By Kalansooriya, A Jennings, P; Haddad, F; Holbrook, I; Whiting, P H
ABSTRACT The association between urine microalbumin, alpha^sub 1^- rtnicroglobulin concentration (alpha1MG) and the urinary enzyme activities of alanine aminopeptidase (AAP), N-acetyl-beta-D- glucosaminidase (NAG), alpha-glutathione-S-transferase (alphaGST) and pi-glutathione-S-transferase (piGST) is investigated in 36 type 2 diabetic and 15 age- and sex-matched non-diabetic subjects. Diabetic subjects were grouped into those with microalbuminuria
KEY WORDS: Diabetic nephropathies.
Enzymuria.
Glutathione transferase.
Introduction
Diabetic nephropathy is characterised structurally both by glomerular lesions and changes to the tubulo-interstitial compartment of the kidney, and functionally by increasing severity of microalbuminuria and altered glomerular filtration rate (GFR), the latter usually being assessed in the laboratory by measurements of serum or plasma creatinine concentrations.1-3 Furthermore, end- stage renal disease (ESRD) in diabetics is increasing and now accounts for approximately 40% of treated ESRD by either transplantation or dialysis.4 Although incipient diabetic kidney disease is usually characterised functionally by the presence of microalbuminuria, serum creatinine is of limited value in the early detection of renal insult due to its poor sensitivity to early nephron insult and renal dysfunction.5
It is now well established that chronic hyperglycaemia, and consequently effective glycaemic control, is the main metabolic determinant associated with irreversible kidney damage in diabetes mellitus.6 Although microalbuminuria has been used for many years as a predictor of incipient diabetic nephropathy, reflecting the loss of glomerular selectivity,2 estimation of renal tubular function and integrity may yet provide an early indication of renal insult and thus identify those at risk of developing kidney dysfunction.7 Consequently, decreased renal tubular reabsorption capacity is characterised by elevated low molecular weight protein (e.g. alpha^sub 1^-microglobulin and beta2-microglobulin) levels in the urine, and early renal tubular insult is characterised by increased proximal tubular enzymuria.7-9
In this regard, elevated urine alanine aminopeptidase (AAP) and N- acetyl-beta-D-glucosaminidase (NAG) activities have been used to indicate the early onset of proximal renal tubular insult in several clinical states in general (e.g., drug-induced nephrotoxicity),10 and the diabetic state in particular.11-18 Other studies indicate that the isoforms of glutathione S-transferase (GST), alphaGST and piGST, are located in the cells of the proximal and distal renal tubules, respectively, and their urine measurement has allowed the identification of site-specific renal insult following nephrotoxin- induced renal damage.19-21
However, renal dysfunction in general and glomerular dysfunction in particular are observed only rarely in early diabetic nephropathy, and the identification of early renal insult and those at risk of developing renal dysfunction is important so that corrective therapies can be applied at an early stage. Consequently, the aim of this study is to investigate the association between the urinary enzyme activities of NAG, AAR alphaGST and piGST in subjects with type 2 diabetes and the severity of microalbuminuria, the standard laboratory indicator of the onset of nephropathy.
Materials and methods
Thirty-six type 2 diabetic subjects and 15 age- and sex-matched non-diabetic control subjects were recruited. Mean age (standard deviation [SD]) was 48 (10) years and duration of diabetes was between eight and 10 years. Diabetic subjects with renal disease characterised by proteinuria, hypertension (with or without antihypertensive treatments), dyslipidaemia or cardiovascular disease, evidence of renal insufficiency, chronic urinary tract infection, renal stones and renal humours were excluded from the study. Diabetic subjects were grouped according to urine protein concentration into those with microalbuminuria
Fasting blood and urine samples (mid-stream) were taken and plasma creatinine and microalbuminuria concentrations were quantitated by standard laboratory methods, while urine activities of AAP and NAG and urine alpha^sub 1^-microglobulin (alpha1MG) levels were measured, as previously described,11,22,23 and alphaGST and piGST urine activities were measured using a commercial enzyme immunoassay (EIA) kit (Biotrim, Dublin, Ireland). Enzyme activities were expressed on the basis of urinary creatinine to allow for inter- individual variations in urine flow rate. Results, expressed as mean (SD), were analysed using two-way analysis of variance (ANOVA) with post hoc analysis performed using Dunnett’s test. P values
Results
All the diabetic subjects had serum creatinine concentrations within the laboratory reference range (
Urine activities of NAG, a lysosomal hydrolase found predominantly in the renal proximal tubule, were significantly increased (three-fold) in diabetic subjects with microalbuminuria 3mg/L. Similar and significant four-fold elevations in AAP activity were noted in groups B and C, compared to the control values and those obtained from diabetic subjects with microalbuminuria
A 1.3-, two- and three-fold increase in urinary alphaGST activity was observed in groups A, B and C, respectively (P
The proximal tubular enzyme NAG and the small molecular weight protein alpha1MG demonstrated similar patterns of urinary excretion with increasing severity of microalbuminuria, albeit with serum creatinine concentrations within the reference range, but showed elevated values in the absence of significant microalbuminuria. alphaGST and the distal tubular marker piGST were elevated only when significant proteinuria was present in a manner similar to that seen with AAP excretion. Discussion
Incipient diabetic nephropathy is characterised by structural, functional and biochemical changes associated with glomerular and tubular elements of the kidney.3 The results of the present study demonstrated clearly that increased severity of microalbuminuria, the standard clinical predictor of incipient diabetic nephropathy,3 and increased glomerular permeability, are associated with early changes in urinary parameters (i.e. alpha1MG proteinuria, NAG, AAP, alphaGST and piGST enzymuria), indicating renal tubular dysfunction in a cohort of type 2 diabetic subjects. Furthermore, urine NAG activity and alpha1MG concentration were elevated in those diabetic subjects with microalbumuria
In diabetic kidney disease, increased NAG and AAP enzymuria has been observed.7,8,13 Both enzymes are localised predominantly to the proximal renal tubule, with NAG being of lysosomal origin and AAP being of mitochondrial origin in the renal brush border. Both have been shown to be sensitive indicators of renal proximal tubular insult in several disease processes,7 and in early diabetic nephropathy in particular.7-15 In the present study, while increased AAP activity was only observed in individuals with significantly elevated microalbuminuria, elevated NAG enzymuria was seen in the absence of significant microalbuminuria, but with serum creatinine concentrations still within the laboratory reference range. These findings are consistent with altered lysosomal function due to an autophagic response to early epithelial cell injury initiated by chronic hyperglycaemia.
Indeed, while previous studies have suggested that increased NAG enzymuria is a consequence of chronic or fluctuating hyperglycaemia, and not necessarily a reflection of diabetic renal insult,24,25 it is now accepted that hyperglycaemia resulting from poor long-term glycaemic control is a major underlying cause of diabetic complications, fuelling interrelated pathophysiological processes such as increased polyol pathway activity, protein glycation, advanced glycation end-products and free radical effects.26-28
However, while elevated GST activities have been noted in the diabetic state,19,29,30 the present study also demonstrated elevated activity of the cytosolic isoenzymes of GST. These different patterns of GST isoenzyme excretion are especially interesting because alphaGST is only found in the proximal renal tubule, whereas the intrarenal localisation of piGST is to the distal tubule.19-21 Consequently, the presence of elevated piGST suggests the presence of distal tubule insult in diabetics with proteinuria, and suggests that while proximal tubular and glomerular insult may be initial events in the natural history of diabetic nephropathy, as demonstrated by increased alpha1MG excretion, elevated NAG, AAP and alphaGST urine activities and microalbuminuria, this is followed by distal tubular insult indicated by increased piGST activity.
In conclusion, the present study demonstrates clearly that proximal tubular insult, characterised by increased NAG, AAP and alphaGST enzymuria, is present in type 2 diabetics and that generally it is associated with increasing proteinuria, as indicated in other studies.19 However, NAG activity was elevated in patients without significant proteinuria or increased serum creatinine concentration, which is consistent with its role as an early indicator of diabetic renal insult. Furthermore, the results suggest that renal tubular insult may precede increased glomerular permeability in diabetic renal disease. Consequently, as these urinary biomarkers assess different areas of the renal architecture, estimation of renal tubular function and integrity may provide a site-specific and early indication of renal insult in subjects with diabetes.
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A. KALANSOORIYA*, P. JENNINGS[dagger], F. HADDAD[dagger], I. HOLBROOK[dagger] and P. H. WHITING*
* School of Allied Health Sciences, De Montfort University, Leicester LE1 9BH, UK
and [dagger] Department of General Mediane, York District General Hospital, York, UK
Accepted: 20 September 2007
Correspondence to: Professor P. H. Whiting
School of Allied Health Sciences, De Montfort University
Hawthorn Building, The Gateway, Leicester LE1 9BH, UK
Copyright Step Communications Ltd. 2007
(c) 2007 British Journal of Biomedical Science. Provided by ProQuest Information and Learning. All rights Reserved.
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