Urinary Transforming Growth Factor-β Excretion in Patients With Hypertension, Type 2 Diabetes, and Elevated Albumin Excretion Rate: Effects of angiotensin receptor blockade and sodium restriction

Twenty-one patients with type 2 diabetes, hypertension, and elevated urinary AER were studied on an ambulatory basis. A previous study has reported the changes in albuminuria, systemic, and renal hemodynamics in this population (10). Patients were included if seated systolic blood pressure was between 130 and 165 mmHg, AER was between 10 and 200 μg/min, HbA_1c was <11.0%, and 24-h urinary sodium excretion on patients usual diet was >100 mmol/24 h on at least two consecutive measurements 1 month apart. Exclusion criteria were hyperkalemia >5.5 mmol/l, plasma creatinine >200 μmol/l, cardiac failure, or concomitant nitrate therapy. All antihypertensive or diuretic treatments were stopped at least 2 weeks before study entry.

The study protocol has been previously described (10). Patients were randomly assigned in a double blind fashion to receive either the ARB losartan 50 mg daily (n = 11) or matching placebo (n = 10). Medication was taken daily for two 4-week phases with a 4-week washout in between. Patients remained on their usual diets during the first 2 weeks of each phase (run-in) and were then randomly assigned to a 2-week dietary period of either sodium restriction (low-sodium phase) or continuation of regular-sodium diet (regular-sodium phase). In the second phase, there was a crossover in dietary assignment (Fig. 1).

Low-sodium diets were conducted on an ambulatory outpatient basis, with patients having received dietary advice from a clinical nutritionist and subsequently preparing their own food. The dietary sodium target during the 2 weeks of low-sodium diet was 50–70 mmol/day.

Clinical and laboratory parameters were assessed at week 0 and 4 of each phase and included urinary TGF-β excretion, urinary electrolytes, urine volume, plasma TGF-β concentration, clinic blood pressure, albuminuria, and plasma renin activity (PRA).

Blood pressure was measured by an automatic oscillometric digital blood pressure monitor (Omron model HEM-705CP). The average of three measurements taken in the sitting position, 5 min apart and after 5 min of rest, were recorded. The study was approved by the Human Research and Ethics Committee at the Austin and Repatriation Medical Center, and all patients gave informed consent before commencement of the study.

Urine electrolytes were measured on an Hitachi 911 automatic analyzer (Roche Diagnostics, Mannheim, Germany). AER was measured on a 24-h urine collection. Radioimmunoassay for albumin was performed by a double antibody method with intra- and interassay coefficients of variation of 1.8 and 4.8%, respectively, for a concentration of 27 mg/l (11). Specimens for PRA were collected in EDTA tubes on ice, spun, and plasma frozen within 1 h for analysis at a later date. PRA was determined by measuring the rate of generation of angiotensin I by radioimmunoassay after incubating plasma at 37°C for 1 h (12).

For plasma TGF-β, blood was drawn and placed in EDTA-containing tubes and placed on ice. Samples were then sequentially centrifuged to provide platelet-free plasma as previously described (13). In brief, specimens were centrifuged at 800g for 10 min at 4°C, then recentrifuged for 15 min at 2,500g, and then again at 3,600g for 20 min to remove platelets. Complete removal of platelets was confirmed by an automatic cell counter. Aliquots were stored at −80°C.

For urinary TGF-β excretion, a 2.0-ml aliquot from each 24-h urine collection was obtained and stored at −80°C. Before assay, specimens were thawed, placed in a filter unit (Centricon-10 filter; Amicon, Danver, MA), and concentrated 25-fold by centrifugation for 60 min at 6,500 rpm, as previously described (7). Plasma and urinary TGF-β1 were assayed by solid-phase ELISA (enzyme-linked immunsorbent assay) (Quantikine; R&D Systems, Abingdon, U.K.) according to the manufacturer’s instructions. The intra- and interassay coefficients of variation were 7.5 and 12.2%, respectively.

Because of their skewed distribution, plasma TGF-β concentration, urinary TGF-β excretion, albuminuria, and PRA were logarithmically transformed before statistical analysis and expressed as the geometric mean x/÷ tolerance factor. Other results were expressed as means ± SE, unless stated otherwise. Changes in urinary TGF-β excretion and albuminuria are expressed as median percent change (interquartile range). Repeated measures, two-way ANOVA was used to examine the effects of drugs, diet and their interaction on the urinary excretion of TGF-β. Paired t tests were used for comparisons of the two phases within losartan and placebo groups and unpaired t tests for comparisons between treatment groups. The first measurement in each patient on study entry was considered as baseline. Simple linear regression analysis (using log-transformed values where appropriate) was used to determine the relationship between variables using Excel 97. Other analyses were performed using Stata and Statview (SAS Institute, Cary, NC). A P value <0.05 was considered statistically significant.

There were no significant baseline differences in plasma or urinary TGF-β, blood pressure, urinary sodium excretion, AER, BMI, or diabetes duration between placebo- and losartan-treated groups (Table 1).

Equivalent levels of sodium restriction were achieved with urinary sodium excretion of 80 ± 22 and 91 ± 14 mmol/day in placebo and losartan groups, respectively (both P < 0.001 vs. regular-sodium diet). Urine volume did not differ significantly between regular- and low-sodium dietary phases in either group (Table 2). Both losartan treatment and low-sodium diet (both groups) were accompanied by renin angiotensin system activation, as demonstrated by a rise in PRA (Table 2).

Losartan treatment was associated with similar decreases in mean arterial blood pressure (MAP) during the regular- and low-sodium phases (Table 3), and in the placebo group, the changes in MAP were also not significantly different between the regular- and low-sodium phases. However, the changes in MAP in the losartan group were significantly greater than in the placebo group for both the regular- and low-sodium phases (Table 3). In the losartan group, a significant reduction in albuminuria was observed during the low-sodium phase only (Table 3). In the placebo group, a trend toward a reduction in albuminuria during the low-sodium phase was noted (P = 0.06) (Table 3).

At baseline, significant correlations were noted between urinary TGF-β and sodium excretion, fasting plasma glucose and HbA_1c (Figs. 2A–C). No significant baseline correlation was found between urinary TGF-β and MAP or AER.

In the losartan group, urinary TGF-β excretion decreased by 23.2% (−39.2 and 13.6) [median (interquartile range)] and 38.5%(−46.8 and −6.1) in the regular- and low-sodium phases, respectively. In the placebo group, median changes of 0.0% (−12.1 and 44.4) and 0.0% (−29.2 and 110.7) occurred in the regular- and low-sodium phases, respectively. When the groups were analyzed using two-way ANOVA (repeated measures), there was a significant difference for an overall drug effect (P = 0.042). However, there was no significant difference for an overall diet effect (P = 0.54), and no evidence of a significant interaction between drug and diet (P = 0.29) (Fig. 3).

Change in urinary TGF-β excretion in the losartan group did not correlate with change in MAP (r = 0.02, P = 0.65) or AER (0.39, P = 0.23). No significant changes in plasma TGF-β were noted in losartan- or placebo-treated subjects during either the regular- or low-sodium phases (Table 2).

The present study demonstrates several findings in relation to urinary TGF-β and incipient nephropathy in subjects with type 2 diabetes. First, urinary TGF-β excretion was reduced by the ARB losartan but was unaffected by dietary sodium restriction, suggesting that reduction in renal TGF-β production may contribute to the renoprotective effects of this class of drug. Second, urinary TGF-β excretion at baseline correlated closely with indexes of glycemic control, consistent with the role of hyperglycemia in the pathogenesis of diabetic kidney disease.

Over the past several years, experimental evidence has suggested a key role for TGF-β in the pathogenesis of the extracellular matrix accumulation that characterizes diabetic nephropathy (5) and correlates closely with declining renal function (14). The mechanisms for this fibrogenic or prosclerotic action of TGF-β are multiple and include both stimulation of extracellular matrix synthesis and inhibition of its degradation (15). Several studies have documented that not only is TGF-β expression increased in the diabetic kidney but that it is also present in a biologically active form (16). Moreover, inhibition of TGF-β action with neutralizing antibodies has recently been shown to attenuate mesangial expansion and declining GFR in experimental diabetic nephropathy (17).

In patients with diabetes, renal production accounts for the majority of the TGF-β that appears in the urine (6). Blockade of the renin angiotensin system by either ACE inhibition (1) or ARBs (2,3) has been shown to attenuate the rate of progression of renal dysfunction in patients with type 1 and type 2 diabetes, respectively. These important clinical trials have been accompanied by experimental data that angiotensin II, the effector molecule of RAS, stimulates extracellular matrix protein synthesis through induction of TGF-β in mesangial cells (18), renal interstitial fibroblasts (19), and proximal tubular epithelial cells (20). Thus, the reduction in urinary TGF-β excretion with losartan, demonstrated in the present study, may reflect attenuated TGF-β synthesis by all of these renal cell types.

In addition to being a manifestation of renal disease, increased urinary TGF-β has also been implicated in the pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy. Indeed, as recently reviewed (21), there are several mechanisms whereby increased urinary TGF-β may lead to progressive renal disease. First, TGF-β in tubular fluid may act directly on tubular epithelial cells to induce the expression of extracellular matrix proteins by receptor-mediated mechanisms. Second, TGF-β–mediated activation of tubular epithelial cells induces these cells to elaborate platelet-derived growth factor, another prosclerotic growth factor that leads to matrix production by neighboring interstitial fibroblasts. Third, TGF-β may induce chemokine expression leading to macrophage infiltration and acceleration of tissue injury. Thus, the reduction in urinary TGF-β excretion with losartan, as demonstrated in the present study, may explain, at least in part, the beneficial effects of RAS blockade on tubulointerstitial disease in experimental diabetic nephropathy (22).

In the current study, the decrease in urinary TGF-β excretion in the losartan group was linked to a reduction in MAP. Despite no significant correlation between reductions in MAP and urinary excretion of TGF-β in the losartan group, the relatively small sample size in the present study does not preclude an underlying relationship between these two parameters. Previous in vitro studies have linked pressure and TGF-β, demonstrating that pulsatile mechanical stress applied to mesangial cells (23) and vascular smooth muscle cells (24) enhances TGF-β expression. Although it is likely that the decreases in MAP and urinary TGF-β are related, the relative importance of the local renin-angiotensin system versus systemic blood pressure is difficult to determine. In a study using a model of progressive diabetic nephropathy, despite equivalent blood pressure reduction by angiotensin and endothelin receptor antagonists, only the ARB was associated with a reduction in renal TGF-β mRNA levels and severity of diabetic renal structural changes (25).

Although AER and urinary TGF-β both declined with losartan treatment during the low-sodium phase, there was no significant correlation between these changes in individual patients. Although there may be specific regulating mechanisms for urinary TGF-β excretion over and above the excretion of albumin, they were not determined in this study.

In the present study, while losartan treatment reduced urinary TGF-β excretion, plasma levels remained unchanged. In contrast, in a previous study, captopril was reported to reduce serum TGF-β in patients with type 1 diabetes and nephropathy who participated in the Collaborative Study Group Trial (26). There are several reasons that may account for these differences. In the latter study, serum rather than plasma TGF-β was measured. Platelets contain abundant TGF-β, stored within their α-granules (27). A low-force step-wise centrifugation process was therefore used in the present study to avoid platelet contamination and degranulation (12). Other differences include study duration, diabetes type, and drug treatment used.

Previous experimental studies have suggested that high dietary sodium intake may induce cardiac and renal fibrosis via TGF-β–dependent pathways (9) and have also demonstrated high dietary sodium to increase urinary TGF-β excretion within a 2-week period (28). In the present study, although a close correlation between urinary sodium and TGF-β excretion was noted at baseline, a low-sodium diet over 2 weeks did not significantly influence urinary TGF-β in either losartan- or placebo-treated patients. For the effect of a low-sodium diet on the change in urinary TGF-β excretion, this study had 80% power (assuming P = 0.05) to detect an overall mean difference of ∼31%. The findings in the present study raise the possibility that the beneficial effects of reducing dietary sodium intake in diabetic humans may develop over prolonged periods, as seen, for instance, with the effects of improved glycemic control (29).

In the present study, no significant changes in urine flow rate were found between the regular- and low-sodium phases, and it is therefore unlikely that urinary flow rates impacted on the urinary excretion of TGF-β. In addition, a previous experimental study demonstrated that increasing urinary flow rate with diuretics did not increase the urinary excretion of TGF-β (28).

Baseline urinary TGF-β excretion was found to correlate with HbA_1c and fasting plasma glucose in the current study. In type 2 diabetes, glycemic control has been shown to be a risk factor for development of microvascular complications (30). The association of urinary TGF-β with these markers of glycemic control supports the concept that urinary TGF-β may be a pathogenetic risk factor for diabetic nephropathy.

In summary, losartan treatment reduced urinary TGF-β in patients with hypertension, type 2 diabetes, and incipient nephropathy. Further longitudinal intervention studies will be required to determine whether serial measurements of urinary TGF-β may serve as a surrogate marker for the development of diabetic nephropathy and its response to therapeutic interventions.

These studies were supported by a grant from the Austin Hospital Medical Research Foundation and a Merck Medical School Grant and a NovoNordisk Diabetes Research Grant. During the period of this work, C.A.H. was supported by grants from the Austin Hospital Medical Research Foundation and the National Health and Medical Research Council. R.E.G. is the recipient of a career development award from the Juvenile Diabetes Research Foundation International.

We thank Judy Winikoff for her technical assistance and Amynta Baxter for advising and overseeing patient diets throughout the study.