Curr Diab Rep (2014) 14:513 DOI 10.1007/s11892-014-0513-1
MICROVASCULAR COMPLICATIONS—NEPHROPATHY (B ROSHAN, SECTION EDITOR)
Novel Urinary Biomarkers in Early Diabetic Kidney Disease Atsuko Kamijo-Ikemori & Takeshi Sugaya & Kenjiro Kimura
# Springer Science+Business Media New York 2014
Abstract In diabetic kidney disease, detection of urinary albumin is recommended to aid in diagnosis, evaluate disease severity, and determine effects of therapy. However, because typical histopathologic changes in diabetic kidney disease or early progressive renal decline may occur in patients with normoalbuminuria, urinary albumin may not be sufficient to identify patients with early-stage diabetic kidney disease or to predict its progression. Therefore, intensive efforts have been made to identify novel noninvasive urinary biomarkers to discriminate patients with a higher risk of end-stage renal failure. Because diabetic kidney disease progression is associated with the extent of histologic changes in the glomeruli and the degree of tubulointerstitial changes, urinary biomarkers that accurately reflect the degree of histopathologic damage may be excellent biomarkers. This review article summarizes the clinical significance of new urinary biomarkers in the early detection of diabetic kidney disease.
Keywords Urinary biomarker . Diabetic kidney disease . Glomerular damage . Tubulointerstitial damage
This article is part of the Topical Collection on Microvascular Complications—Nephropathy T. Sugaya : K. Kimura (*) Department of Nephrology and Hypertension, Internal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-Ku, Kawasaki 216-8511, Japan e-mail: [email protected] A. Kamijo-Ikemori Department of Anatomy, St. Marianna University School of Medicine, Kanagawa, Japan
Introduction Diabetic kidney disease is the leading cause of end-stage renal failure (ESRD) [1] and contributes to the onset of cardiovascular disease [2–5]. Although the number of patients with diabetic kidney disease is increasing worldwide, there are no specific curative treatments yet. Therefore, diagnostic markers for predicting the prognosis of early diabetic kidney disease are needed to identify patients with a higher risk of progression to ESRD so that renal protective therapy, such as inhibition of the renin–angiotensin–aldosterone system (RAAS), can be started to prevent progression to ESRD. Typical renal histopathologic findings of human diabetic kidney disease include the presence of nodular lesions, doughnut lesions, and exudative glomerular lesions [6, 7], which are associated with the decline of renal function [8, 9•, 10]. Therefore, detection of urinary albumin, which reflects impairment of the glomerular filtration barrier, is recommended for diagnosing diabetic kidney disease, determining its severity, and evaluating the effects of treatment [11–13]. Macroalbuminuria or microalbuminuria is an accepted risk factor for ESRD, cardiovascular disease, and death [14–19]. However, the reliability of urinary albumin as an indicator of early diabetic kidney disease and as a predictor of its progression has been questioned [20•, 21], because normal histopathology also may be observed and progression to microalbuminuria or macroalbuminuria also may occur in patients with normoalbuminuria [8, 20•, 22•, 23]. Therefore, more reliable biomarkers are needed to identify patients at high risk for progression to ESRD and to begin therapeutic intervention in patients with early diabetic kidney disease. In addition to glomerular changes, tubulointerstitial injury has an important impact on diabetic kidney disease progression [24–26]. In both type 1 and type 2 diabetes, progression of renal dysfunction is related to the degree of tubulointerstitial damage with vascular changes, such as
513, Page 2 of 9
arteriolar hyalinosis and arteriosclerosis [8, 22•, 27]. In the early phase of diabetic kidney disease with normoalbuminuria, what are the aggravating factors of tubulointerstitial damage? Chronic tubulointerstitial hypoxia is known to be the final common pathway to ESRD [28]. Chronic hyperglycemia provokes microvascular damage [29]. Oxidative stress induced by hyperglycemia is related to augmentation of oxygen consumption and impairment of oxygen tension in the tissues [30]. Angiotensin II produced by activation of the intrarenal renin– angiotensin system in diabetes [31] decreases peritubular capillary blood flow as a result of contraction of the efferent arterioles. Hyperfiltration that occurs in diabetes increases sodium delivery to tubular cells and imposes excessive tubular sodium reabsorption beyond the oxygen supply [25], resulting in functional tubular hypoxia. Microvascular injury, oxidative stress, a decrease in peritubular capillary blood flow and overwork of tubular cells may lead to tubular hypoxia and, finally, tubulointerstitial damage (Fig. 1). Renal hypoxia was observed even in the early phase of diabetic kidney disease by blood oxygenation level–dependent (BOLD) MRI [32, 33]. Therefore, tubular biomarkers, in addition to urinary albumin, may be useful for monitoring diabetic kidney disease progression, especially in patients with early diabetic kidney disease. Although a review article recently reported the clinical usefulness of various biomarkers in diabetic kidney disease [34•], the present review summarizes recent findings in the early phase of diabetic kidney disease in patients with normal urinary albumin levels and the clinical significance of a urinary glomerular biomarker, urinary podocalyxin, and 2 urinary tubular biomarkers, urinary liver-type fatty acid–binding
Fig. 1 Mechanism of tubulointerstitial damage in diabetes without urinary albumin. Chronic hyperglycemia provokes microvascular damage. Oxidative stress induced by hyperglycemia is related to augmentation of oxygen consumption and impairment of oxygen tension in the tissue. Angiotensin II produced by activation of the intrarenal renin-angiotensin system (RAS) in diabetes decreases peritubular capillary blood flow as a result of contraction of the efferent arteriole. Hyperfiltration that occurs in
Curr Diab Rep (2014) 14:513
protein (L-FABP), which was approved as a tubular injury biomarker in clinical practice by the Ministry of Health, Labour and Welfare in Japan in 2010, and urinary connective tissue growth factor (CTGF) (Table 1); neither of these biomarkers was discussed thoroughly in the earlier review. Limitation of Urinary Albumin Urinary albumin has been used not only to diagnose and categorize diabetic kidney disease, but also to evaluate various management strategies. In many large-scale clinical trials, an increase or a decrease in urinary albumin levels was defined as progression or remission of diabetic kidney disease, respectively [35–38]. However, as described earlier, progressive renal decline and renal histologic changes also were found in patients with normoalbuminuria. In diabetic kidney disease, it has been assumed that an increase in the urinary albumin excretion rate (AER) precedes the development of renal dysfunction. However, several studies, including a recent one [20•], showed a disconnect between increased urinary albumin levels and decreased glomerular filtration rates (GFRs). In patients with type 1 diabetes and normoalbuminuria (n=286) recruited to the second Joslin Kidney Study [20•], who were followed up for a median (range) of 8 (6–9) years, the estimated GFR (eGFR) was calculated by a formula using parameters of both serum creatinine and cystatin C [39]. Progressive renal decline, defined as a continuous loss of eGFRcr-cys greater than 3.3 % per year, was present in 10 % of patients (n = 28) with normoalbuminuria. In another clinical study, which emphasized the importance of microalbuminuria compared with normoalbuminuria in type 1 diabetes [40], progressive renal dysfunction was found in 9 % of all patients with
diabetes causes an increase in sodium delivery to tubular cells and imposes excessive tubular sodium reabsorption beyond oxygen supply, resulting in functional tubular hypoxia. Microvascular injury, oxidative stress, a decrease in peritubular capillary blood flow, and overwork of tubular cells may lead to tubular hypoxia and, finally, tubulointerstitial damage.
Curr Diab Rep (2014) 14:513
Page 3 of 9, 513
Table 1 Characteristics of urinary biomarkers focused at the present review Biomarkers
Expression site
Podocalyxin Podocyte L-FABP
Proximal tubules
CTGF
Glomerulus and tubules
Clinical significance Urinary podocalyxin level increased due to podocyte injury and was higher in the patients with type 2 diabetes and normoalbuminuria compared with healthy control subjects [51•]. Urinary L-FABP level increased with the progression of diabetic kidney disease, and levels were high even in patients with normoalbuminuria in the patients with type 1 and type diabetes [59•, 60•, 61••]. Higher urinary L-FABP level was associated with not only the progression of diabetic kidney disease in the patients with type 1 and type 2 diabetes [59•, 61••, 64•, 66••], but also the onset of cardiovascular events in the patients with type 2 diabetes [66••]. Urinary CTGF levels in type 1 diabetes increased in the patients with macroalbuminuria compared with those with normoalbuminuria or microalbuminuria [71]. Urinary CTGF indicated a response to inhibitors of renin-angiotensin system [73, 74].
normoalbuminuria (n=268). With regard to type 2 diabetes, patients with biopsy-proven diabetic kidney disease were stratified by albuminuria and eGFR calculated using the parameters of serum creatinine and age [41] at the time of renal biopsy and were followed up for a mean of 8.1 years [22•]. The outcomes of this study were the first occurrence of a renal event (requirement of dialysis or a 50 % decline in eGFR from baseline), cardiovascular events (cardiovascular death, nonfatal myocardial infarction, coronary interventions, or nonfatal stroke), and all-cause mortality. These outcomes occurred even in the patients with normoalbuminuria (n=43), and there were no significant differences in the frequency of these outcomes between patients with normoalbuminuria and those with microalbuminuria (n = 217), regardless of eGFR category. Although not conducted routinely, renal biopsy, and histopathologic evaluation have been performed in patients with type 1 and type 2 diabetes who had no abnormality in urinary albumin. In the patients with type 1 diabetes and normoalbuminuria (n=71), a greater width of the glomerular basement membrane (GBM) and higher levels of glycated hemoglobin were independent predictors of progression to massive proteinuria and ESRD, but not AER [9•]. In those with type 2 diabetes [22•], diffuse lesions of glomeruli, interstitial fibrosis, tubular atrophy, interstitial inflammation, and arteriosclerosis were found in patients with normoalbuminuria and maintained eGFR (n=28). Furthermore, in the patients with normoalbuminuria and low eGFR (n=15), glomerular, tubulointerstitial, and vascular lesions were more advanced compared with those in patients with normoalbuminuria and maintained eGFR (n=28). Novel biomarkers that can detect these renal histopathologic changes are expected to reveal the detailed pathophysiology of early diabetic kidney disease, which urinary albumin cannot detect. Some studies in the 1990s suggested the possibility that the upper limit of normoalbuminuria was too high, thus underestimating the diagnosis of early diabetic kidney disease. Normal urinary AER in type 1 diabetes was reported to be 13 μg/min [42]. Another study reported that the mean AER in type 1 diabetes was approximately 5 μg/min and rarely
exceeded 15 μg/min in completely healthy individuals [43]. In type 2 diabetes, an AER close to the upper limit of normoalbuminuria was a predictor of progression to microalbuminuria [44]. Further, it was reported that any degree of measurable urinary albumin bore a significant risk for cardiovascular events in a large cohort of hypertensive patients with type 2 diabetes and baseline AER less than 20 μg/ min who were included in the Bergamo Nephrologic Diabetes Complication Trial and were followed up for a median of 9.2 years [45]. For each 1-μg/min excess in baseline albuminuria, there was a progressive incremental risk of cardiovascular events up to an AER of 13 to 14 μg/min [45]. However, at present, normoalbuminuria is defined as less than 20 μg/min or 30 mg/g creatinine [1]. Urinary Glomerular Biomarker: Urinary Podocalyxin Podocytes cover the outer aspect of the GBM, function as a glomerular filtration barrier, and prevent leakage of various molecules derived from blood [46]. In the early phase of diabetic kidney disease, podocyte detachment from the GBM is observed [47, 48]. Therefore, a biomarker for podocyte injury may be promising for detection of early diabetic kidney disease. Podocalyxin, a sialomucin most closely related to CD34 and endoglycan, is expressed on the surface of podocytes [49]. It contributes to the maintenance of podocyte shape and distortion of the slit diaphragm [49]. Podocalyxin is shed into the urine from podocytes during various kidney injuries [50], and urinary podocalyxin may reflect the degree of podocyte injury [51•]. A cross-sectional study showed that urinary podocalyxin levels significantly increased in type 2 diabetes patients with normoalbuminuria (n = 39) compared with healthy controls (n=69) [51•]. Another group measured urinary podocalyxin-positive element by flow cytometry using a monoclonal antibody against podocalyxin and reported that urinary levels of podocalyxin-positive element were significantly higher in patients with type 2 diabetes patients and normoalbuminuria (n=21) than in the controls (n=28) [52]. Although immunofluorescence revealed no podocytes in
513, Page 4 of 9
patients with type 2 diabetes and normoalbuminuria (n=10) [53], the sensitivity of immunofluorescence assay for detection of urinary podocytes may be lower than that of the flow cytometry technique. Urinary podocalyxin or urinary podocalyxin-positive element may be a useful biomarker for the early detection of diabetic kidney disease in type 2 diabetes. However, longitudinal studies and intervention trials have not been performed yet to evaluate the correlation between urinary podocalyxin and the prognosis of diabetic kidney disease in type 1 diabetes patients. Further studies are needed to confirm the diagnostic and prognostic potential of this marker in diabetic kidney disease. Urinary Tubular Biomarker: Urinary L-FABP L-FABP, expressed in the proximal tubules of the human kidney [54], is an effective endogenous antioxidant during oxidative stress generated in pathophysiologic conditions [55]. Because L-FABP is not expressed in mouse kidneys, we generated human L-FABP chromosomal transgenic (Tg) mice in which human L-FABP was expressed in the proximal tubules of the cortex by microinjection of human L-FABP genomic DNA, including its promoter region, to evaluate the dynamics of human L-FABP [56]. Expression of human LFABP in the proximal tubules of the Tg mice is thought to be regulated by the same transcriptional factors as those in humans. Results using a streptozotocin (STZ)-induced diabetic model showed that human L-FABP gene expression in the kidney was up-regulated and that urinary excretion of human L-FABP was increased by hyperglycemia at 8 weeks after STZ injection [57]. These dynamics of L-FABP found in the proximal tubules of the diabetic Tg mice might be reproduced in those of diabetic patients. Although we recently summarized the clinical significance of urinary L-FABP in diabetic kidney disease [58•], the present review focuses on the importance of urinary L-FABP in diabetic kidney disease with normoalbuminuria. Urinary L-FABP in type 1 and type 2 diabetes was investigated clinically by cross-sectional and longitudinal analyses. The former studies showed that urinary L-FABP level increased in a stepwise manner with the progression of diabetic kidney disease, and levels were high even in patients with normoalbuminuria [59•, 60•, 61••] (Fig. 2). These results indicate that urinary L-FABP accurately reflects the severity of diabetic kidney disease and may be a suitable biomarker for its early detection. The mechanism by which urinary L-FABP levels during the normoalbuminuria stage are higher than those of healthy volunteers is still unknown. Another study reported that only an increase in urinary L-FABP was significantly correlated with a decrease in peritubular capillary blood flow; other urinary tubular markers, such as α1-microglobulin, β2-
Curr Diab Rep (2014) 14:513
microglobulin, and N-acetyl-β-D-glucosaminidase (NAG) were not [62]. Further, a relationship was reported between urinary L-FABP levels and anemia [63]. Anemia reduces the renal oxygen supply and leads to tubular hypoxia. An increase in urinary L-FABP level was significantly associated with anemia severity. We speculate that tubular hypoxia, which is induced in the early phase of diabetic kidney disease, triggers the upregulation of L-FABP in the proximal tubules, resulting in a rise in urinary excretion of L-FABP from the proximal tubules [62], because the promoter region of the L-FABP gene contains a binding site for hypoxia-inducible factor 1 (HIF-1), which is activated by tubular hypoxia [28]. Is a higher level of urinary L-FABP a risk factor for progression of early diabetic kidney disease? Prospective observational follow-up studies in type 1 diabetes show that urinary L-FABP was an independent predictor of progression from normoalbuminuria in 2 studies [61••, 64•]. In the type 1 diabetes patients (n=165) with normoalbuminuria who were followed up for a median (range) of 18 (1–19) years, 39 patients developed persistent microalbuminuria, and of these, 8 patients had further pro gression to persistent macroalbuminuria and 24 patients died [64•]. Urinary LFABP levels at the start of this study tended to be higher in the patients with progression to abnormal urinary albumin levels than in those with persistent normoalbuminuria. Based on a Cox regression model, continuous urinary L-FABP levels predicted microalbuminuria when adjusted for known risk factors (age, sex, glycated hemoglobin [HbA1c], blood pressure, urinary albumin, serum creatinine, and smoking) with an odds ratio (OR) of 2.3 (95 % CI, 1.1–4.6). Based on a Kaplan– Meier plot, urinary L-FABP divided into quartiles predicted the development of microalbuminuria. Moreover, high levels of urinary L-FABP were associated with a significantly increased risk of mortality (OR, 3.0; 95 % CI, 1.3–7.0). With regard to progression to macroalbuminuria, the number of patients was small (n=8), thus a significant result was not obtained. After this study, a mega-study was performed with patients enrolled in the Finnish Diabetic Nephropathy Study [61••]. At baseline, 1549 patients with type 1 diabetes with normoalbuminuria were followed up for a median (range) of 5.8 years (95 % CI, 5.7–5.9), and 112 patients had progression to microalbuminuria. Urinary L-FABP levels at baseline were significantly higher in the progression group (0.075 μg/μmol) than in the nonprogression group (0.014 μg/μmol; P
MICROVASCULAR COMPLICATIONS—NEPHROPATHY (B ROSHAN, SECTION EDITOR)
Novel Urinary Biomarkers in Early Diabetic Kidney Disease Atsuko Kamijo-Ikemori & Takeshi Sugaya & Kenjiro Kimura
# Springer Science+Business Media New York 2014
Abstract In diabetic kidney disease, detection of urinary albumin is recommended to aid in diagnosis, evaluate disease severity, and determine effects of therapy. However, because typical histopathologic changes in diabetic kidney disease or early progressive renal decline may occur in patients with normoalbuminuria, urinary albumin may not be sufficient to identify patients with early-stage diabetic kidney disease or to predict its progression. Therefore, intensive efforts have been made to identify novel noninvasive urinary biomarkers to discriminate patients with a higher risk of end-stage renal failure. Because diabetic kidney disease progression is associated with the extent of histologic changes in the glomeruli and the degree of tubulointerstitial changes, urinary biomarkers that accurately reflect the degree of histopathologic damage may be excellent biomarkers. This review article summarizes the clinical significance of new urinary biomarkers in the early detection of diabetic kidney disease.
Keywords Urinary biomarker . Diabetic kidney disease . Glomerular damage . Tubulointerstitial damage
This article is part of the Topical Collection on Microvascular Complications—Nephropathy T. Sugaya : K. Kimura (*) Department of Nephrology and Hypertension, Internal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-Ku, Kawasaki 216-8511, Japan e-mail: [email protected] A. Kamijo-Ikemori Department of Anatomy, St. Marianna University School of Medicine, Kanagawa, Japan
Introduction Diabetic kidney disease is the leading cause of end-stage renal failure (ESRD) [1] and contributes to the onset of cardiovascular disease [2–5]. Although the number of patients with diabetic kidney disease is increasing worldwide, there are no specific curative treatments yet. Therefore, diagnostic markers for predicting the prognosis of early diabetic kidney disease are needed to identify patients with a higher risk of progression to ESRD so that renal protective therapy, such as inhibition of the renin–angiotensin–aldosterone system (RAAS), can be started to prevent progression to ESRD. Typical renal histopathologic findings of human diabetic kidney disease include the presence of nodular lesions, doughnut lesions, and exudative glomerular lesions [6, 7], which are associated with the decline of renal function [8, 9•, 10]. Therefore, detection of urinary albumin, which reflects impairment of the glomerular filtration barrier, is recommended for diagnosing diabetic kidney disease, determining its severity, and evaluating the effects of treatment [11–13]. Macroalbuminuria or microalbuminuria is an accepted risk factor for ESRD, cardiovascular disease, and death [14–19]. However, the reliability of urinary albumin as an indicator of early diabetic kidney disease and as a predictor of its progression has been questioned [20•, 21], because normal histopathology also may be observed and progression to microalbuminuria or macroalbuminuria also may occur in patients with normoalbuminuria [8, 20•, 22•, 23]. Therefore, more reliable biomarkers are needed to identify patients at high risk for progression to ESRD and to begin therapeutic intervention in patients with early diabetic kidney disease. In addition to glomerular changes, tubulointerstitial injury has an important impact on diabetic kidney disease progression [24–26]. In both type 1 and type 2 diabetes, progression of renal dysfunction is related to the degree of tubulointerstitial damage with vascular changes, such as
513, Page 2 of 9
arteriolar hyalinosis and arteriosclerosis [8, 22•, 27]. In the early phase of diabetic kidney disease with normoalbuminuria, what are the aggravating factors of tubulointerstitial damage? Chronic tubulointerstitial hypoxia is known to be the final common pathway to ESRD [28]. Chronic hyperglycemia provokes microvascular damage [29]. Oxidative stress induced by hyperglycemia is related to augmentation of oxygen consumption and impairment of oxygen tension in the tissues [30]. Angiotensin II produced by activation of the intrarenal renin– angiotensin system in diabetes [31] decreases peritubular capillary blood flow as a result of contraction of the efferent arterioles. Hyperfiltration that occurs in diabetes increases sodium delivery to tubular cells and imposes excessive tubular sodium reabsorption beyond the oxygen supply [25], resulting in functional tubular hypoxia. Microvascular injury, oxidative stress, a decrease in peritubular capillary blood flow and overwork of tubular cells may lead to tubular hypoxia and, finally, tubulointerstitial damage (Fig. 1). Renal hypoxia was observed even in the early phase of diabetic kidney disease by blood oxygenation level–dependent (BOLD) MRI [32, 33]. Therefore, tubular biomarkers, in addition to urinary albumin, may be useful for monitoring diabetic kidney disease progression, especially in patients with early diabetic kidney disease. Although a review article recently reported the clinical usefulness of various biomarkers in diabetic kidney disease [34•], the present review summarizes recent findings in the early phase of diabetic kidney disease in patients with normal urinary albumin levels and the clinical significance of a urinary glomerular biomarker, urinary podocalyxin, and 2 urinary tubular biomarkers, urinary liver-type fatty acid–binding
Fig. 1 Mechanism of tubulointerstitial damage in diabetes without urinary albumin. Chronic hyperglycemia provokes microvascular damage. Oxidative stress induced by hyperglycemia is related to augmentation of oxygen consumption and impairment of oxygen tension in the tissue. Angiotensin II produced by activation of the intrarenal renin-angiotensin system (RAS) in diabetes decreases peritubular capillary blood flow as a result of contraction of the efferent arteriole. Hyperfiltration that occurs in
Curr Diab Rep (2014) 14:513
protein (L-FABP), which was approved as a tubular injury biomarker in clinical practice by the Ministry of Health, Labour and Welfare in Japan in 2010, and urinary connective tissue growth factor (CTGF) (Table 1); neither of these biomarkers was discussed thoroughly in the earlier review. Limitation of Urinary Albumin Urinary albumin has been used not only to diagnose and categorize diabetic kidney disease, but also to evaluate various management strategies. In many large-scale clinical trials, an increase or a decrease in urinary albumin levels was defined as progression or remission of diabetic kidney disease, respectively [35–38]. However, as described earlier, progressive renal decline and renal histologic changes also were found in patients with normoalbuminuria. In diabetic kidney disease, it has been assumed that an increase in the urinary albumin excretion rate (AER) precedes the development of renal dysfunction. However, several studies, including a recent one [20•], showed a disconnect between increased urinary albumin levels and decreased glomerular filtration rates (GFRs). In patients with type 1 diabetes and normoalbuminuria (n=286) recruited to the second Joslin Kidney Study [20•], who were followed up for a median (range) of 8 (6–9) years, the estimated GFR (eGFR) was calculated by a formula using parameters of both serum creatinine and cystatin C [39]. Progressive renal decline, defined as a continuous loss of eGFRcr-cys greater than 3.3 % per year, was present in 10 % of patients (n = 28) with normoalbuminuria. In another clinical study, which emphasized the importance of microalbuminuria compared with normoalbuminuria in type 1 diabetes [40], progressive renal dysfunction was found in 9 % of all patients with
diabetes causes an increase in sodium delivery to tubular cells and imposes excessive tubular sodium reabsorption beyond oxygen supply, resulting in functional tubular hypoxia. Microvascular injury, oxidative stress, a decrease in peritubular capillary blood flow, and overwork of tubular cells may lead to tubular hypoxia and, finally, tubulointerstitial damage.
Curr Diab Rep (2014) 14:513
Page 3 of 9, 513
Table 1 Characteristics of urinary biomarkers focused at the present review Biomarkers
Expression site
Podocalyxin Podocyte L-FABP
Proximal tubules
CTGF
Glomerulus and tubules
Clinical significance Urinary podocalyxin level increased due to podocyte injury and was higher in the patients with type 2 diabetes and normoalbuminuria compared with healthy control subjects [51•]. Urinary L-FABP level increased with the progression of diabetic kidney disease, and levels were high even in patients with normoalbuminuria in the patients with type 1 and type diabetes [59•, 60•, 61••]. Higher urinary L-FABP level was associated with not only the progression of diabetic kidney disease in the patients with type 1 and type 2 diabetes [59•, 61••, 64•, 66••], but also the onset of cardiovascular events in the patients with type 2 diabetes [66••]. Urinary CTGF levels in type 1 diabetes increased in the patients with macroalbuminuria compared with those with normoalbuminuria or microalbuminuria [71]. Urinary CTGF indicated a response to inhibitors of renin-angiotensin system [73, 74].
normoalbuminuria (n=268). With regard to type 2 diabetes, patients with biopsy-proven diabetic kidney disease were stratified by albuminuria and eGFR calculated using the parameters of serum creatinine and age [41] at the time of renal biopsy and were followed up for a mean of 8.1 years [22•]. The outcomes of this study were the first occurrence of a renal event (requirement of dialysis or a 50 % decline in eGFR from baseline), cardiovascular events (cardiovascular death, nonfatal myocardial infarction, coronary interventions, or nonfatal stroke), and all-cause mortality. These outcomes occurred even in the patients with normoalbuminuria (n=43), and there were no significant differences in the frequency of these outcomes between patients with normoalbuminuria and those with microalbuminuria (n = 217), regardless of eGFR category. Although not conducted routinely, renal biopsy, and histopathologic evaluation have been performed in patients with type 1 and type 2 diabetes who had no abnormality in urinary albumin. In the patients with type 1 diabetes and normoalbuminuria (n=71), a greater width of the glomerular basement membrane (GBM) and higher levels of glycated hemoglobin were independent predictors of progression to massive proteinuria and ESRD, but not AER [9•]. In those with type 2 diabetes [22•], diffuse lesions of glomeruli, interstitial fibrosis, tubular atrophy, interstitial inflammation, and arteriosclerosis were found in patients with normoalbuminuria and maintained eGFR (n=28). Furthermore, in the patients with normoalbuminuria and low eGFR (n=15), glomerular, tubulointerstitial, and vascular lesions were more advanced compared with those in patients with normoalbuminuria and maintained eGFR (n=28). Novel biomarkers that can detect these renal histopathologic changes are expected to reveal the detailed pathophysiology of early diabetic kidney disease, which urinary albumin cannot detect. Some studies in the 1990s suggested the possibility that the upper limit of normoalbuminuria was too high, thus underestimating the diagnosis of early diabetic kidney disease. Normal urinary AER in type 1 diabetes was reported to be 13 μg/min [42]. Another study reported that the mean AER in type 1 diabetes was approximately 5 μg/min and rarely
exceeded 15 μg/min in completely healthy individuals [43]. In type 2 diabetes, an AER close to the upper limit of normoalbuminuria was a predictor of progression to microalbuminuria [44]. Further, it was reported that any degree of measurable urinary albumin bore a significant risk for cardiovascular events in a large cohort of hypertensive patients with type 2 diabetes and baseline AER less than 20 μg/ min who were included in the Bergamo Nephrologic Diabetes Complication Trial and were followed up for a median of 9.2 years [45]. For each 1-μg/min excess in baseline albuminuria, there was a progressive incremental risk of cardiovascular events up to an AER of 13 to 14 μg/min [45]. However, at present, normoalbuminuria is defined as less than 20 μg/min or 30 mg/g creatinine [1]. Urinary Glomerular Biomarker: Urinary Podocalyxin Podocytes cover the outer aspect of the GBM, function as a glomerular filtration barrier, and prevent leakage of various molecules derived from blood [46]. In the early phase of diabetic kidney disease, podocyte detachment from the GBM is observed [47, 48]. Therefore, a biomarker for podocyte injury may be promising for detection of early diabetic kidney disease. Podocalyxin, a sialomucin most closely related to CD34 and endoglycan, is expressed on the surface of podocytes [49]. It contributes to the maintenance of podocyte shape and distortion of the slit diaphragm [49]. Podocalyxin is shed into the urine from podocytes during various kidney injuries [50], and urinary podocalyxin may reflect the degree of podocyte injury [51•]. A cross-sectional study showed that urinary podocalyxin levels significantly increased in type 2 diabetes patients with normoalbuminuria (n = 39) compared with healthy controls (n=69) [51•]. Another group measured urinary podocalyxin-positive element by flow cytometry using a monoclonal antibody against podocalyxin and reported that urinary levels of podocalyxin-positive element were significantly higher in patients with type 2 diabetes patients and normoalbuminuria (n=21) than in the controls (n=28) [52]. Although immunofluorescence revealed no podocytes in
513, Page 4 of 9
patients with type 2 diabetes and normoalbuminuria (n=10) [53], the sensitivity of immunofluorescence assay for detection of urinary podocytes may be lower than that of the flow cytometry technique. Urinary podocalyxin or urinary podocalyxin-positive element may be a useful biomarker for the early detection of diabetic kidney disease in type 2 diabetes. However, longitudinal studies and intervention trials have not been performed yet to evaluate the correlation between urinary podocalyxin and the prognosis of diabetic kidney disease in type 1 diabetes patients. Further studies are needed to confirm the diagnostic and prognostic potential of this marker in diabetic kidney disease. Urinary Tubular Biomarker: Urinary L-FABP L-FABP, expressed in the proximal tubules of the human kidney [54], is an effective endogenous antioxidant during oxidative stress generated in pathophysiologic conditions [55]. Because L-FABP is not expressed in mouse kidneys, we generated human L-FABP chromosomal transgenic (Tg) mice in which human L-FABP was expressed in the proximal tubules of the cortex by microinjection of human L-FABP genomic DNA, including its promoter region, to evaluate the dynamics of human L-FABP [56]. Expression of human LFABP in the proximal tubules of the Tg mice is thought to be regulated by the same transcriptional factors as those in humans. Results using a streptozotocin (STZ)-induced diabetic model showed that human L-FABP gene expression in the kidney was up-regulated and that urinary excretion of human L-FABP was increased by hyperglycemia at 8 weeks after STZ injection [57]. These dynamics of L-FABP found in the proximal tubules of the diabetic Tg mice might be reproduced in those of diabetic patients. Although we recently summarized the clinical significance of urinary L-FABP in diabetic kidney disease [58•], the present review focuses on the importance of urinary L-FABP in diabetic kidney disease with normoalbuminuria. Urinary L-FABP in type 1 and type 2 diabetes was investigated clinically by cross-sectional and longitudinal analyses. The former studies showed that urinary L-FABP level increased in a stepwise manner with the progression of diabetic kidney disease, and levels were high even in patients with normoalbuminuria [59•, 60•, 61••] (Fig. 2). These results indicate that urinary L-FABP accurately reflects the severity of diabetic kidney disease and may be a suitable biomarker for its early detection. The mechanism by which urinary L-FABP levels during the normoalbuminuria stage are higher than those of healthy volunteers is still unknown. Another study reported that only an increase in urinary L-FABP was significantly correlated with a decrease in peritubular capillary blood flow; other urinary tubular markers, such as α1-microglobulin, β2-
Curr Diab Rep (2014) 14:513
microglobulin, and N-acetyl-β-D-glucosaminidase (NAG) were not [62]. Further, a relationship was reported between urinary L-FABP levels and anemia [63]. Anemia reduces the renal oxygen supply and leads to tubular hypoxia. An increase in urinary L-FABP level was significantly associated with anemia severity. We speculate that tubular hypoxia, which is induced in the early phase of diabetic kidney disease, triggers the upregulation of L-FABP in the proximal tubules, resulting in a rise in urinary excretion of L-FABP from the proximal tubules [62], because the promoter region of the L-FABP gene contains a binding site for hypoxia-inducible factor 1 (HIF-1), which is activated by tubular hypoxia [28]. Is a higher level of urinary L-FABP a risk factor for progression of early diabetic kidney disease? Prospective observational follow-up studies in type 1 diabetes show that urinary L-FABP was an independent predictor of progression from normoalbuminuria in 2 studies [61••, 64•]. In the type 1 diabetes patients (n=165) with normoalbuminuria who were followed up for a median (range) of 18 (1–19) years, 39 patients developed persistent microalbuminuria, and of these, 8 patients had further pro gression to persistent macroalbuminuria and 24 patients died [64•]. Urinary LFABP levels at the start of this study tended to be higher in the patients with progression to abnormal urinary albumin levels than in those with persistent normoalbuminuria. Based on a Cox regression model, continuous urinary L-FABP levels predicted microalbuminuria when adjusted for known risk factors (age, sex, glycated hemoglobin [HbA1c], blood pressure, urinary albumin, serum creatinine, and smoking) with an odds ratio (OR) of 2.3 (95 % CI, 1.1–4.6). Based on a Kaplan– Meier plot, urinary L-FABP divided into quartiles predicted the development of microalbuminuria. Moreover, high levels of urinary L-FABP were associated with a significantly increased risk of mortality (OR, 3.0; 95 % CI, 1.3–7.0). With regard to progression to macroalbuminuria, the number of patients was small (n=8), thus a significant result was not obtained. After this study, a mega-study was performed with patients enrolled in the Finnish Diabetic Nephropathy Study [61••]. At baseline, 1549 patients with type 1 diabetes with normoalbuminuria were followed up for a median (range) of 5.8 years (95 % CI, 5.7–5.9), and 112 patients had progression to microalbuminuria. Urinary L-FABP levels at baseline were significantly higher in the progression group (0.075 μg/μmol) than in the nonprogression group (0.014 μg/μmol; P
