Abstract
Background: In patients with heart failure plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) levels are correlated to urine neutrophil gelatinase-associated lipocalin (NGAL) levels. We prospectively evaluated the relationship among glomerular filtration rate (eGFR), urine albumin-to-creatinine ratio (ACR), urine and serum NGAL and NT-proBNP levels in 20 type II diabetic patients with macroalbuminuria at 4-month intervals. Results: Compared with 20 age, gender-matched healthy controls, diabetic patients had higher urine and serum NGAL, serum NT-proBNP and lower eGFR. The eGFR of the patients at the baseline, the 4th and the 8th month were 29.6 ± 12.0, 27.8 ± 13.7 and 22.9 ± 10.4 mL/min/1.73 m2, respectively. No significant change in urine NGAL levels was detected (p > 0.05), whereas there were significant increases in NT-proBNP, serum NGAL and urine ACR and significant decrease in eGFR as the study progressed (p < 0.05). Both the baseline and the 4th month urine ACR were positively correlated to NT-proBNP levels measured at the same periods (r: 0.451; p: 0.046; r: 0.489; p: 0.029 respectively). In all measurements, urine ACR was negatively correlated to serum albumin levels measured at the same periods (r: −0.792; p: 0.000; r: −0.716; p: 0.000; r: −0.531; p: 0.016 respectively). None of eGFR measurements was correlated with NT-proBNP (p > 0.05). Neither serum NGAL nor urinary NGAL levels are associated with NT-proBNP (p > 0.05). Conclusion: Our findings show an association between NT-proBNP and proteinuria in type II diabetic patients with macroalbuminuria but not with serum and urine NGAL.
Introduction
Once proteinuria occurs and/or chronic renal failure develops, the rate of decline of renal function in diabetic nephropathy is considerably higher than the rate in other renal diseases.Citation1,Citation2
Studies evaluating total body exchangeable sodium in diabetic patients showed increased sodium content, 10% higher than in nondiabetic subjects.Citation3–5 B-type natriuretic peptide (BNP) belongs to a family of natriuretic proteins whose physiological role is maintenance of sodium homeostasis and protection of the cardiovascular system from volume overload. BNP was synthesized by ventricular myocytes in response to physiological signals such as stretching of the ventricular wall, changes in systemic blood pressure, sodium levels or extracellular volume.Citation6,Citation7
Neutrophil gelatinase-associated lipocalin (NGAL), a small 25-kDa protein belonging to the lipocalin family is normally secreted in low amounts in lung, trachea, stomach, colon and kidney. NGAL is produced by the nephron in response to tubular epithelial damage.Citation8,Citation9 It has been well established that serum and urine NGAL (u NGAL) levels rise in acute kidney injury (AKI). Some studies have reported altered NGAL levels in patients affected by some chronic kidney disease (CKD) associated conditions, such as autoimmune, polycystic and proteinuric diseases, suggesting the possibility that under these circumstances NGAL production from tubular cells may reflect the entity of active renal damage.Citation10,Citation11
Some studies reported that in diabetic nephropathy renal impairment is not only associated with decreased glomerular filtration rate (GFR) and increased urinary albumin excretion, but also with the presence of structural tubular damage, as measured by increased urinary concentrations of specific tubular marker proteins such as NGAL.Citation12–14
While BNP is a marker of ventricular stretch, and therefore a surrogate of volume overload, NGAL is a marker of renal tubular injury that is both diagnostic and prognostic. Recently, it was hypothesized that venous congestion might also be a determinant of renal damage.Citation15 It was reported that in patients with heart failure plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were correlated to NGAL levels.Citation16
The main aim of this pilot study is to investigate the relationship among eGFR, urine albumin-to-creatinine ratio, urinary NGAL levels and NT-proBNP in diabetic patients with macroalbuminuria at 4-month intervals.
Material and methods
Twenty clinically stable type II diabetic patients with macroproteinuria, aged ≥18 years, were asked to participate. In addition, 20 healthy, age- and sex-matched controls were studied. All subjects gave informed consent to participate in the study, which was approved by the ethics review committee of the study center.
All patients were on angiotensin-converting enzyme inhibitors and/or angiotensin II receptor blockers, and all medication had been stable for at least 1 month. The dose of angiotensin-converting enzyme inhibitors and/or angiotensin II receptor blockers were adjusted according to blood pressure during the study.
The demographic information and medical history were obtained at baseline by interview and a review of the medical records. Clinical measurements including weight, height, systolic and diastolic blood pressure and blood tests including blood urine nitrogen (BUN), serum creatinine, albumin, urine and serum NGAL, NT-proBNP levels, urine albumin and urine creatinine were measured at the baseline, at the 4th month and at the 8th month. Serum and urine samples were collected at baseline and at 4th and 8th month and stored in aliquots at −70 °C.
All subjects enrolled in the study underwent detailed clinical examination, including measurements of height, weight and blood pressure. Blood pressure was determined using a Standard aneroid sphygmomanometer (Merk Perfect Aneroid, Bad Toelz, Germany) and cuffs adapted to arm circumference. The systolic blood pressure was taken as the point of appearance of Korotokof sounds, and the diastolic blood pressure as the point of disappearance of the sounds.
Urinary albumin and creatinine concentration were determined the same morning of the clinical examination on an early morning first void sterile urine sample. The urinary albumin-to-creatinine ratio (ACR) was then calculated. Macroalbuminuria was defined as an ACR > 0.3, a level that approximates an albumin excretion of 300 mg/24 h, considered as the upper limit of microalbuminuria.Citation17
Levels of BUN, creatinine, serum albumin were measured using the Abbott system Architect C 16000 model instrument (Abott Laboratories, 100 Abott Park Road, Chicago, IL). Urinary albumin (mg/dL) was measured by the immunoturbidimetric method. In the spot urine specimens urinary creatinine (mg/dL) was measured by the Jaffe rate method (kinetic alkaline picrate).
The concentrations of NGAL in urine and serum NGAL were measured by ELISA method (Basic radim Immunoassay Operator, Radim Spa, Pomezia, Italy).
Estimated GFR was calculated using the simplified modification of diet in renal disease formula (186.3 × serum creatinine−1.154 × age−0.203 (×0.742 if female)) as validated in the patients.Citation18
Statistical analysis
Data are presented as mean ± standard deviation. Differences between patients and controls were tested using Mann–Whitney U or Student’s T testing where appropriate. Differences between repeated measurements were analyzed with Paired-Samples T test or Wilcoxon test and were appropriate. Correlations were performed using Spearman’s correlation coefficients. All reported probability values are two-tailed, and a p-value <0.05 was considered statistically significant. Categorical data were expressed as percentages and compared using X2 analysis or Wilcoxon test. The non-normal variables (i.e., NT-proBNP) were log transformed for analytical purposes.
Results
details the general characteristics and clinical parameters of the study groups. Compared with the control group, diabetic patients had higher urinary NGAL, serum NGAL, serum NT-proBNP and lower eGFR.
Table 1. The general characteristics and clinical parameters of the study groups.
The GFR of the patients at the baseline and the 4th and the 8th month were 29.6 ± 12.0, 27.8 ± 13.7 and 22.9 ± 10.4 mL/min/1.73 m2, respectively (). The monthly change in GFR (ΔGFR) was −0.8 ± 1.1 mL/min/1.73 m2. Sixteen patients (80%) showed a decrease in GFR during study period. We could not find any statistically significant change in urine NGAL levels (p > 0.05), whereas there were significantly increases in NT proBNP, serum NGAL and urine ACR and significant decrease in eGFR (p < 0.05). There was no change in diastolic blood pressure (p > 0.05) whereas systolic blood pressure was decreased (p < 0.05).
Table 2. Comparison of the repeated measurements.
Both the baseline and the 4th month urine ACR were positively correlated to NT-proBNP levels measured at the same periods (r: 0.451; p: 0.046; r: 0.489; p: 0.029, respectively). The baseline, the 4th month and the 8th month urine ACR were negatively correlated to serum albumin levels measured at the same periods (r: −0.792; p: 0.000; r: −0.716; p: 0.000; r: −0.531; p: 0.016, respectively) (). The 4th month urine ACR was positively correlated with serum NGAL (r: 0.478; p: 0.033) and diastolic blood pressure (r: 0.620; p: 0.006).
Table 3. The correlation analysis results.
The 8th month eGFR was negatively associated with urine NGAL (r: −0.471; p: 0.039).
At any measurement eGFR was not correlated with NT-proBNP (p > 0.05). Neither serum NGAL nor urinary NGAL levels are associated with NT-proBNP at any measurement (p > 0.05).
The 4th month eGFR was negatively associated with systolic blood pressure (r: −0.471; p: 0.048). The 8th month eGFR was negatively correlated with diastolic blood pressure (r: −0.488; p: 0.040).
Discussion
We found significantly higher serum NT-proBNP levels in patients with diabetic nephropathy than those of healthy controls. NT-proBNP levels increased in our study subjects as the study progressed.
Spanaus et al.Citation19 examined the relevance of BNP and NT-proBNP as predictors of CKD progression. Of 227 nondiabetic patients with mild-to-moderate renal insufficiency, 177 patients ages 18–65 years were followed in a prospective multicenter cohort study for a period of ≤7 years. These authors found that increased BNP and NT-proBNP concentrations indicate an increased risk for accelerated progression of CKD to end-stage renal disease (ESRD) and may prove to be valuable biomarkers for the assessment of prognosis in patients with CKD.Citation19
Desai et al.Citation20 showed that higher levels of NT-pro-BNP were associated independently with higher rates of ESRD. These authors reported that measurement of NT-pro-BNP may improve the identification of patients with CKD who are likely to require renal replacement therapy, supporting a link between cardiac injury and the development of ESRD.
Locatelli et al.Citation21 showed that in CKD patients with mild-to-moderate anemia, elevated baseline plasma NT-proBNP levels are associated with a higher risk of cardiovascular events and an accelerated progression toward ESRD. Yasuda et al.Citation22 reported that elevation of BNP level is associated with an increased risk for accelerated progression of CKD ultimately to ESRD. Monitoring the BNP level could be helpful in the management of combined heart and kidney disease.
An association between progression of CKD and impaired salt regulation and extracellular fluid volume expansion was reported.Citation23 The increased activity of the renin–angiotensin–aldosterone system (RAAS) in CKD may also influence NT-proBNP plasma concentrations. Increased concentrations of NT-proBNP in renal failure reflect not only impaired glomerular filtration but also a counterregulatory response of the heart to changes in hemodynamics and water homeostasis.Citation6,Citation7
Hypervolemia in diabetes is aggravated by abnormalities in several sodium and volume regulating systems such as the renin–angiotensin–aldosterone system, insulin, natriuretic peptides, sodium transport pathways, vessel compliance, intrarenal dopamine production and renal function.Citation24,Citation25
In our study, both the baseline and the 4th month NT-proBNP levels were positively correlated to urine ACR measured at the same periods. One clamp study has shown that BNP infusion increases urine albumin excretion in patients with type I diabetes.Citation26 It was reported that this may be due to a downregulation of A-type guanylate cyclase-coupled natriuretic peptide receptors in renal tubules or due to elevated NT proBNP levels leading to higher glomerular hydraulic pressure or higher capillary permeability and possibly increased albumin excretion.Citation27 Zietse et al. and McKenna et al.Citation26,Citation28 have shown that during ANP infusion, albumin excretion increases in diabetic patients and suggested that ANP increases the transcapillary escape rate of albumin. Because both ANP and BNP bind to the same receptor and have the same biological activity, it is conceivable that abnormally elevated levels of BNP also cause increased glomerular hydraulic pressure and thus induce increased albumin excretion in diabetic patients.
Urine ACR and serum NGAL levels increased and eGFR decreased in our study subjects as the study progressed. However, the urine NGAL levels did not change significantly throughout the course of the study.
Kim et al.Citation12 reported that urinary tubular markers may be independently associated with albuminuria in the early stage of nephropathy in type II diabetics and may reflect inflammatory processing and the activation of the intrarenal RAS. Yang et al.Citation14 showed that serum and urine NGAL are sensitive for predicting the progression of type-II diabetic nephropathy. Increased urinary NGAL levels in type II diabetic patients with low glomerular hyperfiltration than the normal GFR and control groups were reported by Fu et al.Citation13
However, some studies have shown conflicting results concerning the correlation between increased tubular markers, including urinary NGAL, and disease progression in diabetic patients. Nielsen et al.Citation29 reported a lack of an independent correlation between these biomarkers and GFR in type I diabetic patients with overt nephropathy.
We found significantly higher serum and urine NGAL levels in patients with diabetic nephropathy than those of healthy controls. Serum NGAL levels increased whereas the urine NGAL levels did not change significantly throughout the course of the study. It seems that there was a tubular injury in diabetic patients with macroproteinuria having higher serum and urine NGAL levels. However, the severity of injury was stable because there was no change in urine NGAL levels. It is possible that there was increase in serum NGAL, due to decreased renal excretion.Citation8 Serum NGAL levels were positively associated with urine ACR only at the 8th month. Urine NGAL levels were negatively correlated with eGFR only at the 8th month. It seems that the increase in urine ACR might be mainly mediated by glomerular injury rather than increased tubular damage.
In conclusion, our findings provide evidence of an association between NT-proBNP and proteinuria in type II diabetic patients with macroalbuminuria but not with urine NGAL. In light of what has been discussed above, patients with high NT-proBNP may have more tubular injury; however, examination of small groups in our study entails loss of statistical power and therefore the present results require confirmation in larger groups of patients. Additional studies are needed to clarify relationship between eGFR, urine ACR, serum and urine NGAL and BNP.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
This study was supported by Inonu University Medical Research Center.
References
- Takazakura E, Nakamoto Y, Hayakawa H, Kawai K, Muramoto S. Onset and progression of diabetic glomerulosclerosis: a prospective study based on serial renal biopsies. Diabetes. 1975;24(1):1–9
- Feest TG, Dunn EJ, Burton CJ. Can intensive treatment alter the progress of established diabetic nephropathy to end-stage renal failure? QJM. 1999;92(5):275–282
- Stern N, Tuck ML. Mechanisms of hypertension in diabetes mellitus. In: Laragh JH, Brenner BM, eds. Hypertension: pathophysiology, diagnosis, and management. New York: Raven; 1995:2301–2314
- Feldt-Rasmussen B, Mathiesen ER, Deckert T, et al. Central role for sodium in the pathogenesis of blood pressure changes independent of angiotensin--aldosterone and catecholamines in type 1 (insulin-dependent) diabetes mellitus. Diabetologia. 1987;30:610
- Weidmann P. Pathogenesis of hypertension accompanying diabetes mellitus. Contrib Nephrol. 1988;73:73
- Liang F, Gardner DG. Autocrine/paracrine determinants of strain-activated brain natriuretic peptide gene expression in cultured cardiac myocytes. J Biol Chem. 1998;273:14612–14619
- Richards AM, Crozier IG, Holmes SJ, Espiner EA, Yandle TG, Frampton C. Brain natriuretic peptide: natriuretic and endocrine effects in essential hypertension. J Hypertens. 1993;11:163–170
- Mori K, Nakao K. Neutrophil gelatinase-associated lipocalin as the real time indicator of active kidney damage. Kidney Int. 2007;71:967–70
- Bolignano D, Donato V, Coppolino G, Campo C, Lacquaniti A, Buemi M. Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage. Am J kidney Dis. 2008;52:595–609
- Brunner HI, Mueller M, Rutherford C, et al. Urinary neutrophil gelatinase-associated lipocalin as a biomarker of nephritis in child-hood-onset systemic lupus erythematosus. Arthritis Rheum. 2006;54(8):2577–2584
- Bolignano D, Lacquaniti A, Coppolino G, et al. Neutrophil gelatinase-associated lipocalin (NGAL) and progression of chronic kidney disease. Clin J Am Soc Nephrol. 2009;4(2):337–344
- Kim SS, Song SH, Kim IJ, et al. Clinical implication of urinary tubular markers in the early stage of nephropathy with type 2 diabetic patients. Diabetes Res Clin Pract. 2012;97(2):251–257
- Fu WJ, Li BL, Wang SB, et al. Changes of the tubular markers in type 2 diabetes mellitus with glomerular hyperfiltration. Diabetes Res Clin Pract. 2012;95(1):105–109
- Yang YH, He XJ, Chen SR, Wang L, Li EM, Xu LY. Changes of serum and urine neutrophil gelatinase-associated lipocalin in type-2 diabetic patients with nephropathy: one year observational follow-up study. Endocrine. 2009;36(1):45–51
- Damman K, Navis G, Smilde TD, et al. Decreased cardiac output, venous congestion and the association with renal impairment in patients with cardiac dysfunction. Eur J Heart Fail. 2007;9:872–878
- Palazzuoli A, Beltrami M, Pellegrini M, Nuti R. Natriuretic peptides and NGAL in heart failure: does a link exist? Clin Chim Acta. 2012;413(23–24):1832–1838
- Klein R, Klein BE, Linton KL, Moss SE. Micro-albuminurian a population-based study of diabetes. Arch Intern Med. 1992;152:153–158
- Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of diet in renal disease study group. Ann Intern Med. 1999;130:461–470
- Spanaus KS, Kronenberg F, Ritz E, et al. Mild-to-Moderate Kidney Disease Study Group. B-type natriuretic peptide concentrations predict the progression of nondiabetic chronic kidney disease: the Mild-to-Moderate Kidney Disease Study. Clin Chem. 2007;53(7):1264–1272
- Desai AS, Toto R, Jarolim P, et al. Association between cardiac biomarkers and the development of ESRD in patients with type 2 diabetes mellitus, anemia, and CKD. Am J Kidney Dis. 2011;58(5):717–728
- Locatelli F, Eckardt KU, Macdougall IC, et al. Cardiovascular risk reduction in early anaemia trial with epoetin beta investigators and coordinators. Value of N-terminal brain natriuretic peptide as a prognostic marker in patients with CKD: results from the CREATE study. Curr Med Res Opin. 2010;26(11):2543–2552
- Yasuda K, Kimura T, Sasaki K, et al. Plasma B-type natriuretic peptide level predicts kidney prognosis in patients with predialysis chronic kidney disease. Nephrol Dial Transplant. 2012;27(10):3885–3891
- Vasavada N, Agarwal R. Role of excess volume in the pathophysiology of hypertension in chronic kidney disease. Kidney Int. 2003;64:1772–1779
- Ditzel J, Brochner-Mortensen J. Tubular reabsorption rates as related to elevated glomerular filtration in diabetic children. Diabetes. 1983;32(Suppl 2):28
- Tuck ML, Corry D, Trujillo A. Salt-sensitive blood pressure and exaggerated vascular reactivity in the hypertension of diabetes mellitus. Am J Med. 1990;88:210
- McKenna K, Smith D, Moore K, Glen A, Tormey W, Thompson CJ. Brain natriuretic peptide increases urinary albumin and alpha-1 microglobulin excretion in Type 1 diabetes mellitus. Diabet Med. 2001;18:973–978
- Siebenhofer A, Ng LL, Plank J, Berghold A, Hödl R, Pieber TR. Plasma N-terminal pro-brain natriuretic peptide in Type 1 diabetic patients with and without diabetic nephropathy. Diabet Med. 2003;20(7):535–539
- Zietse R, Derkx FH, Weimar W, Schalekamp MA. Effect of atrial natriuretic peptide on renal and vascular permeability in diabetes mellitus. J Am Soc Nephr. 1995;5:2057–2066
- Nielsen SE, Andersen S, Zdunek D, Hess G, Parving HH, Rossing P. Tubular markers do not predict the decline in glomerular filtration rate in type 1 diabetic patients with overt nephropathy. Kidney Int. 2011;79:1113–1118