Transforming Growth Factor Beta and Excess Burden of Renal Disease

Abstract

End-stage renal disease (ESRD) is more frequent in African Americans (blacks) compared to whites. Because renal fibrosis is a correlate of progressive renal failure and a dominant feature of ESRD, and because transforming growth factor beta 1 (TGF-β1) can induce fibrosis and renal insufficiency, we hypothesized that TGF-β1 hyperexpression is more frequent in blacks compared to whites. We measured circulating levels of TGF-β1 in black and white patients with ESRD, hypertension, and in normal patients. We demonstrated that circulating levels of TGF-β1 are higher in black ESRD patients, hypertensive patients, and normal control patients compared to their white counterparts. Our preliminary genetic analyses suggest that TGF-β1 DNA polymorphisms are different in blacks and whites. Our observations of hyperexpression of TGF-β1 in blacks suggest a mechanism for the increased prevalence of renal failure and hypertensive target organ damage in this population.

In the last decade, there has been a steady increase in the prevalence and incidence of kidney failure (¹). The number of patients in the United States requiring renal replacement therapy with either dialysis or transplantation was 209,000 patients in 1991 and 472,000 in 2004 (²).

The prevalence of chronic kidney disease is far greater. As many as 26 million Americans have chronic kidney disease (CKD), and the prevalence rate has increased from 10.0% (95% CI, 9.1–10.9) to 13.1% (95% CI, 12.0–14.1) in the last decade (¹). Kidney disease is clearly a major global public health burden, and because of the high cost of both dialysis and transplantation, only those countries with robust economies can even begin to meet the challenges of treating patients with end stage renal disease (ESRD).

There are multiple risk factors for the initiation as well as the progression of renal disease, particularly diabetes, hypertension, obesity and proteinuria. Kidney disease clusters in families, suggesting a role for genetic factors. Racial differences in the prevalence of kidney disease and the risk for progression to end stage have also been observed (^{2, 3}). The incidence of ESRD in about 3-fold higher in blacks compared to whites in the US, and the lifetime cumulative risk for ESRD in blacks is 1 in 12, compared to 1 in 45 for whites (⁴).

The basis for the increased prevalence of kidney failure in blacks is multifactorial, including a higher prevalence of renal risk factors such as diabetes and hypertension, genetic factors, unequal access to care and lower socioeconomic status. Additionally, recent studies have emphasized that CKD progresses more rapidly to ESRD in African Americans compared to other ethnic/racial groups (³).

It is well recognized that regardless of the initial cause of injury, renal disease often progresses to end stage by a final common pathway involving a complex interplay of hemodynamic as well as inflammatory, cellular and immune mechanisms which ultimately result in glomerulosclerosis (⁵). There is compelling evidence that TGF-β, a multifunctional cytokine, is a major mediator of glomerulosclerosis (⁶). TGF-β induces fibrosis during the process of tissue repair, and contributes to the pathogenesis of a variety of renal diseases (⁷). TGF-β is hyperexpressed in human glomerular diseases including immunoglobulin A (IgA) nephropathy, lupus nephritis, focal and segmental glomerulosclerosis (⁸). There is considerable evidence that increased production of TGF-β plays an important role in the development of diabetic nephropathy (⁹). We reported that progressive failure of renal allografts (chronic rejection) is associated with TGF-β hyperexpression (¹⁰). Recent data also suggest that TGF-β may have a direct pathogenic role in elevated blood pressure (¹¹). TGF β stimulates the expression of mRNA encoding endothelin-1 in vascular endothelial cells (¹²) and also increases renin release from juxtaglomerular cells in the kidney (¹³).

We therefore hypothesized that excess TGF-β is a candidate mechanism for the increased burden of ESRD in African Americans, that hyperexpression of TGF-β is in part genetically determined, and that hyperexpression of TGF-β may identify those at risk for hypertensive organ damage.

Materials and Methods

We performed two clinical studies of circulating TGF-β1 in black and white subjects (^{14, 15}).

Study of TGF β1 levels in black and white subjects with ESRD.

Fifty-two black and 46 white patients with ESRD, treated with chronic hemodialysis therapy, were studied for level of expression of TGF-β1 protein in their peripheral blood. In addition, circulating levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were also determined in the ESRD cohort. Interleukin-6 and TNF-α were selected for study because of their potent immunoregulatory and remodeling properties and in consideration of earlier reports of their dysregulated expression in ESRD patients (¹⁶). All patients were clinically stable and were free of any active infection. Blood was also drawn from 30 healthy white and 32 healthy black individuals for cytokine analysis. These control subjects did not have any history of hypertension, diabetes, renal, cardiovascular, peripheral vascular or cerebrovascular disease. Peripheral venous blood was obtained from the patients, and the sera were isolated and stored at −70°C until assayed for cytokines. The blood samples were drawn prior to the dialysis procedure. Pre- as well as post-dialysis blood specimens were obtained on the same day to calculate urea reduction ratio. The biologically active TGF-β1 protein concentration was determined using a solid phase TGF-β1-specific sandwich ELISA (Promega, Madison, WI, USA), as described (¹⁷). The sera were activated by acidification and tested at 1:300 dilution, as recommended. A TGF-β 1 standard curve was constructed using 1000, 500, 250, 62.5, 31.25, and 15.6 pg/ml of recombinant human TGF-β1 protein, and a curve-fitting software program was used to quantify TGF-β1 protein concentration in the sera. The minimum level of detection of TGF-β1 with the sandwich ELISA was 25 pg/ml.

Study of TGF-β1 levels in black and white subjects with and without hypertension.

Hypertensive subjects were recruited from the outpatient clinic at the Hypertension Division of Weill Medical College of Cornell University. Subjects were included if blood pressure (average of three determinations) was greater than 140/90 mmHg on at least two occasions. Patients with renal insufficiency and/or diabetes (serum creatinine ≥2 mg/dl) were excluded. Normal subjects were recruited from hospital employees and were required to have a blood pressure of <140/90 mmHg on three determinations. Blood pressure was measured in the seated position according to American Heart Association guidelines. Individuals with significant comorbidity (cancer, autoimmune disease, or active infection) were excluded. The biologically active TGF-β1 protein concentration was determined using a solid phase TGF-β1-specific sandwich ELISA (Promega, Madison, WI, USA), as described (¹⁷).

Results

Circulating Levels of TGF β1 in Patients with ESRD.

Diabetes, hypertension and glomerulonephritis were the most frequent causes of ESRD in our study cohort, as it is in the general U.S. dialysis population. There were no significant differences between black and white patients with respect to the frequencies of the different diseases responsible for their ESRD (P=0.05 by Fisher's exact test). There were also no significant differences between black patients and white patients with respect to age, sex, years on dialysis, serum electrolytes (Na, K, Cl, HCO₃⁻, Ca²⁺, PO₄⁻), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase, albumin, cholesterol, glucose and hematocrit. Dialysis efficacy, as assessed by urea reduction ratio, was also similar in both groups. Plasma creatinine concentration and systolic, diastolic and mean arterial blood pressures were higher in the black cohort compared to the white ESRD cohort.

TGF-β1 protein was detected in the peripheral blood of all 98 ESRD patients studied, and the mean ± SEM TGF-β1 concentration for the entire study group was 174 ± 7 ng/ml (median value=169 ng/ml). The TGF-β1 protein level was higher in the black patients compared to their white counterparts (Fig. 1). The mean ± SEM TGF-β1 protein concentration was 207 ± 10 ng/ml in the 52 black ESRD patients (median value=199 ng/ml); the concentration was 137 ± 8 ng/ml in the 46 white ESRD patients (median value=138 ng/ml). The race-dependent difference in the circulating level of TGF-β1 protein in the ESRD group was significant at P=0.0001 by Student's t-test.

Fig. 1.

A. Differential level of expression of transforming growth factor-β1 (TGF-β1) protein in black end-stage renal disease (ESRD) patients (shaded bars) and white ESRD patients (clear bars). B. The frequency distribution of TGF-β1 levels in the ESRD patients, distinguished by race, is shown. A total of 96% of the TGF-β1 values from black ESRD patients were within 2 SD of the mean value, and the coefficient of skewness and the coefficient of kurtosis were 0.20 and 0.24, respectively; 93% of the TGF-β1 values from white ESRD patients were within 2 SD of the mean value, and the coefficient of skewness and the coefficient of kurtosis were 0.38 and 0.78, respectively. In view of the normal distribution of TGF-β1 values in the black as well as white ESRD patients, Student's t-test was used to derive the P=0.0001 in (A). Reproduced with permission from Kidney International, Vol. 53 (1998), pp. 639–644.

In a multivariate linear regression model, TGF-β1 was considered as the dependent variable, and race and five other variables (sex, age, albumin, cholesterol, glucose) that did not correlate with each other (by correlation analysis) were considered as independent variables. The general linear model procedure of SAS was used, and race was a significant (P=0.0001) determinant of circulating TGF-β1 protein level in this ESRD cohort. The general linear model procedure also revealed that additional renal related variables, such as blood urea nitrogen, urea reduction ratio, serum creatinine, type of dialyzer, or years on dialysis, were not significant determinants of circulating TGF-β1 levels in the ESRD cohort.

Hyperexpression of transforming growth factor-β1 in black end-stage renal disease patients was cytokine-specific. In contrast to the significant difference between black and white ESRD patients with respect to TGF-β1 protein levels, circulating IL-6 levels and TNF α were not significantly different between the black ESRD patients and the white ESRD patients.

Circulating TGF beta levels in Normotensive and Hypertensive Black and White Subjects.

Quantification of TGF-β1 levels in sera obtained from hypertensive subjects and normotensive controls demonstrated that TGF-β1 levels were higher in hypertensives compared with normotensives. The mean ± SEM TGF-β1 protein concentration was 261 ± 9 ng/ml in hypertensive subjects (n=61) and was 188 ± 7 ng/ml in the normotensive controls (n=90) (P, 0.0001, Mann-Whitney two-sample test, Fig. 2A). Fig. 2B compares TGF-β1 protein levels across diagnosis (hypertensive or normotensive) and race (black or white). TGF-β1 levels were the highest in black hypertensives, and comparison of the mean TGF-β1 levels across the four groups demonstrated that the null hypothesis of equal group means should be rejected (P=0.0001, ANOVA, Fig. 2B). Bonferroni P values were calculated to correct for type 1 experiment-wise error rate in view of comparisons across multiple groups. The multiple comparisons showed that the TGF-β1 protein levels in black hypertensives (322 ± 16 ng/ml, n=18) were higher than those in white hypertensives (235 ± 9 ng/ml, n=43, P=0.001), black normals (221 ± 12 ng/ml, n=39, P=0.001), and white normals (165 ± 6 ng/ml, n=51, P=0.001). The analysis also showed that TGF-β1 protein levels in black normals were significantly higher than those in white normals (P=0.001), but not different from those in white hypertensives (P=0.05). The frequency distribution of TGF-β1 levels in the black and white normotensive and black and white hypertensive subjects is shown in Fig. 2C. Whereas 66.6% of the black hypertensives had TGF-β1 levels greater than 300 ng/ml, only 16.2% of white hypertensives had levels greater than 300 ng/ml (P=0.00009, Fisher's exact test). A multiple regression analysis was used to estimate the independent effects of race (black vs. white) on TGF-β1 protein levels, controlling for gender and age. Race, but not gender or age was a highly significant predictor of TGF-β1 levels. A multiple logistic regression analysis then was used to estimate the effect of TGF-β1 protein levels on hypertension status controlling for race, gender, and age. This analysis demonstrated that TGF-β1 protein concentration is a significant predictor of diagnosis (P=0.004). The estimated odds of an individual at the 75th percentile of TGF-β1 distribution (285 ng/ml) being hypertensive was 234% greater than that of an individual at the 25th percentile (187 ng/ml).

Fig. 2.

Serum TGF-β1 protein levels in normotensive and hypertensive subjects. TGF-β1 protein levels distinguished by diagnosis (hypertensive vs. normotensive) are shown in A. TGF-β1 protein levels, distinguished by diagnosis as well as by race, are illustrated in B. Normotensive whites (NW), normotensive blacks (NB), hypertensive whites (HTW), and hypertensive blacks (HTB), respectively (P, 0.0001, ANOVA). The frequency distribution of TGF-β1 levels, distinguished by race and diagnosis, is shown in C. Reproduced with permission from Proc. Natl. Acad. Sci. USA, March 28, 2000 vol. 97 no. 7 pp. 3479–3484.

Basis for TGF-β1 hyperexpression.

We have begun to explore the basis of TGF-β1 hyperexpression in African Americans. A potential mechanism for heterogeneity in TGF-β1 levels is polymorphisms in the TGF-β1 gene. Seven single nucleotide polymorphisms in the human TGF-β1 gene have been identified, with strong linkage disequilibrium among six of the seven polymorphisms (¹⁸). ARMS-PCR, an allele-specific PCR, is a powerful new tool for the ready detection of single nucleotide polymorphisms (¹⁹). We have genotyped 255 healthy African- Americans and 298 healthy Caucasians for TGF-β1 SNPs at positions −800, −509 and codon 263. Our data demonstrating that the allele frequencies at −800 (P=0.0001), −509 (P < 0.0001) and codon 263 (P < 0.001) are significantly different are shown in Table 1 These genotyping data demonstrate differences in black and white subjects. The allele frequencies of the Caucasian subjects in our study are similar to that of Caucasian subjects investigated in the ECTIM study (¹⁸) comprised of European population (Northern Ireland and France)

TABLE 1.

TGF-β1 −800, −509 and codon 263 allele frequencies in African Americans and Caucasians.

Position	Cornell Study		ECTIM Study
	African-Americans N=255	Caucasians N=298	Caucasians N=629
−800 G/A	0.97/0.03	0.93/0.07	0.92/0.08
−509 C/T	0.73/0.27	0.61/0.39	0.65/0.35
Codon 263	0.99/0.01	0.96/0.04	0.96/0.04

Discussion

Our data demonstrate that circulating levels of TGF-β1 are higher in African American ESRD patients, hypertensive patients, and normal control patients compared to their white counterparts. Our preliminary genetic analyses suggest that TGF-β1 DNA polymorphisms are different in blacks compared with whites. In view of the potential of TGF-β1 to induce renal fibrosis and progressive renal disease, as well as its association with peptides involved in blood pressure regulation such as endothelin-1 and angiotensin II, we offer here the hypothesis that heightened TGF-β1 expression is a mechanism for the increased prevalence of ESRD in the United States African American population. We propose that TGF-β1 hyperexpression is not necessarily the cause of renal disease; it is, rather, an important cofactor that results in accelerated progression of renal failure in subjects with a variety of renal diseases (Fig. 3). Our data demonstrating higher levels of TGF-β1 in African American hypertensives compared with whites also suggests that TGF-β1 may contribute independently to the pathogenesis of hypertension, or it may contribute to the vascular complications of increased blood pressure. Further studies exploring polymorphisms in the TGF-β1 gene and their functional consequences might help to resolve the existing enigma of the increased risk of ESRD in the United States African American population. Investigation of additional TGF-β1 isoforms, TGF-β1 receptors, and TGF-β1 signal transduction may provide insights into the role of this important cytokine in renal and hypertensive disease.

Fig. 3.

Hypothetical model of transforming growth factor beta 1 (TGF-β1) hyperexpression as a contributory factor for progressive renal insufficiency. In this hypothesis for the pathogenesis of progressive renal insufficiency, patients expressing high levels of TGF-β1 in response to renal injury are at an increased risk for progression. In this formulation, TGF-β1 expression is engendered by the primary insult (e.g., diabetes), and progressive renal insufficiency results from the heightened expression of TGF-β1. Abbreviations are: DM, diabetes mellitus; GN, glomerulonephritis; HT, hypertension; ESRD, end-stage renal disease. Reproduced with permission from Kidney International, Vol. 53 (1998), pp. 639–644.

Our results have important clinical implications. Angiotensin II stimulates production of TGF-β1 via the AT-1 receptor (²⁰), and both angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor antagonists have been demonstrated to reduce TGF-β1 in patients with renal transplants and in those with diabetic nephropathy (^{21, 22}). Given the evidence for excessive target organ damage in African Americans, and in particular, the increased incidence of ESRD, which may in part be related to TGF-β1 excess, judicious use of ACE inhibitors and angiotensin receptor antagonists should be considered in African American hypertensive patients, particularly when target organ damage is already manifest. In fact, the AASK Study recently demonstrated the superiority of ACE inhibitors over calcium channel blockers and beta-blockers in slowing the progression of hypertensive nephrosclerosis in African Americans (²³). Lowering blood pressure, however, is of paramount importance, and should be a major goal of therapy as well.

DISCUSSION

Mackowiak, Baltimore: I am not an expert on hypertension, but my understanding is that not only do African Americans differ from white Americans, but African Americans differ from African blacks. I wonder if you would hypothesize as to what happened in terms of the problem of hypertension when blacks migrated from African to North America?

August, New York: That's a whole subject and not one that I have really gone into, but you probably know the hypothesis about the middle crossing and about how stressful that was in terms of salt conservation; and perhaps the African Americans who survived in the United States are particularly avid salt handlers; and that is one hypothesis for the increased prevalence of salt sensitivity in African American hypertensives. As far as hypertension around the world and in other parts of Africa, depending on where you go in Africa and again, this is not one of my areas of expertise, there are different observations about prevalence of hypertension versus normal retention. My knowledge of it seems to be that it has a lot to do with urbanization versus rural living perhaps.

Wesson, Temple, Texas: Phyllis, great studies and good, intriguing hypothesis! I wondered, have you compared TGF beta levels in whites and blacks at similar levels of nephropathy or depressed GFR to try to get at the question as to whether the high TGF is related more to ethnicity or related to nephropathy.

August, New York: The first slide that I showed you of our data was in end-stage kidney failure dialysis patients, and they were higher in blacks compared to whites; and so we haven’t looked at patients with CKD.

Luke, Cincinnati: Having spent a decade in Alabama where we transplanted more African Americans than anywhere else in the world, I certainly believe in renal susceptibility genes; and I think TGF beta has the best evidence. I wondered if you were familiar with the work of Harriet Dustin, a very famous, though deceased unfortunately, cardiologist who was interested in hypertension, and she did some older work which, of course, has fallen off the cliff, because it was pre-electronic, which is an experience many older authors have had; but she cultured the fibroblasts from keloid scars which are more common in African Americans and which may have had some evolutionary advantage in terms of strong wound healing; and the TGF beta production from these fibroblasts was much higher than regular fibroblasts, because it does support what you are talking about.

August, New York: Yes, actually when we published the first paper on end-stage renal failure, Harriet wrote us a letter and informed us of her previous work, and periodically over the years, people have tried to look at keloid scars; and the biology of TGF beta is complicated, but we always remember her early work; and certainly, she was a pioneer in this area.

Hochberg, Baltimore: So a comment and a question: The comment is that there is another disorder that you can add to your list of conditions that are associated with TGF beta, but also is more prevalent and actually has higher a incidence among African Americans than Caucasians—and that's osteoarthritis of the knee. You can block the development of knee osteoarthritis in preclinical models by blocking TGF beta, because it is involved in endochondral ossification. The question is, whether you have looked at any specimens from longitudinal cohorts to test the hypothesis that's been shown in the cross-sectional data prospectively in terms of either serum levels or cohorts that have biomarkers available through cells that you could look at RNA or DNA?

August, New York: Thank you for informing me about the osteoarthritis. I am going to add that to my list. We are trying to get money to do that study, and we are planning those longitudinal studies in looking at blood and urine in prospective cohorts.

Glassock, Laguna Naguel: Phyllis, wonderful talk, great work! Robin Luke has anticipated my question. It is about keloids in African Americans; and is there any epidemiologic evidence that you know of that keloid formation in African Americans is associated with a higher risk of progression to end-stage renal disease?

August, New York: Not that I know of, but it is certainly a great study to do for a fellow I think. Somebody could look into that.

Weir, Baltimore: Great job, Phil! You focus on dietary sodium in your neighborhood study. Did you also measure urinary potassium? The only reason I say that is, that perhaps urinary sodium to potassium ratios or urinary potassium may also be a very powerful regulator of these types of hormonal responses.

August, New York: Great question, Matt! We have the data, and we have asked our statistician for it; and he has yet to give us the results, but we are about to analyze it as soon as he comes up with the print-outs.

Wilson, Durham: I have a comment and a question. First I note that your TGF beta levels were associated with BMI, and your African American patients had a high BMI. Secondly, the question of whether ethnicity is simply a marker for something else, such as psychosocial stress, looking at some of the studies from Steele and others from Stanford and other places. My question, is TGF beta associated at all with psychosocial stressors? Again, from the genetic standpoint, looking at most African Americans in this country, that they are obviously genetically diverse, and certainly, if you were looking at a neighborhood study in New York or any other large city, you would be looking at African Americans of multiple backgrounds.

August, New York: Great question! Our statistician for this study is very interested in socioeconomic status and things like that, and we do have some data which we haven’t analyzed yet; but I can’t tell you per se today whether there is a relationship between TFG beta and things like that. We haven’t really correlated with markers of stress, but its certainly an important dimension to this whole story that shouldn’t be overlooked.