Serum Concentrations of Markers of TNF␣ and Fas-Mediated Pathways and Renal Function in Nonproteinuric Patients with Type 1 Diabetes Monika A. Niewczas,*†‡ Linda H. Ficociello,* Amanda C. Johnson,* William Walker,* Elizabeth T. Rosolowsky,*§ Bijan Roshan,* James H. Warram,* and Andrzej S. Krolewski*† *Research Division of Joslin Diabetes Center, Boston, Massachusetts; †Department of Medicine, Harvard Medical School, Boston Massachusetts; ‡Department of Immunology, Transplant Medicine and Internal Diseases, Warsaw Medical University, Warsaw, Poland; and §Division of Endocrinology, Children’s Hospital, Boston, Massachusetts Background and objectives: The aim of our study was to examine serum markers of the TNF and Fas pathways for association with cystatin-C based estimated glomerular filtration rate (cC-GFR) in subjects with type 1 diabetes (T1DM) and no proteinuria. Design, setting, participants, & measurements: The study group (the 2nd Joslin Kidney Study) comprised patients with Impaired renal function (cC-GFR <90 .(304 ؍ or microalbuminuria (MA) (n (363 ؍ T1DM and normoalbuminuria (NA) (n ml/min) was present in only 10% of patients with NA and 36% of those with MA. We measured markers of the tumor necrosis factor ␣ (TNF␣) pathway [TNF␣, soluble TNF receptor 1 (sTNFR1), and 2 (sTNFR2)], its downstream effectors [soluble intercellular and soluble vascular adhesion molecules (sICAM-1 and sVCAM-1), interleukin 8 (IL8/CXCL8), monocytes chemoattractant protein-1 (MCP1), and IFN␥ inducible protein-10 (IP10/CXCL10)], the Fas pathway [soluble Fas (sFas) and Fas ligand (sFasL)], CRP, and IL6. Results: Of these, TNF␣, sTNFRs, sFas, sICAM-1, and sIP10 were associated with cC-GFR. However, only the TNF receptors and sFas were associated with cC-GFR in multivariate analysis. Variation in the concentration of the TNF receptors had a much stronger impact on GFR than clinical covariates such as age and albumin excretion. Conclusions: Elevated concentrations of serum markers of the TNF␣ and Fas-pathways are strongly associated with decreased renal function in nonproteinuric type 1 diabetic patients. These effects are independent of those of urinary albumin excretion. Follow-up studies are needed to characterize the role of these markers in early progressive renal function decline. Clin J Am Soc Nephrol 4: 62–70, 2009. doi: 10.2215/CJN.03010608 he traditional model of the development of end-stage Low-grade chronic inflammation is thought to be involved in renal disease in type 1 diabetes mellitus (T1DM), in the pathogenesis of diabetic nephropathy (3,4). Tumor necrosis T which microalbuminuria (MA) leads to proteinuria and factor alpha (TNF␣/TNF) is a key mediator of inflammation then proteinuria is followed by renal function loss, has been and plays a role in apoptosis. In animal models, its effects on challenged recently. Increase in urinary albumin excretion is an kidneys include reduced glomerular filtration rate (GFR) and important determinant of diabetic nephropathy progression, increased albumin permeability (3). It mediates its signal via but it does not entirely explain this phenomenon. For example, two distinct receptors, TNF receptor 1 (TNFR1/TNFRSF1A) the loss of renal function commences earlier than previously and TNF receptor 2 (TNFR2/TNFRSF1B), which are mem- recognized and precedes the onset of proteinuria (1). In our brane-bound and also present in soluble form in serum (5). longitudinal study of T1DM (The First Joslin Study of Natural TNF␣ mediates its inflammatory effects by induction of a broad History of Microalbuminuria), renal function decline began spectrum of chemokines, including interleukin 8 (IL8/CXCL8); with the onset of MA in about one-third of patients and pro- monocyte chemotactic protein-1 (MCP-1/CCL2); IFN-␥ induc- gressed in a linear fashion from normal kidney function to renal ible protein-10 (IP-10/CXCL10); and adhesion molecules such insufficiency. Also, renal function decline occurred in a notice- as intercellular adhesion molecule-1 (ICAM-1) and vascular able proportion of patients with T1DM and normal albumin adhesion molecule-1 (VCAM-1) (6,7). excretion (1,2). The Fas pathway mediates apoptosis and may play a role in the progression of diabetic nephropathy (8–11). The binding of Received June 18, 2008. Accepted September 8, 2008. Fas ligand (FasL) to Fas, its membrane-bound receptor that is also present in serum in soluble form (sFasL, sFas), leads to an Published online ahead of print. Publication date available at www.cjasn.org. apoptotic response (12,13). Correspondence: Dr. Andrzej S. Krolewski, Section on Genetics and Epidemiol- ␣ ogy, Joslin Diabetes Center, One Joslin Place, Boston, MA, 02215. Phone: 617-732- Most studies on serum markers of TNF -mediated inflam- 2668; Fax: 617-732-2667; E-mail: [email protected] mation and apoptosis in diabetic nephropathy have explored Copyright © 2009 by the American Society of Nephrology ISSN: 1555-9041/401–0062 Clin J Am Soc Nephrol 4: 62–70, 2009 Markers of TNF, Fas Pathways and Renal Function 63 Table 1. Characteristics of the study group according to albuminuria statusa Characteristics Normoalbuminuria (n ϭ 363) Microalbuminuria (n ϭ 304) P AERb (␮g/min) 15 (11 to 21) 69 (45 to 131) By design Age (yr) 39 Ϯ 12 41 Ϯ 12 Ͻ0.05 Male (%) 44% 61% Ͻ0.0001 Diabetes duration (yr) 20 Ϯ 923Ϯ 10 Ͻ0.0001 HbA1cc (%) 8.3 Ϯ 1.2 8.6 Ϯ 1.5 Ͻ0.01 cC-GFRd (ml/min/1.73 m2) 118 Ϯ 24 99 Ϯ 27 Ͻ0.0001 cC-GFR categories: Ͼ130 ml/min 30% 10% 90 to 130 61% 54% 60 to 89 9% 28% Ͻ60 1% 8% aData are mean Ϯ SD, median (quartiles), or percent. bAER: median albumin excretion rate during the preceding 2-yr window cHbA1c: mean hemoglobin A1c during the preceding 2-yr window dcC-GFR: estimated GFR based on serum cystatin-C their association with MA and proteinuria rather than with be more effective if implemented 5 to 10 yr earlier in the disease GFR (14). course. The goal of this large cross-sectional study was to investigate In this study, the GFR was estimated by a cystatin C-based whether serum concentrations of markers mediated by TNF␣ formula (cC-GFR), previously shown as an accurate way of (sTNFR1, sTNFR2, sICAM-1, sVCAM-1, IL8, MCP-1, IP-10) or evaluating renal function in patients with diabetes (15,16) involved in Fas-related apoptosis (sFasL and sFas) are associ- ated, independently from albuminuria, with variation in renal Materials and Methods function in patients with T1DM who do not have proteinuria or The Committee on Human Subjects of the Joslin Diabetes Center advanced renal function impairment. This knowledge should approved the protocol and informed consent procedures for this study. facilitate the development of new diagnostic tools for identify- The study group was selected from the population attending the ing patients with early renal function decline and help the Joslin Clinic, a major center for the treatment of patients of all ages with search for intervention protocols for high-risk patients that may T1DM or type 2 diabetes mellitus (T2DM). The population is about 90% Table 2. Characteristics of the study group according to albuminuria status and group-specific median cC-GFRa Variable Normoalbuminuria Microalbuminuria Group Contrast Characteristic cC-GFR Ͼ 115 cC-GFR Ͻ 115 cC-GFR Ͼ 101 cC-GFR Ͻ 101 AER cC-GFR N 183 180 152 152 Pb Pc AER (␮g/min) 13 (10-18) 18 (12-23) 56 (42-100) 85 (51-161) By Design Ͻ0.0001 Age (yr) 37 Ϯ 11 40 Ϯ 13 36 Ϯ 12 45 Ϯ 11 Ͻ0.05 Ͻ0.0001d Diabetes duration (yr) 19 Ϯ 921Ϯ 10 20 Ϯ 926Ϯ 9 Ͻ0.0001 Ͻ0.0001d HbA1c (%) 8.3 Ϯ 1.2 8.3 Ϯ 1.2 8.7 Ϯ 1.6 8.4 Ϯ 1.4 Ͻ0.005 NS Body mass index (kg/m2) 25.6 Ϯ 3.6 26.7 Ϯ 4.3 27.2 Ϯ 4.8 27.7 Ϯ 5.2 Ͻ0.0005 Ͻ0.05 Systolic blood pressure 118 Ϯ 12 120 Ϯ 13 124 Ϯ 12 125 Ϯ 15 Ͻ0.0001 NS (mmHg) ACEI or ARB Rx (%)d 18% 21% 49% 55% Ͻ0.0001 NS Antihypertensive Rx (%) 7% 16% 14% 30% Ͻ0.001 Ͻ0.0001 Serum cholesterol 183 Ϯ 29 181 Ϯ 29 190 Ϯ 33 193 Ϯ 30 Ͻ0.0001 NS (mg/dl) Lipid-lowering Rx (%) 24% 34% 31% 42% Ͻ0.05 Ͻ0.005 Current smoker (%) 9% 12% 19% 18% Ͻ0.005 NS aData are mean Ϯ SD, median (quartiles), or %. bP-value for the albuminuria main effect in an ANOVA cP-value for the cC-GFR main effect in an ANOVA. dACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; Rx, treatment. 64 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 4: 62–70, 2009 Table 3. Serum concentrations of markers of inflammation or apoptosis according to AER group and cC-GFR above or below mediana Normoalbuminuria Microalbuminuria Group Contrast Variable cC-GFR Ͼ 115 cC-GFR Ͻ 115 cC-GFR Ͼ 101 cC-GFR Ͻ 101 AER cC-GFR (n ϭ 182) (n ϭ 181) (n ϭ 152) (n ϭ 152) Pb Pc TNF-mediated pathway TNF␣ ( pg/ml) 3.6 (2.3, 4.8) 3.9 (2.8, 5.8) 4.0 (2.6, 5.4) 4.8 (3.3, 6.4) NS Ͻ0.005 sTNFR1 (pg/ml) 1.2 (1.0, 1.4) 1.4 (1.2, 1.7) 1.4 (1.2, 1.6) 2.0 (1.6, 2.5) Ͻ0.0001 Ͻ0.0001 sTNFR2 (ng/ml) 2.1 (1.7, 2.6) 2.6 (2.1, 3.6) 2.3 (1.9, 2.9) 3.2 (2.5, 5.4) Ͻ0.0001 Ͻ0.0001 Potential downstream effectors Chemokines IL8 (pg/ml) 4.4 (2.4, 10.4) 6.1 (3.4, 13.3) 7.6 (3.8, 18.3) 7.0 (4.0, 15.5) Ͻ0.05 NS IP-10 (pg/ml) 107 (79, 136) 122 (88, 171) 102 (75, 141) 115 (80, 158) NS Ͻ0.001 MCP-1 (pg/ml) 124 (75, 184) 120 (77, 184) 113 (78, 191) 105 (77, 174) NS NS Adhesion molecule sICAM-1 (ng/ml) 133 (109, 152) 137 (119, 169) 149 (123, 173) 152 (123, 191) Ͻ0.0005 Ͻ0.005 sVCAM-1 (ng/ml) 386 (301, 481) 389 (303, 489) 376 (295, 467) 394 (330, 495) NS NS Fas-mediated pathway sFasL (pg/ml) 0.00.12 (0.08, 0.19) 0.13 (0.07, 0.20) 0.12 (08, 0.18) 0.11 (0.06, 0.16) NS NS sFas (ng/ml) 3.8 (3.0, 4.7) 4.5 (3.7, 5.5) 4.5 (3.6, 5.6) 5.4 (3.7, 6.9) Ͻ0.0001 Ͻ0.0001 Other inflammatory markers CRP (␮g/ml) 1.2 (0.5, 3.2) 1.1 (0.6, 2.7) 1.4 (0.5, 3.9) 1.6 (0.8, 3.2) Ͻ0.05 NS IL6 (pg/ml) 0.8 (0.6, 1.4) 0.9 (0.7, 1.5) 0.8 (0.4, 1.3) 0.9 (0.6, 2.2) NS NS aData are medians (quartiles); analyses were done on concentrations transformed to their logarithms.