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AbstractMicroalbuminuria represents a condition wherein the urinary albumin excretion is in the range of 30 mg/24 hrs -300 mg/24 hrs. Recently microalbuminuria is gaining attention as more than just a surrogate marker of kidney damage. Although apparently representing passage of a rather trace amount of protein excretion, this has great implication in terms of clinico-pathological association with diabetic nephropathy and other cardiovascular complications in subjects with or without diabetes. Early detection of microalbuminuria is considered to be the aim of clinicians in context to patient’s health perspective so as to change the treatment strategy accordingly. Therefore quite reasonably, understanding of exact patho-physiological significance of microalbuminuria and its prognostic impact in kidney diseases should be of immense help in providing better clinical outcome. To this end, an approach from the clinical biochemistry perspective might provide a better overview. The present commentary is an endeavor to address several relevant issues in this context for development of a better insight.

1Asst. Professor, 2Professor, Dept. of Biochemistry and JBTDRC, MGIMS, Sevagram, MaharashtraReceived: 18.12.2013; Revised: 03.04.2014; Re-revised: 12.01.2015; Accepted: 23.04.2015

Preamble

A c o n s i s t e n t p o s i t i v e relationship between urinary

albumin excretion and adverse clinical conditions of kidney like diabetic nephropathy, hypertensive nephropathy, end-s tage renal d i s e a s e s a n d c a r d i o va s c u l a r diseases in patients with or without diabetes is well-known. Normal reference range of excretion of urinary albumin is considered b e l o w 3 0 m g i n 2 4 h o u r s ; microalbuminuria signifies the minor quantity of albumin ranging between 30 mg/24 hrs - 300 mg in 24 hours to pass out of renal filtration barrier to appear in urine.1

BiochemistryAlbumin; the Versatile Molecule

Albumin, is a small globular non-glycosylated protein with a

molecular mass of around 66.3 kDa. Its relative abundance along with its high solubility rendered it to be an effective carrier in circulation. Suitable pK value (4.6) owing to its charged surface makes it easily dissociable at physiological pH; this factor coupled with its colloidal nature has been contemplated in its significant role for maintenance of plasma oncotic pressure.2 It also has significant nutritional value for the renal tubular cells3 and its high thiol content endows it with great antioxidant prowess. This versatility probably explains the parsimonious approach for conserving this molecule by kidney.Synthesis of Albumin and its Regulation

Daily 10-15 gm of albumin is

synthesized predominantly in hepatocytes accounting for 10% of the total protein synthesis . Synthesis of albumin is constantly regulated by changes in interstitial colloidal pressure.2 Besides that, nutri t ional status and various pathophysiological condit ions inc luding hormonal inf luence l ike thyrox in , cor t i cos tero ids and insulin also affect albumin synthesis.3 Synthetic capacity can be increased up to 2-2.7 fold of its normal level as per the requirement of individual.4 Lower synthetic rate in diabetic subjects might be recovered with insulin infusion.3 Conversely, as major negative acute phase reactant there is plummet in its level during pro-inflammatory s ta te . 2 Albumin escapes f rom plasma at rate 4.5% / hr and from vascular space through lymphatics at same rate by its absorption. Interestingly, albumin synthesis is very much linked and governed by its fractional catabolic rate, may be the most significant fact during dietary protein restricted state or at the deficient synthetic state.2,4 The catabol ic fate of a lbumin is determined mostly through utilization by vascular endothelium (almost 90%) and the rest is by renal tubular epithelial cells.5 Therefore, th i s in t r i ca te and apparent ly paradoxical relationship possibly suggests a subtle balanced nexus between catabolic and anabolic processes.

Microalbuminuria: A Mere Marker or An Ominous Sign?Pranita Waghmare1, Kalyan Goswami2

R e v i e w a R t i c l e

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Renal Membrane and Filtration of Albumin

G l o m e r u l a r f i l t r a t i o n i s considered to result in formation of an essentially albumin-free filtrate due to remarkable sieving through a serially arranged fenestrated endothelium with its glycocalyx, the glomerular basement membrane and podocyte s l i t d iaphragm that almost completely prevents large and negat ive ly charged macromolecules from reaching Bowman’s capsule.6 Albeit certain reports suggested post-filtration albumin retrieval through tubular reabsorption,7 however both logical analysis as well as supportive e v i d e n c e s r e f u t e s u b s t a n t i a l impact of the later process as a determinant of urinary albumin excretion. Classically, the proposed mechanism for rigorous restriction of albumin passage is based on the exclusion of albumin from the glomerular capillary wall through size selectivity and probably more owing to charge repulsion; which would be further clarified in the context of the clinical correlates described later. As opposed to es t imated g lomerular s iev ing coefficient (GSC) of 0.02 to 0.1 by inert polysaccharide probes with a similar hydrodynamic radius like albumin (36 Å), the GSC for albumin determined by micro-puncture was found to be markedly low at the level of 0.0006 to allow free passage. However, renal filter matrix trickle albumin only in trace amount . The hypothesis put forward to account for this apparent paradox is the charge selectivity, whereby negatively charged albumin is repelled by a theoretical, fixed, negative charge of the glomerular capillary wall at physiological pH.8 Endothelial cells express negatively charged glycoprotein capsule containing sialic acid residues.9 The basement membrane, which is a porous matrix of negatively charged proteins including type-4 collagen, laminin, and proteoglycans (agrin and perlecan) and fibronectin further

forms an important f i l t ra t ion barrier to plasma proteins.6,10

B e s i d e s f i l t r a t i o n b a r r i e r , another plausible checkpoint of albumin release in urine is through a catabolic mechanism for albumin degradation through megalin-cubul in complex 11 to conserve the amino acids rather than a ful l - f ledged retr ieval process . The process is mostly completed by proximal convoluted tubule followed by its degradation in the luminal cells. These forms basis of the debate between two hypotheses: a l b u m i n u p t a k e d e f i c i e n c y versus filtration barrier defect. If considered to be significant, albumin retrieval premise which have been revived due to the observed higher GSC for albumin in animal experiments essentially need to be substantiated with commensurate albumin uptake along with i ts del ivery to the circulation. However because of its size, albumin cannot leave the tubular lumen on the paracellular route across the tight junctions. F u r t h e r m o r e , f o l l o w i n g t h i s hypothesis, albumin is not cleaved in the tubular lumen and therefore does not cross the apical membrane of the proximal tubular cell in the form of free amino acids.12

Thus the possible mechanism is shown to mediate a lbumin reabsorption through endocytosis, f o l l o w e d b y t h e p r o p o s e d mechanism of transcytosis for its passage to circulation.9,13 However c e r t a i n t e c h n i c a l l i m i t a t i o n s coupled with the lack of sufficient evidences from human experiments keep this question to be resolved yet.

In pathological conditions e.g. massive proteinuria in nephrotic syndrome, probably results from marked reduction in the charge-selective properties of the filtration barrier and also alteration in the pore size in the slit diaphragms formed by inter-digitating foot processes of adjacent podocytes (epithelial cells) . 6 However, in context of glomerulonephri t is

cases , a higher probabi l i ty of loss of charge factor seemed to be operative with lesser extent of the pathology, whereas relative higher edge of alteration of pore size was contemplated in more severe condition.10

Clinical ChemistryRationale

Analysis of healthy individuals’ urine shows that only little albumin ( u p t o 3 0 m g / d a y ) u n d e r g o excretion.1 Although kidney is a major portal of excretion, the passage of any substance in urine may not necessarily indicates renal damage but actually display a mere reflection of certain systemic disorders like metabolic diseases, with or without renal pathology. Therefore, albumin in urine is considered not just as a surrogate marker of renal damage but also as a reflection of various systemic pa thophys io logy . 1 Given the great physiological significance of albumin, teleologically special s t ra tegy has been devised to prevent its loss; thus traces of albumin in urine may actually be considered as a mark of deviation from physiological homeostasis.

In healthy subjects, filtration and reabsorption of albumin are in equilibrium in which <1% of filtered albumin reaches finally in the urine. Pathophysiological states with increased renal albumin excretion, the so-called albuminuria or more generalized proteinuria therefore may be explained by any of the three basic mechanisms currently available: 1) glomerular injury resulting in excess filtration of protein e.g nephrotic syndrome; 2 ) tubular in jury resul t ing in excessive production and excretion of tubular proteins (e.g., Tamm-Horsfall protein); and 3) over-filtration of plasma proteins due to high plasma concentration, e.g., in multiple myeloma. Diagnostic Dilemma-albuminuria versus Microalbuminuria

E a r l y d e t e c t i o n o f

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m i c r o a l b u m i n u r i a a s a r i s k p r e d i c t i o n m a r k e r d e m a n d s high sensitivity test . The term “proteinuria” refers to increased urinary excretion of any protein w i t h o u t a n y s p e c i f i c t y p e ; “albuminuria” refers specifically to increased urinary excretion of albumin only. “Microalbuminuria” speci f ica l ly refers to a lbumin excretion above the normal range but below the level of detection by the conventional tests for total protein. In fact since such routine tests fail to detect very early stage proteinuria (albuminuria, to be precise) therefore this so called term ‘microalbuminuria’ came into existence, which might be confusing to represent incorrectly the excretion of certain minor version of albumin in terms of size and not the factual lower rate of excretion.

Popularly known dry chemistry based dipstick methods although prove to be quite sensitive test however lack des irable lower detection threshold.14 For regular s c r e e n i n g o f a l b u m i n u r i a i n random sample, certain improvised rapid format semi-quantitative dip-stick method with advantage of reasonably good sensitivity and specificity may be considered. On the other hand, lack of ability for exact quantification, variation wi th d i lu ted or concentra ted sample coupled with the need of confirmation of specific amount of albumin for proper assessment of damage and consequent prognostic impact , l imits i ts use. 15 Hence owing to t echnica l d i f f i cu l ty o f c o n v e n t i o n a l e s t i m a t i o n approach coupled with its clinical s ign i f i cance , i t i s impera t ive to cons ider h igher resolut ion immune-chemica l techniques . Recent addition of HPLC method in the diagnostic repertoire may provide higher sensitivity.14

Guidelines for detection and m o n i t o r i n g o f p r o t e i n u r i a i n adults and children also differ because o f d i f ferences in the prevalence and type of chronic

k idney disease . 16 Large intra-individual and diurnal variation along with lack of available data about any contributing biological factors may also account for lack of precision in available lab results. Furthermore, there is inconsistency between laboratories regarding sample type, units of reporting, and reference intervals or cut points. Recent guideline16 should bring in consensus to address these issues for setting the precise recommendations to define urine sampling technique, detect ion method for albuminuria, and a specific upper and lower limit for microalbuminuria suitable for diabetes, hypertension, and cardiovascular disorders.

Therefore off late, approximate estimation of excretion of urine albumin in mg/ gm of creatinine i s r o u t i n e l y f o l l o w e d . 1 7 F o r measurement of the ratio between albumin to creatinine, test can be done with spot collection of urine sample al lowing simultaneous assessment of albumin against creatinine for a projected effect of renal clearance; therefore, it gives an indication of total albumin excretion in 24 hours being unaffected by the variation in concentration. The cut-off urine albumin to creatinine ratio for microalbuminuria are: ≥3.5 mg/mmol of creatinine and ≥ 2 . 5 m g / m m o l c r e a t i n i n e i n females and males respectively.17 A s p e r T h e N a t i o n a l K i d n e y Foundation guidelines screening for microalbuminuria is needed for in all patients with diabetes, hypertension, family history of chronic kidney disease with age above 60 years, and for certain racial and ethnic minorities.16

Clinico-pathological Correlate Diabetic Nephropathy and Cardio-metabolic Consequences

Diabetes mellitus appears the most common cause of end-stage renal diseases. Approximately 50% of diabetes type-1 individuals

wi th in 10 years and in >75% by 20 years develop in diabetic nephropathy and 20- 40% of type-2 diabetes patients eventually may develop in to end-stage renal d i s e a s e s . 1 5 G l y c a t i ve c h a n g e a s s o c i a t e d l o s s o f c h a r g e o f filtration membrane and binding of advanced glycated end product ( A G E ) o n p o d o c y t e r e c e p t o r which are selectively expressed in g lomerular epi the l ia l ce l l s may lead to increased glomerular p e r m e a b i l i t y w i t h r e s u l t a n t microalbuminuria in Diabetes m e l l i t u s . 1 8 M i c r o a l b u m i n u r i a is general ly f irst c l inical s ign o f s u c h r e n a l d y s f u n c t i o n i n diabetes.9 Development of diabetic nephropathy is characterized by progressive increase in protein excretion, particularly albumin, with early rise in systemic blood p r e s s u r e a n d l a t e d e c l i n e i n glomerular filtration. Passage of protein molecules through kidney may prove detr imental to the ultra-structure of the renal tissue giving rise to perpetual damage w h i c h c u m u l a t e s t o d e ve l o p into frank diabetic nephropathy and ult imately may culminate towards total renal failure.13 Hence, American Diabetes Association (ADA) recommends screening for microalbuminuria annually for patients with type 1 diabetes of more than 5 years duration and for patients with type 2 diabetes from the first time of diagnosis as a prognost i c indica tor for development of CVD.15 Screening for diabetic nephropathy involves monitoring for at least yearly for urinary albumin excret ion > 3 0 m g p e r d a y . 1 5 H o we ve r , confounding factors should be cons idered in case o f such a marker l ike microalbuminuria due to the obvious reason of its association with a wide spectrum of pathology. For example factors like hyperinsulinemia and associated insulin resistance, polymorphism of the D allele of ACE gene might be associated with higher prevalence of microalbuminuria. One should

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also consider the baseline excretion of urinary albumin of a subject. In subjects with hypertension, antihypertensive drugs also can change the course of developing microalbuminuria.19 Besides these, any suspected aetiology of vascular pathology inc luding smoking habit20 might have possible impact on such process. So they need to be considered before diagnosis of the diabetic nephropathy using this marker.

Both glycemic control (HbA1c <7%) and blood pressure control (<130/80 mm of Hg) especially after the onset of renal damage is recommended as per guidelines of ADA,15 presumably because of hypertension induced glomerular damage as reported earlier from both animal as well as in human studies.21

Vasculature and renal glomerulus have a lot in common structurally and functionally.22 According to Steno hypothesis, albuminuria might reflect a general vascular dysfunction and associated leakage of other plasma molecules such as low density lipoproteins into the vessel wall that may lead to inflammatory response which in turn perpetuate atherosclerotic pathology.22 Consequently the ADA recommendation for LDL level to be maintained below 100 mg/dl for the patients with renal disease is a justified proposition to prevent any such vascular condition to be superimposed.15 Many studies concluded that microalbuminuria can be an independent risk factor for all cause and cardiovascular mortality and coronary vascular events within groups of patients with diabetes or hypertension and also in general population.23

The hallmark of cardiovascular diseases in diabetes is accelerated atherosclerosis of medium and large-s ized ar ter ies might be contributed by multiple factors like binding of advanced glycated end products on receptors of endothelial cells of glomerulus and generation of various growth

factors and cytokines favoring pro-inflammatory milieu to accentuate such process.24 Moreover, diabetes a s s o c i a t e d c h a n g e s i n b l o o d viscosity might lead to hypertensive features.24 Reportedly, a positive co-relation between high blood pressure and microalbuminuria exists; hypertension contributes to renal dysfunction by increasing glomerular vascular pressure and capillary damage along with possible tubular defect.21 The mesangial cell proliferation and related matrix modi f i ca t ion in d iabe tes and hypertension associated induction of angiotensin-2 is also responsible for the loss of renoprotect ive effect . 25 Addit ionally, diabetic n e p h r o p a t h y a s s o c i a t e d h i g h level of glucose in the f i l trate gets reabsorbed along with excess sodium in the renal tubule that can enhance local angiotensin26

with a feasible indirect effect on atrial natriuretic peptide release. It is well established that there i s s t rong corre lat ion between degree of hypertension and the rate of progression of overt diabetic nephropathy in type-1 as well as type-2 diabetes mellitus.27 In corroboration with this view, the successful use of ACE inhibitor, which decrease such pressure are actually proved to be beneficial. An earlier study28 demonstrated that use of ACE inhibitors delay progression of less severe selective albuminuria to severe non-selective proteinuria; further combination of ACE inhibitors with angiotensin receptor antagonist have intensified blood pressure control to the kidney pathology.29 These proofs are enough for establishing the key pathogenic role of vascular effect on kidney damage which is mirrored by microalbuminuria as a tel l - tale evidence of such pathology. Overall this pathology may be translated into accentuation o f r e n a l d a m a g e l e a d i n g t o deteriorating renal function. Hence alteration in albumin excretion should be considered not merely as a surrogate marker but as

an important risk predictor for progress ing card io -metabol i c disorders leading cardiovascular events. Therefore, quite obviously, m i c r o a l b u m i n u r i a i s g a i n i n g attention as a predictive as well as prognostic marker of atherogenesis. Moreover the excretion of this protein have noteworthy damaging potential30 which opens up a new perspective and makes us stand before an intr iguing di lemma of cause and effect relationship between vascular pathology and such protein excretion.

Therefore a l l pat ients wi th microalbuminuria (except which are self-limiting) should be screened for macro-vascular diseases and if detected, aggressive interventions should be taken to reduce cardio-metabolic complications.

The Overall Impact and Future Prospect

Wi t h t h e a b o ve m e n t i o n e d perspective, prognostic importance of microalbuminuria for predicting m i c r o va s c u l a r c o m p l i c a t i o n s and also its progression towards i m m i n e n t m a c r o v a s c u l a r pathologic stage leading to serious fallout of diabetic nephropathy and cardiovascular diseases21,25,28 might be well appreciated. However, due to impact of biological and analytical variations, microalbuminuria as a prognostic parameter although quite significant but may not be suff icient alone to predict the progress of renal damage.31 On the other hand, based on the evidence i t might not be unjust i f ied to extrapolate its importance as a risk predictor in several other diseases with common association of vascular pathology. Excretion of albumin in ‘microalbuminuric’ range is definitely an ominous sign which should be checked for with great degree of suspicion through standardized technique having reasonably good resolution and also need to interpret the result with just i f ied cl inical acumen to ensure timely intervention to

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regress the pathology. It might be quite tempting to speculate its certain direct pathognomonic role beyond i ts present status as a mere r isk predictor . This would definitely uplift its status as a versatile marker of vascular pathology.

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