CHAPTER 8

Proteinuria

Anne Dawnay

CHAPTER OUTLINE

INTRODUCTION

For over 150 years, proteinuria has been recognized as one of the cardinal signs of renal disease and it continues to be used for the diagnosis and management of patients with nephropathy. However, the use of sensitive immunoassay methods to measure urine albumin has demonstrated pathologically significant proteinuria well below the detection limit of chemical methods. Since the discovery, several decades ago, that low-level albuminuria (microalbuminuria) identifies diabetic patients with incipient nephropathy, further large-scale studies have revealed that microalbuminuria is also a risk factor for macro- and microvascular disease, in both diabetic and non-diabetic populations. Studies over the last ten years have confirmed that microalbuminuria is not only associated with cardiovascular disease but also with any acute inflammatory condition, and is a reflection of systemic vascular endothelial dysfunction. Of particular importance, is the finding that, in some conditions, microalbuminuria is reversible with interventions that protect the vascular system. Thus modern assessment of proteinuria across the full pathological range has a role not just in the diagnosis and management of primary renal disease, but also as a marker of endothelial dysfunction in a variety of non-renal conditions.

PROTEIN CONSERVATION BY THE KIDNEYS

The kidneys receive approximately 25% of the resting cardiac output, which represents approximately 1.2 L/min of blood or 650 mL/min of plasma. The kidneys’ capacity to conserve protein can be judged from a simple calculation. Every 24 h, approximately 930 L of plasma containing about 70 g/L of protein pass through the kidneys, representing 65 kg of protein, of which < 100 mg (0.00015%) appear in the urine.

The filtration process is dependent on adequate renal blood flow, which is preserved by an autoregulatory system, despite variations in blood pressure (see p. 127). This mechanism allows vasodilatation as perfusion pressure falls and vasoconstriction as pressure rises. The mediators of this process include prostaglandins, kinins and atrial peptides (vasodilators), and angiotensin II, α-adrenergic hormones, thromboxane A₂, noradrenaline (norepinephrine) and vasopressin (vasoconstrictors). In addition, renal arterioles respond within seconds to changes in vessel wall tension; thus, when renal perfusion pressure rises, vessel wall tone increases and, conversely, when renal perfusion pressure falls abruptly, there is a compensatory decrease in vessel wall tone. This phenomenon is called the myogenic reflex and helps to maintain a constant renal blood flow across a range of perfusion pressures.

A minimum intraglomerular pressure, derived from the pumping action of the heart, is required to overcome the two main opposing forces to filtration: the colloidal oncotic pressure and the hydrostatic pressure in the Bowman space. When the renal perfusion pressure falls below 50–60 mmHg, further vasodilatation does not occur and renal blood flow declines in proportion to the reduction in renal perfusion pressure. These mechanisms maintain renal blood flow, and hence glomerular filtration, independently of the normal fluctuations of blood pressure. However, recent studies suggest that chronic hypertension impairs the renal autoregulatory mechanism, which may contribute to hypertension-associated renal damage and proteinuria.

The glomerular capillary wall

The glomerular membrane consists of a modified capillary wall comprising endothelium, an acellular basement membrane and an outer specialized epithelial cell layer (Fig. 8.1). The endothelial cells are thin and fenestrated with 50–100 nm pores. The basement membrane, comprising the lamina rara interna, lamina densa and lamina rara externa, is around 300–350 nm thick and is best considered as a gel-like structure containing 3–5 nm long fibrils but no detectable pores. The numerous foot processes of the epithelial cells (also called podocytes) interdigitate and envelop the outer surface of the glomerular membrane. The foot processes are separated by slit diaphragms about 55 nm in width, which form the final barrier to plasma proteins.

The whole of the glomerular membrane carries a fixed net negative charge, which is partly owing to a glycosialoprotein coat covering both endothelium and epithelium. The charge increases in density from the lamina rara interna towards the lamina rara externa, with the greatest density at the slit diaphragms of the epithelium.

The glomerular capillaries act as high pressure filters, allowing water and low molecular weight solutes to pass through freely and almost completely retaining plasma proteins. The concentration gradient of proteins across the glomerular membrane produces the colloidal oncotic pressure that must be overcome for filtration to take place.

The theory of molecular sieving

The glomerular membrane selectively allows passage of water and low molecular weight solutes into the tubule and restricts passage of larger molecular weight plasma proteins, based on a combination of molecular size, shape and charge. The pore size of the fenestrae of endothelial cells (50–100 nm) is too great to provide a major restriction to the passage of many proteins, which pass through to the glomerular membrane. Like the glomerular membrane, most plasma proteins carry a net negative charge, resulting in their electrochemical retention by the kidneys. The capillary side of the glomerular basement membrane (lamina rara interna) has a charge density of 35–45 mEq/L, so that electrochemical effects alone reduce the concentration of albumin to 5–10% of that in plasma. Thus, the lamina rara interna of the basement membrane is the first impediment to confront charged molecules, with the lamina densa providing the most effective size barrier to macromolecules. Some macromolecules accumulate at the slit diaphragms of the epithelium where the net negative charge is greatest, and there is evidence that some molecules are pinocytosed by the podocytes.

Based on their molecular radius, shape and charge, different proteins penetrate the glomerular membrane to a variable extent, for example, albumin (radius 3.6 nm, isoelectric point 4.7) is restricted at the lamina rara interna, while lactoperoxidase (radius 3.8 nm, isoelectric point 8.0) may reach the slit pores of the epithelial cells.

Despite the protein-retaining properties of the glomerular membrane, some protein does pass into the proximal tubular fluid. In healthy adults, the albumin content of glomerular filtrate is probably about 15 mg/L which, with a glomerular filtration rate (GFR) of 160 L/ 24 h, results in 2–3 g of albumin being presented to the renal tubules each day, most of which is reabsorbed.

Tubular reabsorption of proteins

The amount of protein reaching the proximal tubules is a function of the protein’s ability to cross the glomerular membrane and its plasma concentration. After reabsorption, proteins are catabolized in tubular cells and the amino acids, bound vitamins and trace metals returned to the plasma pool. For example, under normal circumstances the kidneys are responsible for about 10% of albumin catabolism, but this figure can rise to 60% when there is increased glomerular clearance of albumin such as occurs in nephrotic syndrome.

Normally, only a very small proportion of plasma proteins reaches the urine. This is primarily owing to retention of large molecular weight proteins by the glomeruli, and almost complete proximal tubular reabsorption and catabolism of any filtered lower molecular weight proteins that have passed through the glomerular membrane more easily. Thus, the renal clearance of plasma proteins represents a combination of these processes. The relative contributions of glomerular retention and tubular reabsorption to protein clearance by the kidney depend on molecular weight (Table 8.1).

The likely mechanism of proximal tubular protein reabsorption is endocytosis at the tubular epithelial apical cell membrane mediated by two receptors (megalin and cubilin) and the cooperating protein amnionless. Mutations in one or other of these components are responsible for the tubular proteinuria of Imerslund–Grasbeck syndrome (selective vitamin B₁₂ malabsorption) and Donnai–Barrow syndrome (a multisystem disorder affecting many organs that normally express megalin). Following reabsorption, proteins are transferred to lysosomes for degradation while the receptors are recycled from endosomes to the apical cell membrane. Mutations in the lysosomal and endosomal components are responsible for the tubular proteinuria in Dent disease and cystinosis; the molecular mechanism in Lowe syndrome is yet to be resolved (see Chapter 9).

Megalin and/or cubilin bind albumin and low molecular weight proteins but there is some evidence that, of the filtered proteins reaching the proximal tubule, those with lower molecular weights are reabsorbed by different mechanisms from those with higher molecular weights. For example, although increased amounts of low molecular weight proteins are excreted in tubular disease, such large increases are not seen in the urine of patients with massive glomerular proteinuria, when the reabsorptive mechanisms for large molecular weight proteins such as albumin must be saturated. Conversely, experimental induction of massive low molecular weight proteinuria through saturation of reabsorption with other low molecular weight proteins does not induce a similar increase in urine albumin.

Low molecular weight proteins, such as α₁-microglobulin and retinol-binding protein, that are readily filtered at the glomerulus and normally > 99.9% reabsorbed, can be used as sensitive markers of proximal tubular function, since even subtle tubular damage is associated with increased urinary excretion.

Glomerular diseases causing heavy proteinuria induce the release of a multitude of inflammatory and fibrogenic mediators, all of which cause tubulointerstitial fibrosis and renal scarring that contribute to renal impairment. Recent studies suggest that large proteins, such as albumin, which pass through the diseased glomerular membrane, cause tubular cell injury when they reach the proximal tubules. Reabsorption of excessive amounts of these proteins leads to overloading of lysosomes and activation of proximal tubular cells, which produce matrix metalloproteins, cytokines, leukocyte chemoattractants and vasoactive mediators, leading to interstitial inflammation and scarring. The mechanism of injury has been most studied for albumin and it appears likely that compounds attached to the protein, such as fatty acids, are major culprits. In minimal change glomerular disease with heavy proteinuria that is not associated with scarring, the albumin-fatty acid composition appears to be different from that in other glomerulopathies.

Tubular secretion of proteins

Proteins that are too large to be filtered by the normal glomerulus appear in urine either as a result of tubular secretion or from desquamation of tubular epithelial cells as part of the normal cellular turnover. Some large molecular weight proteins may also enter the urine during its postrenal passage along the urinary tract. Measurable activities of enzymes such as N-acetyl β-D-glucosaminidase (150 000 Da), γ-glutamyl transferase (120 000 Da) and lactate dehydrogenase (144 000 Da) can be found in normal urine.

The major urine protein exclusively of renal origin is uromodulin (previously known as Tamm–Horsfall glycoprotein), a heavily glycosylated protein secreted from the thick ascending limb of the loops of Henle. It has a molecular weight of 70 000 Da but is normally present in urine as a large non-covalently linked polymer. Since its isoelectric point is 3.3, it tends to precipitate as a gel during urinary acidification, forming casts by trapping whatever is in the vicinity such as albumin, red blood cells, tubular cells or cellular debris. Numerous mutations in the uromodulin gene have been identified in association with familial juvenile hyperuricaemic nephropathy and medullary cystic kidney disease type 2. These are probably different phenotypes of the same disease and are known collectively as uromodulin storage disease, owing to accumulation of the misfolded mutated protein in cells of the thick ascending limb of the loops of Henle. They are rare, primary tubulointerstitial disorders with autosomal dominant inheritance characterized by chronic progressive kidney disease, hyperuricaemia and gout, with minimal proteinuria.

The normal biological function of uromodulin remains unclear but it probably has a protective role in trapping potentially damaging material in the urinary space, allowing excretion as casts. Microscopic differentiation of the type of casts present in urine can give useful information about pathological processes within the kidney (see Chapter 7). Experimental studies suggest that uromodulin may have a role in protecting against stone formation. Its capacity to bind type-1 fimbriated E. coli has led to the suggestion that its excretion has evolved as a defence against urinary tract infections.

NORMAL URINARY PROTEIN CONTENT

A normal adult excretes about 300 mg/24 h of non-dialysable material, of which < 140 mg/24 h is protein, but published reference ranges for total urinary protein excretion vary considerably with the analytical method used. Plasma proteins represent only some 25 mg/24 h of total urinary protein (Table 8.2), of which about half is albumin: the remaining proteins are of renal origin, uromodulin being the major contributor (70 mg/24 h). For these reasons, and because low-level albuminuria (microalbuminuria) has prognostic value both for renal and non-renal diseases, urine albumin immunoassays should be used to assess glomerular proteinuria when protein excretion rates are low.

Determinants of urine protein excretion

Age, sex and diurnal variation

In neonates, albumin excretion tends to be higher than in older children and adults: this has been attributed to greater permeability of the neonatal glomerulus. Total urine protein tends to fall after birth and rises with increasing age, reaching adult excretion rates by puberty. However, the urine protein/creatinine concentration ratio remains constant from 3 to 15 years of age, since urine creatinine excretion rises with increasing body mass. In children aged 4–16 years, albumin excretion rate, corrected for body surface area, rises with age and is slightly higher in females. Daytime excretion rates are higher than during the night and the sex difference disappears in overnight collections. There appears to be no sex difference in urine albumin excretion in adults, but, when expressed as a ratio to creatinine, the reference range is slightly higher in females owing to their lower muscle mass and hence lower creatinine excretion.

Posture

In both adults and children, ambulatory urine protein excretion is higher than it is overnight or during recumbency, with two- to ten-fold differences being reported for urinary albumin. Orthostatic proteinuria in otherwise healthy subjects has been the subject of controversy for some time. The discussion has been complicated by the variety and differing sensitivities of the protein assays used.

Proteinuria of < 1 g/24 h has been described in 0.6–9% of healthy young adults, in the absence of urinary red cells, white cells or casts, and can be divided into ‘constant’ and ‘postural’ based on its persistence after recumbency. Renal biopsies of patients with postural proteinuria reveal that 8% have unequivocal evidence of well-defined disease and 45% have subtle alterations in glomerular structure. However, more recent studies, using non-invasive Doppler ultrasound to compare recumbent and orthostatic blood flow in the left renal veins of young people with orthostatic proteinuria, have revealed that in > 50% of patients there is reduced blood flow to the left renal vein during standing owing to entrapment of the left renal vein.

The medical management of isolated or postural proteinuria in an otherwise healthy patient tends to be conservative, with annual assessment of proteinuria and renal function; biopsy is reserved for the rare patient who has evidence of progressive renal impairment.

Exercise and diet

Exercise-induced proteinuria was discovered over a century ago in soldiers after marches or drills. Five- to 100-fold increases in the excretion of proteins such as albumin, transferrin and immunoglobulins have been observed following 26-mile marathon runs, with smaller increases after less strenuous activities. The pattern of exercise-induced proteinuria is generally glomerular, although mixed glomerular and tubular proteinuria has also been described, which persists for over 3 h after exercise. The reason for exercise-induced proteinuria is unclear, but some degree of renal ischaemia owing to redistribution of blood during exercise has been suggested as a possible mechanism.

A large protein meal is associated with an increased urine albumin excretion, which appears to be secondary to an associated increase in GFR.

The lowest and most reproducible estimates of urine protein excretion are obtained from an early morning urine specimen after overnight recumbency.

Pregnancy

During normal pregnancy, the urinary albumin excretion rate generally remains within the non-pregnant range, although there is some evidence for a small increase in albumin excretion during the third trimester, which may be related to increased glomerular permeability and/or GFR. Total urine protein excretion increases owing to decreased renal tubular protein reabsorption.

Hypertension in pregnancy is associated with significant maternal and fetal morbidity and mortality. The reliable detection of significant proteinuria is most important in women with new-onset hypertension during pregnancy because it distinguishes between those pregnancies with pre-eclampsia and those with gestational hypertension, the former often requiring admission to hospital owing to the severity of potential complications. In the UK, NICE guidelines for the routine antenatal care of healthy pregnant women recommend blood pressure and urine protein measurement at each antenatal visit; 660 000 women each year will have at least 7–10 such checks.

Gestational hypertension is defined as new hypertension occurring after 20 weeks of pregnancy, but without significant proteinuria. In this group, routine urine protein measurement may be performed using an automated reagent-strip reading device (more reliable than a manual reading) or by a laboratory method. If a reagent strip reading is 1 + or greater, the proteinuria should be quantitated by a laboratory measure in a spot or 24 h urine sample. Women admitted with pre-eclampsia (new hypertension presenting after 20 weeks with significant proteinuria) do not need to have repeated measures of urine protein since there is no strong evidence linking the degree of proteinuria with adverse outcome. Significant proteinuria is defined as > 300 mg/24 h or > 30 mg/mmol creatinine in a random sample. There are insufficient studies of urine albumin excretion to be able to define cut-offs equivalent to those defining significant proteinuria in gestational hypertension or pre-eclampsia.

PROTEINURIA IN KIDNEY DISEASE

Richard Bright, in 1836, is generally credited with noting the association between proteinuria and kidney disease. Proteinuria remains the most frequent clinical finding and quantitation of proteinuria is valuable for diagnosing, monitoring and assessing the prognosis of kidney disease. Normally, total urine protein excretion is < 150 mg/24 h in adults and < 140 mg/m²/24 h in children, depending on the methods employed: normal concentrations are often undetectable by chemical methods. However, sensitive immunoassay of specific proteins has extended the detection limits to urine protein concentrations within the reference range. Measurements of low concentrations of specific proteins such as albumin, predominantly reflecting glomerular function, and α₁-microglobulin or retinol-binding protein, reflecting tubular reabsorptive function, are now used as very sensitive and early markers of primary renal disease (e.g. glomerulonephritis) and secondary renal disease (e.g. in diabetes mellitus or hypertension). Thus, proteinuria can now be regarded as a continuum that extends from the measurable amount of protein normally excreted in urine up to the 1000-fold increases found in nephrotic syndrome.

Conventionally, proteinuria has been classified into glomerular proteinuria; tubular proteinuria; nephrogenic proteinuria (e.g. uromodulin, basement membrane and tubular proteins); proteinuria of prerenal origin (e.g. overflow proteinurias such as light chain disease, myoglobinuria, haemoglobinuria, lysozyme in leukaemia and amylase in pancreatitis), and postrenal proteinuria owing to obstruction of the urinary tract or inflammation such as occurs in urinary tract infection. This is a convenient way of differentiating the principal sites of the renal abnormality, but is an over-simplification because, for example, glomerular disease leading to large amounts of plasma proteins being presented to the renal tubules leads to inflammatory changes within the tubules and renal scarring. Nor does this classification lend itself easily to the multifactorial causes of proteinuria in some disorders, e.g. HIV-associated glomerular and tubulointerstitial disease that may be modulated by co-infection with other viruses and drug-induced interstitial nephritis.

Proteinuria in staging and prognosis of chronic kidney disease

Numerous epidemiological studies have demonstrated the association of proteinuria with poorer prognosis in people in the general population and across all stages of chronic kidney disease (CKD): any renal disease is more likely to progress, there is an increased risk of developing acute kidney injury, and both all-cause and cardiovascular mortality are increased. These outcomes hold true, whether proteinuria is assessed by dipstick testing or by formal laboratory measurement of either total protein or albumin, in timed urine collections or in random samples. The quantitation of proteinuria (in the absence of a symptomatic urinary tract infection and preferably using the first morning urine) is an essential component of CKD staging, where the suffix ‘p’ is used to denote its presence. The decision limit is an albumin/creatinine ratio (ACR) > 30 mg/mmol (~ 300 mg/24 h) or urine protein/creatinine ratio (PCR) > 50 mg/mmol (~ 0.5 g/24 h), although the continuum of risk, albeit lower, extends into the reference range. The presence of proteinuria in CKD is sufficient indication to initiate blockade of the renin–angiotensin–aldosterone system (RAS) with angiotensin-converting-enzyme inhibitors (ACEI) or angiotensin-II receptor blockers (ARB). At higher excretions (ACR > 70 mg/mmol or PCR > 100 mg/mmol), RAS blockade should be titrated to the highest tolerable levels and referral of the patient to specialist care considered.

Glomerular proteinuria and nephrotic syndrome

In the normal adult, the renal tubules reabsorb about 2–3 g of filtered albumin every 24 h. Thus, even total failure of this process cannot explain albuminuria of > 3.0 g/24 h; such losses are usually secondary to increased glomerular permeability associated with glomerular damage.

Nephrotic syndrome can be defined as proteinuria severe enough to cause hypoalbuminaemia and oedema. The degree of proteinuria varies but is generally > 3.5 g/24 h and is accompanied by a plasma albumin < 25 g/L. However, it should be remembered that the amount of protein in the urine may decrease as the plasma protein concentration or the GFR falls. Causes of nephrotic syndrome are listed in Table 8.3. In addition to the nephrotic syndrome, glomerular proteinuria is a feature of several other syndromes of nephron injury, and the severity of proteinuria taken together with other clinical findings can allow useful diagnostic classification (Table 8.4).

Mechanisms underlying glomerular proteinuria

Glomerular injury can occur as a result of either primary or secondary kidney disease and there is no single pathogenetic pathway that will embrace all the possible mechanisms. The term glomerulonephritis is generally reserved for immunologically mediated diseases and excludes other conditions associated with glomerular damage such as diabetes mellitus or amyloidosis.

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