Urinary measurement of transforming growth factor-ß and type IV collagen as new markers of renal injury: application in diabetic nephropathy

¹ Division of Nephrology, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, and
² Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15213.
^a Address correspondence to this author at: Division of Pediatric Nephrology, Children's Hospital of Pittsburgh, 3705 Fifth Avenue at DeSoto Street, Pittsburgh, PA 15213. Fax 412-692-7443.

	Abstract

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Urinary samples were concentrated rapidly and efficiently and were used to develop several protein assays that may be of value in monitoring individuals with progressive renal disorders. Transforming growth factor-ß1 (TGF-ß1) and retinol binding protein (RBP) were measured with modification of commercially available methods used to assay serum specimens; type 3 collagen (T3C) was measured with a new immunonephelometric assay. The precision characteristics of these assays are comparable with those reported for microalbuminuria. The clinical utility of measuring a panel of these markers was demonstrated in urine samples from 16 control subjects and from 46 individuals with insulin-dependent diabetes mellitus (IDDM) with various albumin excretion rates (AERs). TGF-ß1 and T3C were used as markers of cytokine expression and of the renal fibrogenic process, whereas RBP excretion served as a marker of tubular injury or dysfunction. Compared with controls, T3C excretion was significantly increased in 18 normoalbuminuric and further increased in 13 microalbuminuric (AER 20 200 µg/min) IDDM subjects. RBP excretion was increased in macroalbuminuric IDDM subjects (AER >200 µg/min, overt nephropathy). Significant correlations were also found between AER and RBP in all but macroalbuminuric individuals, whereas TGF-ß1 correlated with T3C excretion in controls and in normoalbuminuric diabetic subjects. Urinary RBP but not AER was an excellent predictor of diabetic nephropathy as defined by serum creatinine (P = 0.0001). This underscores the importance of an early tubulopathy in the subsequent development of glomerulopathy and overt nephropathy. The data suggest that longitudinal monitoring of a panel of urinary markers such as that used in the current study may better define their relevance in progressive glomerulosclerosis and may also provide greater insight into the mechanisms underlying such process.

	Introduction

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Measurement of total urinary protein in individuals testing positive by urinary dipstick is a typical method for assessing the presence of potentially serious glomerular disorders. In the absence of such overt proteinuria however, measurement of specific urinary proteins such as ß₂-microglobulin or myeloma proteins may be useful in the diagnosis of tubulopathies or overflow proteinuria, whereas subclinical albumin hyperexcretion may serve as useful early predictor of diabetic nephropathy (1)(2)(3)(4)(5). Sequential monitoring of such markers serves as a guide to progressive renal injury or as a means of gauging the influence of intervention measures on the disease process. Recent experimental studies suggest that newer urinary markers may serve as early indicators of renal injury and may provide greater insight into the pathogenesis of progressive glomerulosclerosis. With this objective we developed methods to measure urinary transforming growth factor-ß1 (TGF-ß1)¹ and type III collagen (T3C) as markers of extracellular matrix synthesis or remodeling (6)(7)(8)(9)(10)(11)(12)(13)(14)(15), and retinol-binding protein (RBP) as a sensitive and reliable indicator of tubular injury or dysfunction (1)(16)(17). Because diabetic nephropathy has been characterized by different stages of albumin excretion rates (AERs), ranging from normoalbuminuria, microalbuminuria (AER 20 200 µg/min), or macroalbuminuria (AER >200 µg/min) (2)(3)(4)(5), which correspond to increasing histopathologic severity (18), this disorder was selected to test the clinical utility of such novel urinary markers.

	Materials and Methods

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subjects
The control group consisted of healthy normotensive volunteers recruited from our center. Each participant underwent a thorough medical history including medication intake, and had urinalysis, serum creatinine, and urinary albumin excretion values within the corresponding reference intervals.

The cases came from the Pittsburgh Epidemiology of Diabetes Complications (EDC) Study, a 10-year follow-up study of childhood-onset Type I diabetes, details of which have been published elsewhere (19)(20). Each subject had complete medical history and physical examination including standardized blood pressure measurements at baseline (Cycle 1, 1986–1988) and every two years subsequently. Also measured at these time intervals were urinary creatinine and AERs in overnight, short daytime, and 24-hour urine collections, as well as plasma hemoglobin A1c, fasting lipids and lipoproteins, and other biochemical variables. The glomerular filtration rate was estimated on the basis of creatinine clearance measurements in validated urine collections. Assignment into normoalbuminuric, microalbuminuric (20 200 µg/min), and macroalbuminuric or overt nephropathy (>200 µg/min) ranges was based on timed urine collections at each biennial exam. Forty-six patients without renal failure (serum creatinine >50 mg/L) who were seen over a 10-month period in Cycle 5 and who fell into one of these three AER groups on the basis of all three current urine collections at both Cycle 5 and at the exam 2 years earlier (Cycle 4, 1992–1994) were studied. Additional details of these 46 subjects are shown in Table 1 and are compared with the other subjects evaluated in Cycle 5 who were not studied during this 10-month period. For practical reasons, only the overnight urine collection was used for the comparisons of albumin, TGF-ß1, T3C, and RBP excretion.

urine preparation
Aliquots of validated overnight urine collections were centrifuged for 5 min to remove cells and particulate matter, and the supernates were stored at -70 °C. Samples were rapidly thawed and centrifuged for 5 min at 2000 rpm to remove any urates or phosphates before use in assays. A small sample portion was analyzed for creatinine and albumin as previously described (21). Because TGF-ß1 and RBP concentrations in urine from healthy individuals are below the detection limits of commercial assays and because T3C concentrations are also low, the following methods using concentrated urine were developed and tested.

A 2.0-mL urine sample was placed in a Centricon-10 filter (Amicon) pretreated with 1 mL/L Tween-20 used to limit adsorption to its polypropylene components. Such filters exclude proteins with molecular weights >10 kDa, including TGF-ß1 and RBP, with molecular masses of 25 kDa and 21 kDa, respectively. The filter-urine units were centrifuged for 60 min at 6500 rpm in a 34° fixed angle rotor (model SA-600, DuPont, Sorvall^®) equipped with appropriate adapter units. This rapid concentration method provides a retentate volume ranging from 40 to 80 µL and a discard volume virtually free of protein. The extent of concentration achieved was calculated from the exact retentate volume measured with a Hamilton pipet. This ranged from 25- to 60-fold, with a median of 40-fold concentration. This prepared sample was utilized in the following assays, which were all performed at room temperature. Similarly concentrated urine samples from the nondiabetic controls were used for the determination of the precision and recovery characteristics of the following assays.

TGF-ß1 ASSAY
Measurement of urinary TGF-ß1 was achieved by modifying a solid-phase enzyme-linked immunosorbent assay (ELISA), which is suitable for measurement of activated TGF-ß1 in biological fluids other than urine (Quantikine^(TM), R & D Systems). A 20-µL aliquot of urinary concentrate was diluted with 130 µL of RD51 diluent supplied in the Quantikine assay. The TGF-ß1 in this sample was then activated by incubation with 30 µL of 1.0 N HCl (final concentration, 0.167 N) for 10 min followed by neutralization with 30 µL of 1.2 N NaOH/0.5 mol/L HEPES buffer. Subsequently, 200 µL of the 210-µL total volume was used as recommended for the Quantikine assay. Seven standards ranging from 31.2 to 2000 ng/L and a zero standard were also prepared using RD51 diluent. Three controls were obtained from R & D Systems. Controls were also made from purified recombinant human TGF-ß1 (R & D Systems) and used with each Quantikine assay. Despite the sample dilutions imposed by this method, the final average urine concentration was 7-fold higher than the original sample. This was taken into consideration in the final calculation of the concentration of TGF-ß1 in individual urine samples. In experiments assessing recovery of TGF-ß1 in urine specimens, purified TGF-ß1 was reconstituted in 1.0 mL of sterile 4.0 mmol/L HCL containing 1.0 g/L bovine serum albumin, producing a TGF-ß1 final concentration of 2000 ng/L. Specific amounts of this stock solution were added to eight urine samples with known concentrations of endogenous TGF-ß1 and reassayed after urinary concentration and TGF-ß1 activation, as described above for the typical assay. The intraassay and interassay coefficients of variation for this assay were 8.5% and 13.1%, respectively. Recovery rates ranged from 92% to 96%.

type iii collagen assay
A completely new immunonephelometric procedure was developed for the quantitation of urinary T3C, using concentrated urine samples. a lyophilized monoclonal IgG antibody against human T3C (Heyltex) was reconstituted with 1.0 mL of double distilled water and purified human T3C protein in 0.5 mol/L acetic acid, pH 2.5 (Chemicon International). Individual antibody dilutions were tested with several concentrations of purified T3C and variable concentrations of polyethylene glycol-8000 (PEG) in phosphate-buffered saline (PBS) to determine the optimal concentration that provided standard curves characteristic of nephelometric kinetics. In the assay chosen for current specimen analysis, the antibody was diluted 75-fold with a 40 g/L PEG solution. After a 45-min incubation, the antibody solution was filtered twice using a 0.2-µm Millipore filter. T3C was diluted with PBS to prepare six standards, ranging in concentration from 6.6 to 104.9 µg/L. Fifty microliters of each standard or 10 µL of the urinary retentate plus 40 µL of PBS were then incubated with 750 µL of the antibody solution for 60 min at room temperature. The amount of light scatter produced by the antigen-antibody complexes in the facilitating buffered PEG solution was detected by a Hyland laser nephelometer PDQ^(TM) (Travenol Laboratories) and converted into a T3C concentration based on a polynomial curve-fitting of the standard values. As with the other assays, the concentration obtained in each urine sample was reduced by the respective degree of urinary concentration. The intraassay and interassay coefficients of variation were 7.8% and 18.6%, respectively, and the recovery rates in eight concentrated urine samples ranged from 90% to 96%.

retinol binding protein
A radial immunodiffusion kit (Nanorid^(TM) Retinol Binding Protein, Binding Site) was used to measure human RBP. Unconcentrated urine aliquots of nondiabetic controls measured well below the low standard (500 µg/L), whereas RBP was readily detected in concentrated samples. Controls used with each radial immunodiffusion plate included the control provided in the kit as well as two controls prepared from ultra-pure human RBP (Cortex Biochem). The latter were also used to obtain recovery data after addition of specific amounts of RBP to urine samples. Recovery was assessed by the comparison of RBP measured in eight such samples before and after urinary concentration, using the Centricon-10 filters. The intraassay and interassay coefficients of variation of this method were 9.3% and 14.5%, respectively. Recovery rates ranged from 90% to 93%.

statistics
Geometric means and median values were determined for each of the four urinary proteins within each group. Tests for linear trend and multiple comparisons (Student–Newman–Keuls test) were used to examine the existence of any linear trend across nephropathic status groups and the differences between groups. To determine the power of urinary proteins to predict renal function, regression analysis was performed after combining the three subgroups of diabetics (n = 46) and utilizing urinary protein as the independent variable and serum creatinine as the dependent variable. Multiple regression analysis was then used to examine the independent predictors of urinary proteins in the combined three subgroups. For this purpose, a stepwise mode was used for the selection of the independent predictors on the basis of risk factor-urinary protein pairs with correlation coefficients having a P value <0.1.

	Results

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Table 1 shows that the current study subjects evaluated at Cycle 5 did not differ substantially in terms of age, gender, disease duration, blood pressure, glycemic control, serum creatinine, or AER compared with the larger number of individuals with the same AER classification during Cycle 5 who were not part of the current study. In addition, comparison of the entire EDC study population and the combined three diabetic subgroups showed similar correlation coefficients between overnight AERs and the following variables: height, body weight, body mass index, waist-to-hip ratio, blood pressure, alcohol intake, insulin dosage, hemoglobin A1, plasma lipids, fibrinogen, serum albumin, and creatinine concentrations.

Both in controls and in subjects with insulin-dependent diabetes mellitus (IDDM), mean AER values commonly reported in µg/min were very similar to AERs expressed in mg/g creatinine. However, among individual subjects within each group there was a greater variability in AER values expressed in µg/min compared with mg/g creatinine. This interindividual variability was especially pronounced in the microalbuminuric group. Hence, statistical analyses were performed utilizing the more conventional units of protein/mg creatinine excreted.

Table 2 summarizes the excretion rates expressed per minute and per g of creatinine for the four proteins measured in overnight urine collections. Median and 10–90 percentile ranges (in parentheses) are provided. A test of linear trend across the three diabetic groups was significant (P <0.003) for each protein. Multiple comparisons obtained by the Student-Newman-Keuls test showed a significant difference in (a) AER between the three IDDM groups, (b) T3C between control and normoalbuminuric as well as between normo- and microalbuminuric diabetics, and (c) RBP between micro and macroalbuminuric groups (all, P <0.05).

Correlations between the four proteins were also studied (vertical lines in Table 2 ). Significant correlations (P <0.05) occurred between AERs and TGF-ß1 in macroalbuminuric subjects (r = 0.49) and between AERs and RBP in control (r = 0.54), normoalbuminuric (r = 0.54), and microalbuminuric (r = 0.60) subjects. Significant correlations also occurred between TGF-ß1 and T3C excretion in controls (r = 0.51) and in normoalbuminuric subjects (r = 0.65) as well as between TGF-ß1 and RBP excretion in microalbuminuric subjects (r = 0.81). The albumin-to-RBP ratios are also shown in Table 2 . A reduced ratio denotes an isolated tubulopathy, whereas an increased ratio denotes either isolated glomerulopathy or both glomerulopathy and advanced tubulopathy.

To determine which of the four urinary proteins was independently associated with renal function as reflected by serum creatinine concentrations in the combined diabetic subgroups, multiple regression was conducted using a stepwise mode. The independent predictors of serum creatinine in the final model were RBP (P = 0.001) and triglycerides (P = 0.05); AERs, TGF-ß1, and T3C were not independent predictors of such renal function.

Table 3 summarizes the multiple regression analyses in which independent potential risk factors for diabetic nephropathy were examined for their ability to predict the magnitude of excretion of each urinary protein. To increase the power of this analysis, the three small IDDM subgroups were combined. Because serum creatinine and the glomerular filtration rate are measures denoting renal injury, they were excluded as appropriate biological predictors of urinary protein markers. Subsequent analysis revealed that increased systolic blood pressure and reduced plasma HDL-cholesterol were significant predictors of AER; insulin dose predicted TGF-ß1 and plasma triglycerides predicted T3C and RBP.

	Discussion

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Although numerous specific causes initiate renal injury, many such disorders appear to share common pathogenetic pathways of disease progression. Measurement of several urinary markers provides information regarding the specific site of injury or renal dysfunction, but the pursuit of such measurements is often predicated on discovering renal dysfunction, abnormal urinalysis, or anatomical disturbances rather than the presence of a silent, preclinical state. An important exception are the 1982 reports of microalbuminuria as an important predictor of diabetic nephropathy (2)(3)(4)(5), which led to recent studies demonstrating the effective role of intensive management with insulin in preventing the progression of diabetic nephropathy (22)(23)(24). Quantitation of urinary markers such as albumin or RBP, however, has not advanced our understanding of pathogenetic processes. On the basis of available evidence obtained since the seminal observations of Border et al. (14), a hypothesis was recently proposed that highlights the key role of persistent and aberrant elaboration of TGF-ß1 in progressive glomerular fibrosis in genetically susceptible individuals with poor control of IDDM (25). Enhanced expression of mRNA for TGF-ß1 and increased secretion of T3C have also been demonstrated in mesangial cells cultured in high glucose media (8)(9)(10). Immunohistochemical and in situ hybridization techniques have been widely used to localize pertubations in TGF-ß1 in renal tissue in diabetes and other forms of experimental and human glomerulonephritis (13)(14)(15). Also, plasma TGF-ß1 may be reliably measured by an ELISA method (Quantikine, see Materials and Methods); there are no simple and reliable quantitative methods, however, for measuring urinary TGF-ß1 or T3C.

The application of newer rapid methods for concentrating urinary proteins and the high efficiency of protein recovery have enabled the subsequent measurement of minute quantities of several proteins in the current study. TGF-ß1 and RBP were measured with modifications of commercially available assays; T3C was measured by a totally new immunonephelometric technique. The latter was made possible by the availability of highly purified T3C analytes and monoclonal antibodies against human T3C. The precision characteristics of all these assays are detailed in Materials and Methods and compare favorably with or are superior to precision data of currently available albumin assays, including our own (21). Although two cumbersome methods for measuring urinary TGF-ß1 include a bioassay (26) and an ELISA method using an enhanced chemoluminescence system with a detection range of 0.1–2.5 µg/L (27), the current method is simpler and more reliable. Similarly, ours is the only study in which T3C has been quantified directly. Apart from urinary albumin, normative data exist only for RBP. Median RBP excretion in the current study is 50.3 ng/min or 6.1 mg/mol creatinine. These values compare well with previous reports of 45 ng/min or 6.2 mg/mol creatinine in control subjects and 87 ng/min or 11.1 mg/mol creatinine in overnight urine collections from normotensive, normoalbuminuric subjects with IDDM (28).

The data in Table 1 indicate that the three smaller AER groups in the current study were representative of the larger AER subgroups in the entire EDC study population evaluated at the same time period. Numerous other variables measured but not shown in Table 1 were also similarly reflective of the individual study populations. The age and gender proportion of the control group were also similar to the sampled diabetic study groups.

A notable finding of the current study are the significant differences in T3C excretion between normoalbuminuric and microalbuminuric subjects (Table 2 ). This implies that T3C may be a discriminating marker of these two groups. Moreover, these data suggest a stimulatory role of TGF-ß1 on T3C excretion only in Group I, in which a small and insignificant increase in TGF-ß1 excretion correlates with a three-fold rise in T3C excretion compared with controls, despite similar AERs in these two groups. It is noteworthy that modest increases in TGF-ß1 excretion in micro- and macroalbuminuric subjects did not correlate with the sixfold increase in T3C excretion in both of these groups. This suggests that factors other than TGF-ß1 may have a regulatory action on T3C excretion or that the region of T3C synthesis and excretion (glomerular or tubular) may shift with advancing glomerulopathy. These data however, do not permit the prediction that normoalbuminuric subjects with higher TGF-ß1 or T3C excretion are more likely to become microalbuminuric. The unique observation in our study that TGF-ß1 excretion is predicted by high insulin dosages but not by glycemic control in the overall IDDM group (Table 3 ), suggests a direct role of insulin in stimulating TGF-ß1 expression. Regardless of the precise mechanisms regulating TGF-ß1 and T3C expression, the current study supports previous reports of enhanced TGF-ß1 expression in glomeruli of diabetic rats (11)(12) and increased T3C in glomerular tissue of individuals with IDDM (13). Other studies in a nondiabetic setting also implicate high concentrations or sustained endogenous release of TGF-ß1 in the development of progressive glomerulosclerosis (14)(15). The high correlation between TGF-ß1 and RBP excretion in Group II (r = 0.81, P <0.05) requires additional study.

A significant increase in excretion in RBP was demonstrated only in the macroalbuminuric group with presumed more advanced histopathologic lesions of diabetic nephropathy (Table 2 ). Previous studies using urinary ß₂-microglobulin as a marker of tubular dysfunction in IDDM have been inconclusive (1)(16)(17). In contrast, measurement of RBP, which is not degraded by acid pH and is not influenced by gender or posture, has provided consistent evidence favoring the presence of tubular dysfunction not only in individuals with IDDM with overt nephropathy (29) but also in those with and without microalbuminuria (28)(30)(31)(32)(33)(34). The notable correlation between AERs and RBP excretion in control, normoalbuminuric, and microalbuminuric subjects lends support to the hypothesis that tubulopathy precedes the development of glomerulopathy and that microalbuminuria may be secondary to impaired tubular reabsorption of physiologic amounts of filtered albumin. The higher, albeit statistically insignificant, upward trend in RBP excretion but similar AER in normoalbuminuric diabetics (Group I) compared with controls, together with a decrease in the albumin-to-RBP ratio from 135 in control to 63 in normoalbuminuric subjects, argues against overflow proteinuria in Group I and suggests an early proximal tubulopathy in the latter. This, however, requires additional confirmation, which may based on the measurement of RBP excretion in larger numbers of diabetic subjects. Such an early tubulopathy, which may be mediated by endothelial cell injury and preferential peritubular as opposed to glomerular vasculopathy (30)(32)(35), may not reduce the tubular reabsorption of larger proteins such as albumin that are minimally filtered but would reduce the reabsorption of smaller proteins such as RBP that, under steady conditions, are freely filtered in glomeruli and are almost completely reabsorbed in the proximal tubule. The higher excretion of both proteins but predominantly of albumin in microalbuminuric and macroalbuminuric subjects is suggestive of a more advanced tubulopathy in conjunction with glomerulopathy.

Using a regression model to detect an independent association between renal function and each of the four proteins measured, we observed that RBP but not AER was an excellent predictor (P <0.0001) of diabetic nephropathy as defined not by albuminuria but by serum creatinine concentrations. These observations also implicate the existence of an early tubulopathy and suggest that this disorder has an adverse effect on renal function.

The ability of the various risk factors to independently predict the magnitude of each urinary protein is shown in Table 3 . Insulin dosage emerged as a predictor of TGF-ß1 and plasma triglyceride concentration as a predictor of RBP excretion. These results in an unselected clinical population of IDDM suggest the intriguing hypothesis that glycemic control, insulin dosage, and triglycerides may influence the development of an early tubulopathy manifested by RBP hyperexcretion and glomerular dysfunction manifested by modest increases in serum creatinine. The current data offer no direct proof, however, that such early disturbances relate to increases in LDL-cholesterol and hypertension, which are more powerful predictors for developing advanced diabetic nephropathy manifested by glomerulosclerosis, overt proteinuria, and severe renal insufficiency.

On the basis of these data, we speculate that the noninvasive monitoring of several urinary proteins may provide clues concerning the modulation of collagen and the glomerular and tubular alterations leading to albumin and RBP excretion, respectively. For instance, the significant association between TGF-ß1 hyperexcretion and insulin dosage suggests a possible cooperative role of these factors, which together with hyperglycemia may stimulate synthesis of mesangial extracellular matrix that is evident in advanced diabetic nephropathy. Longitudinal monitoring of such urinary markers may better define their relevance in disease progression and may also serve as a more precise tool for gauging the influence of various therapeutic regimens on diabetic nephropathy.

In summary, we describe methods for measuring a panel of urinary markers that may be used to monitor the course of progressive renal disorders. The potential benefit of the simultaneous quantitation of such markers was demonstrated in the current study in a well-defined cross-sectional population of individuals with IDDM. The ability to detect abnormal quantities of several interactive molecules in urine at a subclinical stage of IDDM not only improves our understanding of pathogenetic events but also offers the potential of testing intervention measures that may modify the disease process at a very early stage.

	Footnotes

 
¹ Nonstandard abbreviations: TGF-ß1, transforming growth factor-ß1; T3C, type III collagen; RBP, retinol-binding protein; AER, albumin excretion rate; EDC, Epidemiology of Diabetes Complications; PEG, polyethylene gylcol; PBS, phosphate-buffered saline; and IDDM, insulin-dependent diabetes mellitus.

	References

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