Urinary gamma-glutamyl transferase-to-creatinine ratio as an indicator of tubular function in Bence Jones Proteinuria

Abstract

Increased urinary gamma-glutamyl transferase (GGT) activity suggests early renal tubular damage. The aim of this study was to evaluate the urinary GGT activity as a marker of renal injury in different types of Bence Jones Proteinuria (BJP). One hundred and three individuals with BJP were included in the study. Urinary GGT activity, urinary GGT-to-creatinine ratio and urinary protein-to-creatinine ratio were studied. Urine samples were tested by immunofixation agarose gel electrophoresis. Total urinary excretion of kappa and lambda light chains were measured by nephelometric method. There were no significant differences in demographic characteristics of the patients in Lambda BJP, Kappa BJP and Control groups. GGT-to-creatinine ratio of the Lambda BJP group was significantly higher than Kappa BJP group and controls (p = 0.018 and 0.002, respectively). There was no correlation between the quantitative kappa and lambda BJP and urinary GGT-to-creatinine ratio. Our data have demonstrated that urinary GGT-to-creatinine ratio could be a tubular damage marker of lambda light chain proteinuria.

• Acute renal failure
• Bence Jones Proteinuria
• gamma glutamyl transferase
• lambda light chain
• urinary GGT-to-creatinine ratio

Introduction

Light chain amyloidosis, light chain deposition disease and multiple myeloma are immunoproliferative diseases characterized by Bence Jones Protein (BJP) excretion produced as a result of excessive proliferation of monoclonal plasma cells or the accumulation of the immunoglobulin light chains.1

Acute kidney injury remains an important cause of morbidity and mortality in patients with multiple myeloma.2 Severe cases are frequently a direct consequence of the high clonal production rates of immunoglobulin free light chains.2 The resulting high serum concentrations of these proteins often lead to tubular interstitial injuries as endocystic receptors in the proximal tubules are overwhelmed.3 Direct proximal tubule epithelial cell cytotoxicity, tubulointerstitial nephritis and cast nephropathy can occur in isolation or together.3

Several experimental studies have shown that gamma-glutamyl transferase (GGT) is a membranous enzyme that has a critical role in glutathione homeostasis by initiating extracellular glutathione breakdown and it acts as a critical cellular antioxidant defense.4 This enzyme is primarily located on the luminal surface of cellular membranes of proximal tubule epithelial cells, thus its highest activity can be found in kidneys.5 Increased levels of the enzyme in urine can be due to renal damage and it may be possible to determine the primary site of renal damage.6

In this study we aimed to evaluate the relationship between urinary GGT activity – as a marker of renal injury – and quantitative light chain excretion in patients with Bence Jones Proteinuria (BJP).

Subjects and methods

One hundred and three non-diabetic proteinuric individuals were included in the study. About 24-h urine samples were collected before renin–angiotensin system blockage treatment for the assessment of monoclonal light chain excretion as well as total protein, creatinine and GGT excretion. Serum renal functions, albumin, leukocyte and glucose levels were assessed concurrently. All study subjects were non-smokers and did not consume alcohol. None of the subjects received antibiotics, corticosteroids, anti-inflammatory and antihyperlipidemic drugs. Individuals who proved to be in a good state of health except proteinuria and free from any signs of chronic disease after clinical examination were included in the study as the control group. The study was approved by the local ethics committee and all participants gave written informed consent. Based on urinary immunofixation electrophoresis (IFE) results, the cases were divided into three groups: 42 patients without monoclonal light chains but with urinary protein excretion composed as control group, 33 cases with kappa BJP comprised as Kappa BJP group and 28 patients with lambda BJP composed as Lambda BJP group. Urinary light chain measurements were performed by IFE agarose gel electrophoresis technique. Urinary GGT and creatinine levels were measured by colorimetric methods and urinary total protein levels were measured by turbidimetric method on a Cobas Integra 800 analyser. Total excretions of kappa and lambda light chains in the urine were measured by nephelometric method on Siemens BN ProSpec analyzer. Urinary GGT-to-creatinine ratio was calculated as 24-h urine GGT level/24-h urine creatinine level (IU/mg Cre). Urinary protein-to-creatinine ratio was calculated as 24-h urine protein level/24-h urine creatinine level (mg/mg Cre).

Descriptive statistics for continuous variables were expressed as mean ± SD. The Kolmogorov–Smirnov test was used to evaluate whether variables were distributed normally. Kruskal–Wallis test was used for parameters showing non-normal distribution. Chi-squared test was used for inter-group comparisons of qualitative data. Spearman’s correlation test was performed to explore the relationship between urinary GGT-to-creatinine ratio and serum urea, creatinine and 24-h urine kappa and lambda light chain proteinuria. A p value <0.05 was interpreted as statistically significant. All statistical analyses were performed with statistical analysis program (SPSS 20 for Windows).

Results

There were no significant differences in age, body mass index, gender, blood pressure, serum glucose and leukocyte parameters among groups. However, lambda BJP group had higher serum urea and creatinine levels than controls (p = 0.046 and p = 0.034, respectively). Also they had lower serum albumin levels than patients in the control group (p < 0.01) (Table 1).

Table 1. Clinical and demographic characteristics of study groups.

	Control group (n = 42)	Kappa BJP group (n = 33)	Lambda BJP group (n = 28)	p Value
Age (years)	63.8 ± 12.7	67.5 ± 10.2	64.6 ± 10.1	NS
Sex (male/female)	18/24	15/18	18/10	NS
BMI (kg/m^2)	26.24 ± 4.14	25.08 ± 3.26	26.18 ± 6.14	NS
MAP (mmHg)	92.40 ± 15.00	88.52 ± 12.20	90.45 ± 14.60	NS
Glucose (mg/dL)	90.40 ± 17.44	86.59 ± 13.28	89.55 ± 18.06	NS
WBC (10^3/μL)	7.22 ± 1.34	8.16 ± 1.65	8.76 ± 2.30	NS
Urea (mg/dL)	22.08 ± 8.04	46.38 ± 24.08	45.23 ± 18.44	p <0.01^α
Creatinine (mg/dL)	0.77 ± 0.40	1.48 ± 0.67	1.36 ± 0.98	p < 0.01^α
Albumin (g/dL)	4.33 ± 1.21	2.95 ± 1.88	2.46 ± 1.09	p < 0.01^α

Notes: Data is presented as mean ± SD. BMI, body mass index; MAP, mean arterial pressure; WBC, white blood cell; Kappa BJP, Kappa Bence Jones Proteinuria; Lambda BJP, Lambda Bence Jones Proteinuria; NS, non-significant; α, Lambda BJP group versus control group.

The mean 24-h urine levels of GGT are shown in Table 2. The lambda BJP group had significantly higher GGT levels in the urine as compared to the kappa BJP and control groups (p = 0.003). Similarly, the lambda BJP group had significantly higher GGT-to-creatinine ratio in the urine as compared to the kappa BJP and control groups (p = 0.004). When the two groups (lambda and kappa BJP) were pooled for analysis, serum urea (r = 0.417, p < 0.01) and creatinine (r = 0.378, p < 0.01) levels were positively correlated with 24-h urinary GGT-to-creatinine ratio. However when the two groups were separately analyzed, there was a weak positive correlation between 24-h urine levels of GGT-to-creatinine and serum urea (r = 0.272, p = 0.106) and creatinine (r = 0.246, p = 0.072) in the lambda BJP group. There were no significant correlations between 24-h urinary GGT-to-creatinine ratio and renal functions in the kappa BJP group and in control group. There were no correlations between the quantitative kappa and lambda BJP and mean 24-h urinary GGT-to-creatinine ratio (p = 0.752 and p = 0.527, respectively).

Table 2. Biochemical parameters of study groups.

	24-h urinary creatinine (mg/dL)	24-h urinary protein-to-creatinine ratio (mg/mg creatinine)	24-h urinary GGT (U/L)	24-h urinary GGT-to-creatinine ratio (U/mg creatinine)
Control group (n = 42)	51.9 ± 22.6	0.73 ± 1.94	26.80 ± 17.33	52.2 ± 26.9
Kappa BJP group (n = 33)	51.1 ± 25.2	3.60 ± 8.64	23.74 ± 19.58	44.9 ± 25.8
Lambda BJP group (n = 28)	61.9 ± 46.2	3.71 ± 4.90	63.86 ± 34.52	90.3 ± 76.5
p Value	NS	<0.001	0.003	0.004

Notes: Data is presented as mean ± SD. Kappa BJP, Kappa Bence Jones Proteinuria; Lambda BJP, Lambda Bence Jones Proteinuria; NS, non-significant.

Mean 24-h urinary protein-to-creatinine ratio of the lambda and kappa BJP groups were significantly higher as compared to the control group (p < 0.001). There were no significant differences in mean 24-h urinary excretion of creatinine between the study groups (Table 2).

Discussion

In this study, we assessed the urinary GGT activity as an early sub-clinical renal damage biomarker in patients with BJP. The main finding of this study is the demonstration of the increased urinary GGT-to-creatinine ratio in lambda light chain proteinuria.

Several recent studies have suggested that urinary enzymes as well as low molecular weight proteins can be used as early markers of tubular damage and nephrotoxicity in a large number of diseases where renal functions are affected.4,5 We hypothesized that 24-h urinary GGT activity may predict renal tubular damage in patients with BJP. According to our results, patients with Lambda light chain proteinuria have higher urinary GGT-to-creatinine ratio as compared to Kappa light chain proteinuria and controls. Even though urinary GGT levels and urinary GGT-to-creatinine ratio have been studied extensively in patients with acute kidney injury, to the best of our knowledge, this is the first study examining the urinary GGT-to-creatinine ratio in patients with different types of BJP.

According to current literature, urinary GGT-to-creatinine ratio is a reliable biomarker in predicting acute kidney injury in different etiologies.7–9 Gatua et al.7 have reported that alkaline phosphatase (ALP) and GGT enzymes in urine could be early indicators of renal damage caused by diabetes. In addition, De Carvalho et al.8 have demonstrated that urinary GGT and ALP have a potential value as an indicator of nephropathy in type 2 diabetic patients and that urinary GGT is superior to ALP in differentiating diabetic nephropathy. Westhuyzen et al.9 suggested that tubular enzymuria could be helpful in identifying acute renal failure in intensive care unit patients. They further pointed out that the cheapness and wide availability of automated assays for GGT and ALP allows the estimation of these enzymes in random urine samples to be particularly useful in identifying patients at high risk of acute renal failure.9 In another study by Maden et al.,10 the authors showed that urinary GGT-to-creatinine ratio is a sensitive biomarker in determining tubular damage due to gentamicin-induced nephrotoxicity in an experimental model. This study provides evidence for the first time that urinary GGT-to-creatinine ratio – as an evidence of sub-clinical renal damage – increases in patients with lambda light chain proteinuria.

Higher urinary GGT-to-creatinine ratio indicates increased tubular damage in patients with lambda light chain proteinuria as compared to kappa light chain proteinuria and controls. However, the precise mechanisms by which lambda light chain proteinuria results in increased urinary GGT-to-creatinine ratio remain to be studied. It is possible that the interaction of monoclonal free light chains with proximal tubule cells may induce complete or partial kidney ischemia that prevents oxygenation of tubular epithelium and lead to trigger biochemical events responsible for fatal damages and finally cell death.3 We speculate that the molecular structure of lambda light chains facilitates the development of potential renal ischemia. While the lambda light chain is a disulphide-linked covalent dimer, kappa light chain can present as non-covalent dimer, stabile monomer or a combination of both.3 Therefore it may be argued that the covalent linked molecular structure of lambda light chains causes deoxygenation of the tubular epithelium, resulting in increased urinary GGT levels. Future studies are needed to shed more light on this physiological difference.

Due to the cross-sectional design of the study, the results should be interpreted with caution and causal relationship cannot be suggested. It would be interesting to assess urinary GGT-to-creatinine ratios in a long term follow-up study in patients with lambda light chain proteinuria and analyze its relationship with clinical renal dysfunction over time.

As a conclusion, present data show that patients with lambda light chain proteinuria have increased urinary GGT-to-creatinine ratio. Although this is a cross-sectional laboratory study of renal damage prediction in lambda light chain proteinuria, our report provides a proof of concept and have implications for further prospective clinical research in this patient population.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.