Proteinuria: detection and role in native renal disease progression

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Transplantation Reviews 26 (2012) 3 – 13 www.elsevier.com/locate/trre

Proteinuria: detection and role in native renal disease progression Jose Luis Gorriz a,⁎, 1 , Alberto Martinez-Castelao b, 1 b

a Servicio de Nefrologia, Hospital Universitario Dr Peset, Valencia, Spain Servicio de Nefrologia, Hospital Universitario Bellvitge, IDIBELL, Barcelona, Spain

Abstract The presence of albuminuria or proteinuria constitutes a sign of kidney damage and, together with the estimation of glomerular filtration rate, is based on the evaluation of chronic kidney disease. Proteinuria is a strong marker for progression of chronic kidney disease, and it is also a marker of increased cardiovascular morbimortality. Filtration of albumin by the glomerulus is followed by tubular reabsorption, and thus, the resulting albuminuria reflects the combined contribution of these 2 processes. Dysfunction of both processes may result in increased excretion of albumin, and both glomerular injury and tubular impairment have been involved in the initial events leading to proteinuria. Independently of the underlying causes, chronic proteinuric glomerulopathies have in common the sustained or permanent loss of selectivity of the glomerular barrier to protein filtration. The integrity of the glomerular filtration barrier depends on its 3-layer structure (the endothelium, the glomerular basement membrane, and the podocytes). Increased intraglomerular hydraulic pressure or damage to glomerular filtration barrier may elicit glomerular or overload proteinuria. The mechanisms underlying glomerular disease are very variable and include infiltration of inflammatory cells, proliferation of glomerular cells, and malfunction of podocyte-associated molecules such as nephrin or podocin. Albumin is filtered by the glomeruli and reabsorbed by the proximal tubular cells by receptor-mediated endocytosis. Internalization by endocytosis is followed by transport into lysosomes for degradation. The multiligand receptors megalin and cubilin are responsible for the constitutive uptake in this mechanism. Albumin and its ligands induce expression of inflammatory and fibrogenic mediators resulting in inflammation and fibrosis resulting in the loss of renal function as a result of tubular proteinuria. TGF-β, which may be induced by albumin exposure, may also act in a feedback mechanism increasing albumin filtration and at the same time inhibiting megalin- and cubilin-mediated albumin endocytosis, leading to increased albuminuria. Urinary proteins themselves may elicit proinflammatory and profibrotic effects that directly contribute to chronic tubulointerstitial damage. Multiple pathways are involved, including induction of tubular chemokine expression, cytokines, monocyte chemotactic proteins, different growth factors, and complement activation, which lead to inflammatory cell infiltration in the interstitium and sustained fibrogenesis. This tubulointerstitial injury is one of the key factors that induce the renal damage progression. Therefore, high-grade proteinuria is an independent mediator of progressive kidney damage. Glomerular lesions and their effects on the renal tubules appear to provide a critical link between proteinuria and tubulointerstitial injury, although several other mechanisms have also been involved. Injury is transmitted to the interstitium favoring the self-destruction of nephrons and finally of the kidney structure. © 2012 Published by Elsevier Inc.

1. Introduction The presence of variable and persistent amounts of proteins in the urine constitutes a sign of kidney damage, and together with the estimation of glomerular filtration rate ⁎ Corresponding author. Department of Nephrology, Hospital Universitario Dr Peset, Juan de Garay 21, Valencia 4017, Spain. Tel.: +34 96 162 2491; fax: +34 96 162 2456. E-mail address: [email protected] (J.L. Gorriz). 1 GEENDIAB (Diabetic Nephropathy Group, Spanish Society of Nephrology). 0955-470X/$ – see front matter © 2012 Published by Elsevier Inc. doi:10.1016/j.trre.2011.10.002

(GFR), they are based on chronic kidney disease (CKD) evaluation [1]. Proteinuria also identifies a subgroup of patients with high risk of progressive kidney damage [2] and increased cardiovascular (CV) morbidity [3,4]. On the other hand, the presence of protein in urine is a strong indicator that a patient is likely to experience progressive kidney function decline. Even in the setting of a relatively normal GFR, the proteinuric patient has a high chance of s howing a steep progressive slope of GFR loss compared with a patient with low or no urinary protein excretion. The association of proteinuria and estimated GFR improves the

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capacity of prediction of CV morbidity and mortality and renal outcomes [5,6]. Urinary proteins themselves may elicit proinflammatory and profibrotic effects that directly contribute to chronic tubulointerstitial damage. This occurs through multiple pathways, including induction of tubular chemokine expression, complement activation leading to inflammatory cell infiltration in the interstitium, and sustained fibrogenesis [7]. Proteinuria is a marker of renal outcome, independent of other risk factors. The more one lowers the proteinuria in an individual patient, the better the patient is protected against further kidney function loss and eventual kidney failure [8,9]. However, currently used renoprotective therapies were not designed to be antiproteinuric. Rather, they were developed to address other kidney disease risk factors, most notably hypertension. Apart from the blood pressure control, drugs that intervene in the renin-angiotensinaldosterone system (RAAS) are best known for their antiproteinuric effect. The use of angiotensin-converting enzyme inhibitors [10], angiotensin II receptor blockers [11], aldosterone antagonists [12], and renin inhibitors [13] in patients with proteinuria and CKD has shown beneficial effects on the progression of CKD and the incidence of CV events and death. The reduction in proteinuria in the first months of treatment with drugs that blocked the RAAS is associated with long-term kidney function protection, and it is independent from the effect of the drug on blood pressure [14]. Therefore, the decrease of proteinuria is now considered as a therapeutic target [15-17]. Despite treatment with RAAS inhibitors, proteinuric patients have increased risk of progressive renal failure that correlates with the degree of residual proteinuria. Reduction of residual albuminuria or proteinuria to the lowest achievable level should be viewed as a goal for future renoprotective treatments [18]. Recently, other options have been considered as therapeutic targets for proteinuria as endothelin antagonists [19], pentoxifyline [20], and selective activator of the vitamin D receptor, paricalcitol [21] among others (reviewed by Soler et al in this issue). That is the main reason of increasing interest to understand the mechanisms of progression for new therapy targets that would improve the efficacy of antiproteinuric treatment and prognosis on these patients. 2. Evaluation of proteinuria 2.1. Definition of proteinuria The glomerulus produces the primary urine by filtering blood and retains larger proteins including most serum albumin (molecular weight N67 kd). Proteins with a molecular weight below 60 kd usually pass freely through the glomerular basement membrane and are actively reabsorbed within the tubular system. The renal tubule reabsorbs the small molecules and also the small amount of albumin that passes freely through the glomerulus. We usually excrete 40 to 80 mg of proteins per day, from which

10 to 15 mg (30%–40% of them) are albumin and the rest is composed by Tamm-Horsfall protein, derived from epithelial cells from the ascending portion of Henle limb [22], and small quantities of low-molecular-weight proteins (β2-microglobulin, α1-microglobulin, retinol-binding protein, etc). Structural as well as functional defects lead to various distinctive patterns of proteinuria. Previously, abnormal proteinuria was generally defined as the excretion of more than 150 mg of protein per day. However, it is now clear that early renal disease is reflected by lesser degrees of proteinuria, particularly increased amounts of albuminuria. A universal discriminative value for proteinuria does not exist, so other factors such as the kind of specimen used for the evaluation as well as the expression of the results (concentration or excretion) or the age—adults or children—need to be considered. Although for the definition of albuminuria and proteinuria only quantitative parameters have been used, establishing a cutoff, both albuminuria and proteinuria should be considered as a continuous variable, increasing the associated CV and renal risk in parallel to the increase in protein urinary excretion from levels as low as 8 to 10 mg/d [23]. 2.2. Definition of albuminuria An urinary albumin excretion rate of less than 30 mg/d is considered normal. Albuminuria is defined as the elimination of urinary albumin excretion above 30 mg per 24 hours, equivalent to 20 mg/min in urine collected over a determined period or 30 mg/g creatinine in a single sample. Some scientific societies have proposed specific limits for each sex, to minimize the influence of different production and excretion of creatinine. The K/DOQI guidelines suggest limits of 17 mg/g in males and 25 mg/g in females [1]. The ESC-ESH guidelines proposed as normal excretion of up to 22 mg/g in males and 31 mg/g in females [24]. Other guidelines offered their own diagnostic criteria [25-27]. Nevertheless, urinary albumin excretion in a continuous variable and the lower values the better, because even within the range considered normal, the lowest values are associated with lower risk of cardiovascular events. If we express an aleatory specimen, the concentration may be expressed in relation to the urine creatinine concentration (albumin/creatinine ratio, or ACR). The international consensus estimates that more than 2.5 mg/mmol (22 mg/g) in male and N3.5 mg/mmol (26 mg/g) in female can be considered normal. These values, obtained from insulin-dependent diabetic patients [28,29], were extrapolated to the general population [30]. Urinary albumin excretion expresses the presence in the urine of minimal quantities of albumin that are undetectable by dipstick. The quantity varies in relation to the specimen used, the units of measurement, and the criteria for interpreting the results. Some scientific societies recommend taking into account the different creatinine urinary excretion by sex [31]. Some authors express their concern about the terms micro- and macroalbuminuria because of possible confusion

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and suggest abandoning them [32]. Albuminuria has always the same “molecular weight.” So probable should be more accurate oligoalbuminuria than microalbuminuria. We prefer the term albuminuria, or even better urinary albumin excretion. In case of levels higher than 300 mg/g or 300 mg/d, the term is proteinuria. The Bergamo group suggests that the terms microalbuminuria and macroalbuminuria could be replaced by the concepts of albuminuria and proteinuria-associated diseases [33]. The proportion of excreted albumin in relation to different quantities of proteins varies between less than 5% for ACR of less than 2.5 mg/mmol (22 mg/g) and 70% for a protein/ creatinine ratio of more than 90 mg/mmol (784 mg/g) (to convert mg/mmol to mg/g, multiply the value by 8.82) [34,35]. 2.3. Types of proteinuria Urine protein concentration may be the result of different mechanisms [36], being that the quantity and quality of the proteinuria are very different. • Prerenal proteinuria, caused by over production. We can observe that in paroxysmal nocturnal haemoglobinuria rhabdomyolysis (myoglobinuria), mielomonocític leukemia (lysozimuria), and myeloma (free light chain disease). • Glomerular proteinuria, caused by functional or structural modifications of the electronic glomerular base membrane charge, producing a disturbance of the filtration barrier. We can observe that type in glomerulonephritis, infections, systemic erythematous lupus, diabetes, hypertension, neoplasia, and congenital diseases. • Tubular proteinuria: decreased reabsorption of the proteins usually filtered by the glomerulus, caused by alterations of the tubular reabsorption mechanisms. Low-molecular-weight proteins appear in the urine, such as β2-microglobulin, α1-microglobulin, or retinolbinding protein. We can observe this kind of proteinuria in congenital or systemic diseases and in cases of toxicity caused by drugs and toxins. • Postrenal proteinuria: as a consequence of inflammatory, infectious, or hemorrhage processes, epithelial tubular cells can add proteins to the tubular fluid affecting the lower urinary tract. • Orthostatic proteinuria: caused by supine position and disappearing in orthostatism. It is caused by hemodynamic glomerular alterations. 2.4. Measurement of proteinuria 2.4.1. Twenty-four-hour urine collections Twenty-four-hour urine collection is cumbersome, inconvenient for patients, and associated with important errors derived from its incomplete collection. Consequently, 24-hour collections are not recommended as screening in CV disease [37]. It may be helpful in some patients with tubular proteinuria or follow-up of patients with glomerular diseases or kidney transplantation.

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2.4.2. Screening methods 2.4.2.1. Dipstick for the screening of proteins. A cellulose surface with bromotetraphenol at pH 3.0 [38] produces a color change in contact with proteins, varying depending on the protein concentrations. The use of chromatic scale reduces the variability of interpretation and the possibility of errors [39]. Usually, the change of “1+” or superior indicates a concentration between 150 and 300 mg/L [38], but it is not possible to detect concentration less than 300 mg/L. False negatives are very frequent [39]. The sensibility and specificity show great variability and many guidelines, and clinical practice recommendations are in disagreement with the use of dipstick as a screening method to detect the prevalence of proteinuira [25,27,40] and a positive result may be reconfirmed by a quantitative measurement [1]. Some dipstick devices have incorporated a zone to measure the creatinine, making it possible to measure the ACR, but studies are necessary to evaluate the usefulness of this procedure [41]. 2.4.2.2. Dipstick for the screening of albumin. A cellulose surface impregnated with tetrabromosulfoftaleín [38] is able to detect small albumin concentrations (30–40 mg/L) in the urine, and there exists the possibility to measure urine creatinine (peroxidase activity) and to give a semiquantitative measurement of the ACR divided in 3 categories, less than 3.4, 3.4-33.9, and more than 33,9 mg/mmol. There are a scarce number of studies, but these systems can offer good results in CKD patients and the general population [42]. 2.4.3. Quantitative methods 2.4.3.1. Quantitative methods to measure proteins. The protein quantification in urine presents a wide variability because of the possible interference of many nonprotein substances. The most widely used are turbidimetric or benzethonium methods, which are based on the union of the proteins to trichloroacetic acid or to colored fixation methods (Ponceau-S, Coomassie, or pyrogallol molybdate). A unified method of reference does not exist, and the sensibility and specificity are very variable [43]. 2.4.3.2. Quantitative methods to measure albumin. The most usual methods are immunoturbidimetry and nephelometry that are able to detect between 2 years of 10 mg/L of albumin. In recent years, high liquid chromatography can detect nonimmunoreactive albumin. Other forms of albumin—dimeric and glycosilated—can now be detected [44,45]. A new method based on high liquid chromatography and isotopic mass spectometry dilution is a possible reference method in the next years [46]. There is general agreement on the superiority of specific immunoanalysis for albumin determination than for protein quantification [47]. The Laboratory Working Group of the National Kidney Disease Education Program and the International Federation

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of Clinical Chemistry and Laboratory Medicine [32] recommend to consider the preanalytic requirements, the biologic variability, the molecular forms, and degradation of albumin; to develop a reference method for measurement; to search for equations to define differences regarding age, sex, and ethnic groups; and to use very strict quality control programs. 3. Recommendations in clinical practice 3.1. What for screening? Most of guidelines [1,24-27,37] recommend the ACR in a first-morning-void urine sample as the best option for nephropathy screening in diabetic and nondiabetic patients. This is a ratio between milligram of albumin per gram of creatinine in urine (or milligram of albumin/mmol of creatinine in SI units). The drawbacks associated with differences in urine volume or hydration status can be mitigated using the ACR. Twenty-four-hour excretion of creatinine is a constant, not dependent of the urine volume. Therefore, the ACR estimates 24-hour albumin excretion, increasing the diagnostic specificity and sensibility to 85% when compared with 24-hour urine collection [48]. Because intraindividual variability (reproducibility, variability, and circadian time of determination throughout the day) of albuminuria may be up to 40%, 2 of 3 positive samples are required for the diagnosis of a positive albuminuria. By contrast, a normal value in a spot urine sample confirms the normoalbuminuria [49]. 3.2. Factors influencing urinary albumin excretion Some situations can introduce variations in the estimation of the albuminuria along 24 hours or in a spot sample. Physical activity or the rest in supine decubitus can widely vary urine albumin excretion. At night, the urinary albumin excretion is reduced 20% in relation with diurnal excretion because of the decreased renal filtration pressure. Other conditions, such as intense physical exercise, menstruation, urinary tract infections or piuria, hematuria, fever, substantial increases in blood pressure, glycemic disturbances or, heart failure [37], may be considered before a diagnosis of albuminuria can be stated. 3.3. Albumin/creatinine ratio or protein/creatinine ratio? K/DOQI guidelines [1] recommend the urinary ACR for diagnosis and follow-up in adults. This is because albuminuria has been a more sensitive marker for the detection of incipient nephropathy and has been more validated in studies than proteinuria in diabetes, hypertension, and glomerular diseases [1]. However, when the ACR is high (N500 mg/g, which corresponds to a proteinuria N500 mg/d), it increases the removal of other proteins than albumin urine (tubular proteinuria). In these clinical situations, the protein/creati-

nine ratio is fully recommended, with the 24-hour urine collection being unnecessary because of errors that can be introduced in the sample collection [50]. However, other guidelines [25-27] recommend the implementation of protein/creatinine ratio. This is especially recommended in children (PARADE Guidelines) [3] because the causes of kidney disease are often not related to diabetes or hypertension, being the main etiologies associated with abnormal urinary tract or tubular defects, which are characterized by elimination of low-molecularweight proteins [31]. The type of proteins excreted by the urine depends on the etiology of the kidney disease: albumin excretion is more frequent in diabetic, glomerular, o hypertensive kidney disease, whereas the presence of low- molecular-weight proteins is more frequently of tubulointerstitial origin [1,51]. Probably, this may be the reason to recommend preferably ACR in diabetes mellitus, hypertension, and glomerular diseases, whereas protein/creatinine ratio is used in tubulointerstitial diseases. Very recently, Erman et al [52] have proposed a change in the assessment of the ACR in renal transplant recipients, suggesting that the optimal cutoff was 21 mg/g. Their data support the need for a reappraisal of the 30 mg/g cutoff for the detection of microalbuminuria [52]. 3.4. General recommendations [31] 3.4.1. Evaluation of proteinuria and/or albuminuria 1. The presence of proteinuria identifies a subgroup of population at risk for CKD progression and CV morbidity and mortality. 2. In CKD patients and inpatients at risk for CKD, the measurement of albumin/protein excretion in the urine may be periodically practiced 3. The detection and monitorization may be quantitative: a. In adult individuals by the determination of an ACR in a morning urine specimen. A normal protein/ creatinine ratio may be confirmed by an ACR. b. In children without diabetes, an ACR in a specimen of urine may be obtained, because of the frequent presence of nonglomerular proteinuria. c. In children with postpubertal diabetes mellitus from more than 5 years, an ACR in urine specimen may be obtained. In individuals with CKD and significant proteinuria (ACR N56 mg/mmol or N500 mg/g), the monitorization based on an ACR is possible. A value of albumin/ creatinine more than 2.5 mg/mmol (N22 mg/g) in males, more than 3.5 mg/mmol (N30 mg/g) in female, or protein/ creatinine more than 22.6 mg/mmol (N200 mg/g) are indicatives or clinically significant proteinuria. Because of the high proportion of albumin in relation to creatinine in the urine, a conversion factor for the ACR to protein/creatinine ratio is not recommended.

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3.4.2. Specimen First-morning urine is adequate for detection and monitorization because of the less biologic variability and the good correlation with 24-hour albumin/protein excretion. If the specimen is not immediately processed, the urine may be preserved at −70°C or less and thawed at room temperature. The albumin or protein concentration may be referred as concentration of creatinine. The results may be expressed in milligram per gram or milligram per millimole.

4. Proteinuria as a marker of CV disease 4.1. Proteinuria and CKD as risk markers for CV disease Cardiovascular disease is common in the CKD patient and the cause of a high mortality rate. Approximately 30% of all deaths worldwide and 10% of all healthy life lost are accounted by CV disease alone. Mortality of CV origin still remains high in developing countries, in ethnic and socially disadvantaged minorities, or in CKD patients. The presence of CKD significantly increases the risk of CV events in patients at risk, such as diabetic and hypertensive people. However, CKD alone is a strong risk factor for CV disease, independent of diabetes, hypertension, or any other conventional risk factors. This aspect is especially true when proteinuria increases or is present. Albuminuria is a predictor not only of nephropathy but also of CV risk, in diabetic and nondiabetic patients [53]. Its reduction is associated with a decreased risk in renal and CV events [54]. Multiple studies in different patient populations have suggested that, in addition to its relationship with renal disease, albuminuria is an important risk factor for CV disease and early CV mortality in patients with and without diabetes and/or hypertension [55]. In patients with CKD, the level of proteinuria is both the most accurate predictor of renal outcome [18,56] and an independent predictor of mortality [57]. The combination of both proteinuria and GFR estimation is based on the most recent CKD classification. This definition and classification have been widely accepted by most of the scientific societies under the initiative of international Kidney Disease: Improving Global Outcomes [58]. In recent years, the addition of the letter D,T, or P has been proposed to identify dialysis or transplant patients with CKD, and the letter “P” has been added to separate those with proteinuria as a sign of severity of CKD [59,60]. Other consideration when regarding CKD stages has been the subdivision of stage 3 in a 3A (GFR 45–59 mL/min/1.73 m 2) and 3B (30–44 mL/ min/1.73 m 2) [25,58]. Some authors have proposed to eliminate the CKD stages 1 and 2 as considered CKD [60] or to join them in a unique stage [61]. The need for additional evidences of renal lesions could be considered as a prerequisite to estimate GFR values more than 30 or 45 mL/min/1.73 m 2 [62].

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The presence of albuminuria makes the diagnosis of most of CKD stage 1 or 2 cases. Those patients have a CV risk comparable with stage 1 and 2 CKD and had more renal function loss compared with subjects with stage 3 CKD without albuminuria. Subdividing stage 3 CKD according to the presence or absence of albuminuria probably improves risk stratification within this stage [6]. These findings suggest that, with the goal of risk stratification in mind, an evaluation of the current K/DOQI classification of CKD may be mandatory. Very recently, Peralta et al [63], in a prospective cohort study involving 26 643 US adults enrolled in the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study, showed that adding cystatin C to the combination of creatinine and ACR measurements improved the predictive accuracy for all-cause mortality and end-stage renal disease. Compared with those with CKD defined by creatinine alone, the hazard ratio for death in multivariate adjusted models was 3.3 for creatinine and ACR, 3.2 for creatinine and cystatin C, and 5.6 for those with the 3 CKD biomarkers [63]. 4.2. Specific CV risk attributed to proteinuria Although proteinuria is not a conventional CV risk factor, the risk of CV disease is better correlated with albuminuria or proteinuria than with GFR alone. The Prevention of Renal and Vascular End Stage Disease (PREVEND) [64] study, which analyses 40 548 individuals, showed a direct linear relationship between albuminuria and the risk of CV death in the general population, even at albuminuria levels of 15 to 29 mg/d, generally considered within the reference range. The risk of CV death was increased more than 6-fold when albumin excretion was more than 300 mg/d. Additional analysis from this PREVEND study showed that both macroalbuminuria and impaired renal function predict a worse prognosis with respect to CV morbidity and mortality, but macroalbuminuria is a better risk marker than low estimated GFR to identify individuals at risk for accelerated GFR loss [5]. Albuminuria and reduced GFR are independent and continuous risk factors for CV and kidney outcomes in patients with type 2 diabetes, as shown by the Action in Diabetes and Vascular disease: preterAx and diamicroNMR Controlled Evaluation (ADVANCE) study including 10 640 type 2 diabetic patients followed up more than 4 years [65]. Recent data from the US National Health and Nutrition Examination Survey also document an independent effect of albuminuria on risk of both CV disease and all-cause mortality at all levels of GFR. In patients with congestive heart failure without diabetes, hypertension, or reduced GFR, increased urinary albumin excretion predicts both CV and all-cause mortality [66]. Proteinuria also confers a greater risk of mortality than reduced renal failure in patients with coronary disease or

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previous myocardial infarction. The presence of proteinuria at all levels of GFR accelerates the time to development of a CV event [65,67], Multiple studies now confirm that proteinuria is a graded risk factor for CV disease independent of GFR, hypertension, or diabetes. That risk extends down into ranges of albuminuria generally considered normal [23], This increased CV risk has also been demonstrated in studies in which only dipsticks were used to screen for protein excretion. A concern exists about the value of GFR as a diagnostic of CKD in older adults because of age alone. 4.3. Proteinuria as a risk marker for CKD progression The presence of proteinuria is a powerful indicator that the patient has a high probability of renal disease progression both diabetic and nondiabetic [5,18,55], A patient with proteinuria, even with normal GFR, has a high probability of a progressive deterioration of the renal function compared with diabetic patients without albuminuria or excreting minimal quantities of albumin by the urine [5], The progression and risk of terminal renal disease was correlated with the degree of proteinuria. A very important practical aspect is that proteinuria is a modifiable factor, and the more it is reduced, the better protection for the patient from renal progression and to develop end-stage renal disease. Indirectly, while reducing proteinuria, we act on mechanisms that influence the patient's CV risk. Proteinuria is not only a CV risk marker of renal impairment but also has been proposed as a treatment goal. A post hoc analysis of the Reduction in Endpoints in Non–insulin dependent diabetes mellitus with the Angiotensin II Antagonist Losartan (RENAAL) study, stratifying the patients into 3 groups as the intensity of proteinuria increases, showed a clear decrease of the renal end points— doubling of plasma creatinine or dialysis initiation—in patients in whom proteinuria decreased [7]. Patients with baseline proteinuria (N3 g/g) were 5 times more at risk for a renal event and 8-fold increased risk for progression to dialysis against patients with low albuminuria (b1.5 g/g). The reduction of proteinuria in the first 6 months of treatment was the strongest predictor of CV events. Patients from this study were proteinuric (ACR mean of 1 240 mg/g). Each reduction of albuminuria by 50% reduced the risk of CV events by 18%. Patients with greater reduction in albuminuria also had less renal impairment. Patients who developed less renal events were those with a greater reduction in albuminuria [18]. Proteinuria is therefore one of the most important predictors of progression of CKD. Despite that the different etiologies of CKD present different degrees of progression (chronic glomerulonephritis, diabetic nephropathy, polycystic kidney disease, and tubule-interstitial nephritis), the main risk factors influencing disease progression and the degree of control are more important that the cause of the disease itself.

4.4. Other risk factors for CKD progression Along with proteinuria, other clinical predictors of accelerated progression of renal disease are blood pressure control [[68-70], hyperglycemia [71], obesity [72], dislypidemia [73], and tobacco use [74], Control of blood pressure, hyperglycemia, and obesity decrease proteinuria and/or slow renal function deterioration [68-72]. However, it has been difficult to demonstrate in clinical trials that dyslipidemia treatment can reverse the progression of CKD.

5. Mechanisms for proteinuria progression in CKD Proteinuria is a strong marker of CKD progression. Proteinuria may accelerate kidney disease progression to end-stage renal failure, and this process occurs through multiple pathways, including induction of tubular chemokine expression and complement activation that lead to inflammatory cell infiltration in the interstitium and sustained fibrogenesis. 5.1. Where do proteins in the urine come from and how is proteinuria produced? Proteins in the final urine have different origins. First, proteins cross the glomerular barrier and tubular cell reabsorption modifies their final concentration. Second, there is tubular secretion of proteins from the blood. Third, proteins may be synthesized by the cells themselves and released into the urine. Fourth, proteins may be added to the urine at a later stage (eg, by excretion from the prostate gland in men). Independently of the underlying causes, chronic proteinuric glomerulopathies have in common a sustained or permanent loss of selectivity of the glomerular barrier to protein filtration. Glomerular sclerosis is the progressive lesion beginning at the glomerular capillary wall, the site of abnormal filtration of plasma proteins. Injury is transmitted to the interstitium favoring the self-destruction of nephrons and eventually the kidney. Factors involved in the development of albuminuria are as follows: • Molecular size and sieving coefficient: Low-molecularweight proteins (molecular weight, 40 kd) are essentially freely filtered, whereas high-molecular-weight proteins (molecular weight, 4100 kd) are almost completely restricted. However, in diabetic patients with low-grade proteinuria (b300 mg/d), no change of size selectivity has been reported, suggesting that alterations in the charge barrier may be responsible for. As the magnitude of albuminuria increases, there is an increment of large pores and sieving coefficient [75]. • Charge barrier: This aspect remains debated on. Although the site of any anionic charge barrier has been considered to lie within the glomerular basal

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membrane, it has been demonstrated also in podocytes and endothelial glycocalyx [76]. 5.1.1. Alteration in glomerular filtration barrier (glomerular filtration of albumin) The glomerular barrier is by far the most complex biologic membrane, with properties that allow for high filtration rates of water, nonrestricted passage of small and middle-sized molecules, and almost total restriction of serum albumin and larger proteins. In humans, approximately 180 L of primary urine are produced each day at capillary pressures far exceeding those in other organs. Despite this tremendous work load, the glomerulus remains intact year after year. The integrity of glomerular filtration barrier depends on his 3-layer structure: the endothelium with his glycocalyx, the glomerular basement membrane, and podocytes (glomerular epithelial cells). Each layer contributes to the permeability of albumin. Albuminuria/proteinuria results from defects in the glomerular filtration barrier, although abnormalities in tubular albumin reabsorption may also contribute. a. Endothelial cell injury: The mechanisms that contribute to the endothelial permeability in albuminuria are not fully determined, but likely, they included inflammatory cytokines, reactive oxygen spices, activation of renin-angiotensin system, dyslipidemia, and hyperglucemia [75]. b. Defects in glomerular endothelial glycocalyx: Heparin sulfate proteoglycans are largely responsible for the negative charge characteristics of the glycocalyx. Removal of the glycocalyx increases vascular protein permeability, providing evidence that it hinders the passage of macromolecules [76]. c. Glomerular basal membrane: This is a hydrated meshwork of collagens and laminins to which negatively charged heparin sulfate proteoglycans are attached. This only makes a small direct contribution to the barrier to protein passage [75]. d. Podocytes: Although the mesangial cells have been considered to be at least the epicenter of injury in CKD, there is an increasing and convincing body of research that shows that injury in podocytes leads directly to proteinuria/albuminuria, specially in diabetic nephropathy. The mutations in podocyte-specific proteins (eg, nephrin mutations result in congenital nephritic syndrome) indicate the importance of podocytes in resisting the passage of protein [77]. In case of diabetic nephropathy, there are a decrease in podocyte number and density, widening of the foot processes, shortening of the slit diaphragm/loss of slit diaphragm proteins, changes in actin cytoskeleton, and decrease in negative charge. All these changes that occur in endothelium, glomerular basement membrane, and podocytes are triggered by different pathways that produce albuminuria. Key players include proinflammatory cytokines and adipokines. Tumor

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necrosis factor α (TNF-α) directly increases endothelial permeability and disrupts the glycocalyx and increases the production of endothelial cell adhesion molecules. Adipokines, which include leptin and adiponectin, affect the enlargement of fat cells, which in obesity is associated with a proinflammatory state, including increased levels of TNF-α and interleukin 6. Leptin induces vascular permeability and stimulates VEGF. In case of diabetes, hyperglycemia causes dysregulation of mediators including TNF-α and enhances production of reactive oxygen species, which directly damage the glomerular endothelial glycocalyx, and disturbs podocyte–endothelial cell communication, leading to albuminuria [75]. Progression of albuminuria to overt nephropathy is accompanied by predictable structural changes in the glomerulus, including podocyte damage and loss. As a result of these changes, overload proteinuria appears as a consequence of increased glomerular permeability enabling glomerular passage of proteins of molecular mass larger than albumin, which are normally retained in the circulation. Increased filtration is a result of either damage to glomerular basement membrane or increased intraglomerular hydraulic pressure. The mechanisms underlying glomerular disease are very variable and include infiltration of inflammatory cells, proliferation of glomerular cells, and malfunction of podocyte-associated molecules such as nephrin or podocin [78]. 5.1.2. Tubular reabsorption of albumin Albumin is filtered by the glomeruli and reabsorbed by the proximal tubular cells by receptor-mediated endocytosis. Internalization by endocytosis is followed by transport into lysosomes for degradation. Filtration of albumin by the glomerulus is followed by tubular reabsorption, and thus, the resulting albuminuria reflects the combined contribution of these 2 processes. Dysfunction of both these processes may result in increased excretion of albumin, and both glomerular injury and tubular impairment have been implicated in the initial events leading to proteinuria. Proximal tubular reabsorption of albumin by endocytosis was demonstrated almost 40 years ago [79]. Endocytic uptake of proteins has been intensively studied and includes both nonspecific fluid-phase endocytosis as well as receptormediated endocytosis. In the kidney, proximal tubule fluid-phase endocytosis of solutes is negligible. Receptor-mediated endocytosis involves the specific binding of a ligand to receptors in the apical plasma membrane. The receptor-ligand complex is internalized by invagination of the plasma membrane caused by adaptor molecule-mediated formation of a cytoplasmic coat. Several receptors for tubular uptake of albumin have been identified. These include the multiligand receptors megalin and cubilin, responsible for the constitutive uptake of a vast variety of filtered plasma proteins and other substances as polypeptide hormones, vitamins, and some

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drugs [78]. Cubilin plays an important role in normal proximal tubule endocytic reabsorption of filtered albumin and is responsible for cellular trafficking including cell surface expression of cubilin. The cubilin-albumin complex requires megalin to be internalized and transferred to lysosomes for degradation. Interestingly, TGF-β, which may be induced by albumin exposure, may also act in a feedback mechanism increasing albumin filtration and, at the same time, inhibiting megalin- and cubilin-mediated albumin endocytosis, leading to increased albuminuria [80]. Alternative mechanisms of albumin reabsorption have been suggested, including a fast, high-capacity retrieval pathway for nondegraded albumin located distal to the glomerular basement membrane [79]. Inactivation or alterations in all these mechanisms lead to tubular proteinuria.

such as RANTES, monocyte chemotactic protein 1, interleukins, and different growth factors inducing tubular changes and tubulointerstitial damage [79]. In glomerular animal models, the interruption of this protein reabsorption process in the proximal tubule may lead to proteinuria [75]. In diabetes, proximal tubular reuptake of protein may be impaired by high glucose, TGF-β, or angiotensin II. Once proteinuria has developed, tubulointerstitial damage appears, and this injury augments urine protein excretion by impairing the reabsorption of filtered (glomerular) proteins, working as a vicious circle [75].

Proteinuria may accelerate kidney disease progression to end-stage renal failure by multiple pathways, including induction of tubular chemokine expression and complement activation that lead to inflammatory cell infiltration in the interstitium and sustained fibrogenesis. Tubular chemokine expression, complement activation, chemoattractants, NFκB activation, and activation signals from proximal tubular cell receptors for uptake of plasma proteins have been involved in the process [81].

5.2.2. Tubulointerstitial injury It has long been recognized that patients with high degree of proteinuria caused by chronic glomerular disease are more likely to develop chronic renal failure than patients with lowgrade or no proteinuria. There are 2 aspects that make the tubulointerstitial injury as one of the most important factors that influence the renal progression. The first one is that renal functional outcome for patients with chronic glomerulopathy is best predicted histologically by the severity of chronic extraglomerular damage (peritubular capillary loss, tubular atrophy, and interstitial fibrosis). The second one is the evidence that urinary proteins themselves may elicit proinflammatory and profibrotic effects that directly contribute to chronic tubulointerstitial damage.

5.2.1. Glomerular proteinuria Glomerular proteinuria in excess in the tubular lumen leads to the induction of inflammation, possible tubular epithelial-mesenchymal transformation, and interstitial fibrosis. Overload albuminuria in rats and mice causes interstitial inflammation and fibrosis. Albumin exposure in tubular cells induces the expression of a number of inflammatory and fibrogenic mediators, including cytokines

a. Effects of proteinuria on renal tubules: Proteinuria stimulates proximal tubular cells to synthesize chemokines (MCP-1, RANTES) that recruit monocytes, T cells, and interleukins that attract neutrophils and fibrosispromoting molecules (eg, endothelin, angiotensin II, TGF-β). Then, damage to tubular basement membrane facilitates the passage of tubular-derived products into the interstitium and peritubular capillaries spaces. Along

5.2. How does proteinuria cause kidney damage?

Fig. 1. Schematic summary of the mechanisms of proteinuria and decreased GFR in CKD.

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the distal nephron, protein casts may obstruct urinary flow and aggravate tubulointerstitial damage [7]. b. Other pathways from the glomerulus to the interstitium: Although the response of proximal tubules to excessive and/or novel urinary proteins has been strongly involved in the development of proteinuria, interstitial inflammation and additional proinflammatory mediators may originate glomerular inflammation, either produced systemically or locally by inflammatory and resident glomerular cells. The glomerular-derived products may elicit additional tubular responses. As glomerular diseases progress to a chronic phase, regions of sclerosis of the glomerular tuft develop. Often, these sclerotic regions become adherent to and disrupt Bowman capsule. Such lesions provide an additional site for direct leakage of the glomerular ultrafiltrate into the peritubular interstitial space. In summary, high-grade proteinuria is an independent mediator of progressive kidney damage. Glomerular lesions and the effects on renal tubules appear to provide a critical link between proteinuria and tubulointerstitial injury, although other several mechanisms have also been implicated (Fig. 1). The authors report no conflicts of interest. References [1] K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2002;39: S1-266. [2] Ruggenenti P, Perna A, Mosconi L, Pisoni R, Remuzzi G. Urinary protein excretion rate is the best independent predictor of ESRF in nondiabetic proteinuric chronic nephropathies. “Gruppo Italiano di Studi Epidemiologici in Nefrologia” (GISEN). Kidney Int 1998;53:1209-16. [3] Keane WF, Eknoyan G. Proteinuria, albuminuria, risk, assessment, detection, elimination (PARADE): a position paper of the National Kidney Foundation. Am J Kidney Dis 1999;33:1004-10. [4] Brantsma AH, Bakker SJ, de ZD, de Jong PE, Gansevoort RT. Extended prognostic value of urinary albumin excretion for cardiovascular events. J Am Soc Nephrol 2008;19:1785-91. [5] Halbesma N, Kuiken DS, Brantsma AH, et al. Macroalbuminuria is a better risk marker than low estimated GFR to identify individuals at risk for accelerated GFR loss in population screening. J Am Soc Nephrol 2006;17:2582-90. [6] Brantsma AH, Bakker SJL, Hillege HL, de Zeeuw D, de Jong PE, Gansevoort RT, for the PREVEND Study Group. Cardiovascular and renal outcome in subjects with K/DOQI stage 1–3 chronic kidney disease: the importance of urinary albumin excretion. Nephrol Dial Transplant 2008;23:3851-8. [7] Eddy AA. Proteinuria and interstitial injury. Nephrol Dial Transplant 2004;19:277-81. [8] Rossing P, Hommel E, Smidt UM, Parving HH. Reduction of albuminuria predicts beneficial effect on diminishing the progression of human diabetic nephropathy during antihypertensive treatment. Diabeteologia 1994;37:511-6. [9] Apperloo AJ, De Zeew D, De Jong PE. Short term antiproteinuric response to antihypertensive treatment predicts long-term GFR decline in patients with non-diabetic renal disease. Kidney Int 1994(Suppl 45): S174-8.

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