Am J Physiol Renal Physiol 302: F647–F657, 2012. First published December 14, 2011; doi:10.1152/ajprenal.00090.2011. Translational Physiology Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy Maria-Dolores Sanchez-Niño,3 Milica Bozic,1 Elizabeth Córdoba-Lanús,4 Petya Valcheva,1 Olga Gracia,1 Merce Ibarz,2 Elvira Fernandez,1 Juan F. Navarro-Gonzalez,4* Alberto Ortiz,3* and Jose Manuel Valdivielso1* 1Research Laboratory and Nephrology Department, 2Biochemistry Department, Hospital Universitari Arnau de Vilanova, IRBLLEIDA, Lleida; 3Nefrología, IIS-Fundación Jiménez Díaz, Universidad Autonoma de Madrid and Instituto Reina Sofia de Investigaciones Nefrológicas-IRSIN, Madrid; and 4Servicio de Nefrología y Unidad de Investigación, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain Submitted 8 February 2011; accepted in final form 7 December 2011 Sanchez-Niño M, Bozic M, Córdoba-Lanús E, Valcheva P, eventual decrease in GFR. Damage to highly differentiated Gracia O, Ibarz M, Fernandez E, Navarro-Gonzalez JF, Ortiz A, glomerular podocytes is thought to be an early event in DN Valdivielso JM. Beyond proteinuria: VDR activation reduces renal (35). In recent years, a direct association between inflammatory inflammation in experimental diabetic nephropathy. Am J Physiol parameters and clinical markers of glomerular as well as Renal Physiol 302: F647–F657, 2012. First published December 14, tubulointerstitial damage has been demonstrated, suggesting 2011; doi:10.1152/ajprenal.00090.2011.—Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D that inflammation may be a pathogenic factor for the develop- ment and progression of DN (22). Hyperglycemia, angiotensin receptor (VDR) activator paricalcitol has an antiproteinuric effect in ␤ human diabetic nephropathy at high doses. We have explored poten- II, transforming growth factor (TGF)- 1, and proteinuria itself tial anti-inflammatory effects of VDR activator doses that do not all play roles in stimulating renal inflammation and/or fibrosis modulate proteinuria in an experimental model of diabetic nephrop- and contribute to progression of DN. athy to gain insights into potential benefits of VDR activators in those Multiple factors have been implicated in the progression of patients whose proteinuria is not decreased by this therapy. The effect DN, but inflammatory cytokines may be critical in the devel- of calcitriol and paricalcitol on renal function, albuminuria, and renal opment of diabetic complications and nephropathy. Inflamma- inflammation was explored in a rat experimental model of diabetes tory markers such as IL-6, IL-18, monocyte chemoattractant induced by streptozotocin. Modulation of the expression of mediators protein-1 (MCP-1), and TNF-␣ are increased in the serum and of inflammation by these drugs was explored in cultured podocytes. urine of patients with diabetes and DN (22). Furthermore, At the doses used, neither calcitriol nor paricalcitol significantly urinary TNF-␣ is directly related to UAE, and strategies modified renal function or reduced albuminuria in experimental dia- ␣ betes. However, both drugs reduced the total kidney mRNA expres- modulating TNF- activity are associated with antialbuminuric sion of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. effects. This increase in inflammatory parameters occurs early Immunohistochemistry showed that calcitriol and paricalcitol reduced in the disease, correlates with the degree of albuminuria, and MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular even precedes the increase in UAE (18, 37). Resident cells, infiltration by macrophages, glomerular cell NF-␬B activation, apo- such as mesangial, tubular epithelial, and podocytes, can pro- ptosis, and extracellular matrix deposition. In cultured podocytes, duce cytokines and can express molecules that are part of the paricalcitol and calcitriol at concentrations in the physiological and costimulatory pathway (30). Experimental animal models have clinically significant range prevented the increase in MCP-1, IL-6, recently provided evidence that inflammatory molecules, in- renin, and fibronectin mRNA expression and the secretion of MCP-1 cluding TNF-␣ and MCP-1, may have a causative role in the to the culture media induced by high glucose. In conclusion, in development of DN (22). experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria The vitamin D receptor (VDR) is a ligand-activated tran- is not decreased. This may be ascribed to decreased inflammatory scription factor which, after activation, recruits cofactor mol- responses of intrinsic renal cells, including podocytes, to high glucose. ecules and binds to specific DNA binding sites to modify the expression of target genes (34). There is evidence that VDR is glomerular disease; interleukins; microalbuminuria a modulator of glomerular injury. Calcitriol, the endogenous VDR ligand, decreases the glomerulosclerosis index and UAE in rats with subtotal nephrectomy (13, 31), and mice lacking DIABETIC NEPHROPATHY (DN) is the leading cause of end-stage renal disease in the Western world. Diabetes is associated with the VDR are more susceptible to hyperglycemia-induced renal an initial increase in glomerular filtration rate (GFR). DN injury (39). The combination of a VDR activator and an ACE progression is characterized by the development of microalbu- inhibitor protected mice from developing DN (41). In the minuria in patients with normal renal function, followed by a clinical setting, chronic kidney disease (CKD) patients treated progressive increase in urinary albumin excretion (UAE) and with paricalcitol (a selective VDR activator) showed a signif- icant reduction of proteinuria (measured by a semiquantitative dipstick method) after 23 wk of therapy, independently of * J. F. Navarro-Gonzalez, A. Ortiz, and J. M. Valdivielso share senior GFR, blood pressure, or ACE inhibition (1). However, in authorship. clinical trials the proteinuria response to VDR activators has Address for reprint requests and other correspondence: J. M. Valdivielso, Laboratorio de Investigación IRBLLEIDA, Hospital Universitari Arnau de not been uniform, raising the question of whether patients Vilanova, Rovira Roure 80, 25198 Lleida, Spain (e-mail: Valdivielso without reduced UAE might still be benefiting from VDR @medicina.udl.es). activator therapy. Thus, in Fishbane’s trial which included 50% http://www.ajprenal.org 1931-857X/12 Copyright © 2012 the American Physiological Society F647 Translational Physiology F648 VDR ACTIVATION IN DIABETIC NEPHROPATHY Table 1. Weight and biochemical parameters in diabetic nephropathy animal model at the end of the 4-mo follow-up Sham Diabetes DiabetesϩCalcitriol DiabetesϩParicalcitol Weight, g 393 Ϯ 10 345 Ϯ 10* 343 Ϯ 7* 343 Ϯ 13* Serum calcium, mg/dl 10.53 Ϯ 0.1 10.3 Ϯ 0.2 10.6 Ϯ 0.3 10.5 Ϯ 0.2 Serum phosphate, mg/dl 7.4 Ϯ 0.4 7.1 Ϯ 0.5 6.3 Ϯ 0.5 6.5 Ϯ 0.6 Serum creatinine, mg/dl 0.42 Ϯ 0.01 0.43 Ϯ 0.02 0.45 Ϯ 0.04 0.44 Ϯ 0.02 Serum glucose, mg/dl 91.4 Ϯ 2.08 490 Ϯ 5* 472 Ϯ 10* 505 Ϯ 9* Urine volume, ml/day 9.7 Ϯ 1 152 Ϯ 14* 148 Ϯ 19* 132 Ϯ 15* Urinary albumin/creatinine ratio, mg/mg 0.073 Ϯ 0.019 8.3 Ϯ 1.7* 6.7 Ϯ 1.6* 7.1 Ϯ 1.2* Creatinine clearance, ml/min 2.0 Ϯ 0.14 2.0 Ϯ 0.2 1.9 Ϯ 0.1 1.8 Ϯ 0.1 Values are means Ϯ SE. *P Ͻ 0.05 vs. Sham. diabetic subjects, paricalcitol decreased UAE Ͼ10% in only However, despite podocytes being key cells in proteinuria and 58% of patients (vs. 30% in the placebo group) (11). In the DN, potential direct VDR modulation of high glucose-induced VITAL trial of DN patients, differences in the primary out- inflammation in cultured podocytes had not being studied. come (% change in urinary albumin/creatinine ratio in the Thus the aim of our study was to investigate the regulation combined paricalcitol group vs. placebo) just fell short of of renal inflammatory parameters in experimental DN by statistical significance (8). However, even in patients without subantiproteinuric doses of VDR activators and the modulation reduced proteinuria, VDR activators might have beneficial by VDR activators of the inflammatory response elicited in effects. In this regard, another potential beneficial effect of cultured murine podocytes by high glucose. VDR activation is an anti-inflammatory action. In animal models of primary glomerular disease, VDR activators reduces METHODS glomerular infiltration of inflammatory cells (23). Consistently, higher serum vitamin D levels in CKD patients are associated Animal study. Thirty male Sprague-Dawley rats were rendered with decreased systemic inflammation (33). However, no ex- diabetic by intravenous administration of streptozotocin (65 mg/kg) perimental diabetes animal study had previously addressed (7). Experimental methods used with laboratory animals comply with Law 5/1995 of June 21 by “Generalitat de Catalunya” of protection of whether VDR activators may have beneficial effects on renal animals used for experimentation and other scientific finalities and the inflammation when proteinuria is not significantly reduced. In Royal Decree 1201/2005 of October 10 on the protection of animals tubular and mesangial cells, VDR activation inhibits NF-␬B, a used for experimentation and other scientific finalities. Moreover, the key transcription factor in inflammation that regulates cyto- present protocol was approved by the Ethic Animal Experimentation kine, chemokine, and adhesion molecule gene expression (4). Committee of the University of Lleida. Fig. 1. Vitamin D receptor (VDR) activation with paricalcitol or calcitriol downregulates total kidney expression of inflammatory medi- ator mRNA in experimental diabetic nephrop- athy (DN), quantified by qRT-PCR. Values are means Ϯ SE. MCP-1, monocyte chemoattrac- tant protein-1. *P Ͻ 0.05 vs. control. #P Ͻ 0.01 vs. diabetic vehicle. ϩP Ͻ 0.05 vs. dia- betic vehicle. AJP-Renal Physiol • doi:10.1152/ajprenal.00090.2011 • www.ajprenal.org Translational Physiology VDR ACTIVATION IN DIABETIC NEPHROPATHY F649 After 48 h, the presence of hyperglycemia was confirmed and nondiabetic controls.