current evidence that proteinuria is a determinant of progression of renal failure remains circumstantial and is predominantly based on correlations. The correlations cannot exclude the possibility that proteinuria is merely a marker of severe glomerular disease and severe glomerular disease progresses more rapidly. There are also important exceptions that need an explanation. However, plausible hypotheses have been formulated supported by experimental evidence to explain how proteinuria as a whole and individual proteins in particular may influence progression of renal disease, and these await further study. Because proteinuria is associated with a bad prognosis, therapeutic strategies aimed at nonspecifically reducing proteinuria, such as tight blood pressure control, ACE inhibition, and possibly low-protein diets, are logical. Further understanding of how subtle differences in quality of proteinuria may affect the propagation of renal scarring holds the prospect of developing more specific therapies. REFERENCES

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