Applying the Logic Chain to Green Infrastructure in Integrated Schemes and City-wide Development

Tubular proteinuria, or low molecular weight (LMW) proteinuria, is less common than glomerular proteinuria, but is often of clinical significance ^{28, 81.} The kidney function can be assessed by a number of methods. Most patients with tubular dysfunction excrete large amounts of β2-microglobulin, although they excrete normal or only slightly increased amounts of albumin and only moderately increased quantities of total protein²¹. In healthy individuals with normal proximal tubular function <0.4 mg/L β2-microglobulin is excreted in urine¹.

Quantitative determinations of urinary β2-microglobulin and urinary albumin are useful for detecting disorders of the renal handling of proteins²¹. The role of the proximal tubule in the reclamation and conservation of the constituent amino acids of albumin is of fundamental

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importance in the assessment of proteinuria⁸². According to one estimate, based on the concentration of albumin in the glomerular filtrate the daily filtered load in an adult with a glomerular filtration rate of 180 L/day approaches 5.7 g, of which only 30 mg of intact albumin is excreted. Thus, in excess of 99.95% of filtered albumin is reabsorbed, transported and/ or degraded in the proximal tubule¹⁹.

The proximal tubular reabsorption of filtered albumin is accomplished by receptor-mediated endocytosis. A packet of filtrate is engulfed by an invagination of apical membrane at the base of the microvilli, forming an endocytic vesicle. The efficiency of this process is enhanced by the presence of a high-affinity receptor for albumin³. A second pathway channels albumin to the sorting endosomal compartment and is eventually delivered to the lysosomal compartment for degradation to peptides and amino acids. The latter are transported across the basolateral membrane of the proximal tubular cell¹⁶. Some filtration of low molecular weight proteins (eg, β 2 microglobulin, retinol-binding protein, small peptides) occurs.

The proximal tubular cells reabsorb most of these filtered proteins. However, modest, low-grade proteinuria may occur in certain congenital metabolic tubulopathies, such as Fanconi syndrome or X-linked recessive nephrolithiasis with kidney failure that results from a mutation in the CLCN5 gene on chromosome 11 (Dent’s disease)^83. Most chronic nephropathies that appear to be primary glomerular disorders are invariably accompanied by tubulointerstitial disease, which may have a more ominous impact on progression than the glomerular lesion. The pathogenesis of tubulointerstitial mononuclear cell infiltration, inflammation, tubular atrophy, and fibrosis has not been clear^83-85. However, a concept has emerged that the high rate of albumin trafficking in the proximal tubule during the course of proteinuric kidney diseases results in protein overload and injury to the tubular cells. The mechanism of such injury is not

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clear, but generation of reactive oxygen species and consequent oxidative injury has been proposed as one explanation. Other explanations involve the role tubular ischemia and acute cytokine activation play in the progression of tubular injury. Proteinuria enhances the tubular synthesis and secretion of endothelin – 1, a strong chemoattractant for monocytes and macrophages which worsen tubular injury. Filtration of complement has also been implicated in the pathogenesis of secondary tubulointerstitial injury^{84, 85}.

Proteinuria can arise from tubular cell injury by 2 mechanisms: release of tubular epithelial cell proteins into the urine and failure to reabsorb filtered low-molecular-weight proteins^{86, 87}. Release of a wide variety of brush-border enzymes from the proximal tubules into the urine has been studied in various disease states; these enzymes include neutral endopeptidase, dipeptidyl aminopeptidase IV, α-glucosidase, trehalase, leucine aminopeptidase, alkaline phosphatase, γ-glutamyltransferase and alanine aminopeptidase. N-Acetyl-β-D-glucosaminidase, a hydrolytic enzyme present in the lysosomes of proximal tubular epithelial cells, has also been investigated ^{22, 81}. The excretion of all of these proteins is increased by various renal insults, including ischemia, acute interstitial nephritis and the presence of materials such as cisplatin, contrast media, heavy metals and aminoglycoside antibiotics.

In addition, the excretion of tubular proteins may predict the outcome of proteinuric glomerular disease⁸⁸. A number of filtered proteins, including β2-microglobulin, retinol-binding protein and α1-microglobulin, have been used in the diagnosis and follow-up of tubulointerstitial diseases. Neutrophil gelatinase-associated lipocalin and cystatin C have been identified as markers of ischemia-associated acute tubular injury⁷⁸. However some believe that in human diseases, no single urinary component or characteristic of proteinuria has been identified as specific for tubulointerstitial lesions⁷⁸.

37 2.10.1 β2 microglobulin

Many studies showed that tubulointerstitial changes determined the progression of glomerular disease so tubular injury markers were searched for²⁸. Although over 50 enzymes were detected in human urine, only a few have been used for diagnosis in renal disease^{21, 89}. Measurement of urinary excretion of low molecular weight protein is a valuable supplement in estimation of tubulointerstitial system malfunction⁸³. These proteins are readily filtered by normal glomeruli and virtually completely reabsorbed by normal proximal tubules. These proteins are alpha-1-microglobulin, retinol-binding protein and β2microglobulin⁸⁹.

β2microglobulin consists of 99 amino acids with one disulfide bridge and has a molecular weight of 11, 731 Dalton. It is non- covalently bound to the class I major histocompatibility antigen and found on the cell surface of all nucleated cells. Production of β2-microglobulin is known to be between 150 to 250 mg/day in healthy individuals, whereas an increase is observed in some lymphoproliferative and autoimmune diseases^{90, 91}. β2-microglobulin is shed from the cell surface and circulates in serum, 98% as a free form. Most free β2-microglobulin is filtered by the glomeruli and almost all reabsorbed by proximal tubular cells where it is thought to be degraded into peptides/amino acids by lysosomes before reuptake into the circulation²¹. Therefore, in healthy individuals with normal proximal tubular function <0.4 mg/L β2-microglobulin is excreted in urine⁹².

Due to these properties, increased intact urinary β2-microblobulin has been considered an ideal biomarker for proximal tubular dysfunction. However, it was soon realized that intact β2-microglobulin values measured by immunoassays decreased significantly over time in urine with a pH<6, suggesting proteolytic activity, which leads to cleaved β2-microglobulin forms that were not detectable by available immunoassays⁹³. Therefore, the only way to accurately measure

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intact urinary β2-microglobulin is to give patients alkali e.g. sodium bicarbonate systemically to ensure a urine pH≥6 or to analyse only urine samples with pH≥6. By following these steps, the potential of intact urinary β2-microglobulin as a marker for proximal tubular injury has been demonstrated⁹².

The low-molecular-weight proteins are at present the best markers for early detection of tubular dysfunction; other constituents are not as well suited for this, even if the determination of urine enzymes has its proponents⁸³.

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CHAPTER THREE

SUBJECTS, MATERIALS AND METHODS