Age dependent shifts in renal response to injury relate 1 to altered BMP 6 / CTGF expression and signaling 2 3

33 Age is associated with an increased prevalence of chronic kidney disease (CKD), which, 34 through progressive tissue damage and fibrosis, ultimately leads to loss of kidney 35 function. Although much effort is put into studying CKD development experimentally, 36 age has rarely been taken into account. Therefore, we investigated the effect of age on 37 the development of renal tissue damage and fibrosis in a mouse model of obstructive 38 nephropathy (i.e. unilateral ureter obstruction; UUO). We observed that after 14 days, 39 obstructed kidneys of old mice had more tubulointerstitial atrophic damage but less 40 fibrosis than those of young mice. This was associated with reduced connective tissue 41 growth factor (CTGF), and higher BMP6 expression and pSMAD1/5/8 signaling, while 42 TGF-β expression and transcriptional activity were no different in obstructed kidneys of 43 old and young mice. In vitro, CTGF bound to and inhibited BMP6 activity. 44 In summary, our data suggest that in obstructive nephropathy atrophy increases and 45 fibrosis decreases with age, and that this relates to increased BMP signaling, most likely 46 due to higher BMP6 and lower CTGF expression. 47 Lucas L. Falke Abbreviations: 48 BMP (6/7) Bone Morphogenetic Protein (family member 6 or 7) 49 CTGF Connective Tissue Growth Factor 50 TGFβ Transforming Growth Factor β 51 UUO Unilateral Ureter Obstruction 52 CLK Contralateral Kidney 53 OBK Obstructed Kidney 54 CKD Chronic Kidney Disease 55 KW Kidney Weight 56 BW Body Weight 57 Lucas L. Falke Chronic kidney disease (CKD) primarily affects the aging population (11, 37, 42). Decline 58 of kidney function with age might result from multifactorial changes in kidney 59 physiology primarily due to “senescence” itself, and a more adverse response to injury 60 (1, 5, 22, 43, 45, 47). As such, it has been proposed that the aging kidney increasingly 61 accumulates extracellular matrix, leading to glomerulosclerosis and interstitial fibrosis 62 (1, 25) and ultimately to loss of renal mass and reduced glomerular and tubular function 63 (17, 51). Parameters such as diabetes, vitamin D deficiency, alterations in Renin 64 Angiotensin and Aldosteron (RAAS) signaling, oxidative stress by reactive oxygen 65 species (ROS) production and advanced glycation end (AGE) products are thought to 66 underlie these morphological and functional changes of the kidney parenchyma (8). 67 Furthermore, the age associated gradual decline in Klotho, a transmembrane FGF23 co68 receptor expressed in the convoluted distal tubule under physiological condition 69 renders kidneys more susceptible to injury, dysregulation of mineral homeostasis and 70 fibrosis (28). 71 In young mice, kidney response to injury is profoundly influenced by the 72 Transforming Growth Factor β-superfamily (TGF β), including TGFβ1, Bone 73 Morphogenetic Protein 7 (BMP7), and BMP6 (14, 35). Canonical signaling of the TGF β 74 superfamily is accomplished via Activin like kinase (ALK) activation and subsequent 75 downstream SMAD phosphorylation. Activation of ALK4, 5 or 7 by TGFβ leads to fibrosis 76 associated SMAD2/3 phosphorylation. ALK1-3 or 6 activation by BMP’s leads to 77 renoprotection associated SMAD1/5/8 phosphorylation (10). Under specific conditions, 78 TGFβ has been shown to activate ALK1 (36). Upon injury, TGFβ levels rise and BMP 79 levels drop rapidly (34). The shift in these factors is regarded as major early event in 80 ensuing tissue damage with subsequent fibrosis following injury. 81 Lucas L. Falke Connective Tissue Growth Factor is another immediate early factor shown to 82 greatly influence the response to kidney injury. CTGF is a matricellular protein involved 83 in various fibrosis associated phenomena such as extracellular matrix production, 84 proliferation and myofibroblast differentiation (19). Although no exclusive CTGF 85 receptor has been identified, CTGF has been known to interact with TGFβ and BMP7 86 thereby modulating signaling in favor of pro-fibrotic TGFβ whilst inhibiting BMP7 87 signaling (2, 35). As such, TGFβ, BMP6 and 7, and CTGF are regarded as major factors 88 influencing the renal response to injury. The age associated production of reaction 89 oxygen species (ROS) is related to increased levels of pro-fibrotic growth factors (41), 90 but little is known about the impact of aging on the regulation of these factors in the 91 kidney. It has been reported that pro-fibrotic TGFβ signaling generally increases with 92 age, which might at least in part be due to the gradual decline of Klotho (15). 93 The effect of ageing on renal CTGF expression is largely unknown, but CTGF/CCN2 was 94 found to be reduced in aged skin in association with loss of collagen (39). Interestingly, 95 conditional overexpression of CTGF prevented age-related degenerative changes in 96 epiphyseal cartilage of rats (27). In contrast, ageing is associated with increased cardiac 97 CTGF expression in mice (40, 48), suggesting that age related differential expression of 98 CTGF is context dependent. 99 Reports on age-associated changes in expression and signaling of the (anti100 fibrotic) BMPs are scarce, but it has been noted that BMP7 expression declines in aging 101 cartilage (3), while increased expression of BMP6 was found in Alzheimer brains (12), 102 and early aging in Klotho-deficient mice was associated with increased BMP-signaling 103 and vascular calcification (23). To the best of our knowledge, there are no previous data 104 on age-related changes in renal BMP expression and signaling. Based on our find in the 105 Lucas L. Falke current study, we hypothesize that old and young kidneys respond differently to injury 106 in association with differential TGFβ/BMP/CTGF signaling. 107 Unilateral Ureter Obstruction (UUO) is a commonly used model of renal injury 108 characterized by inflammation, extensive morphological damage and fibrosis (7, 29). 109 Upon obstruction, the quick rise in TGFβ levels and subsequent phosphorylation of 110 SMAD2/3 and PAI1 upregulation are regarded as key events ultimately leading to 111 fibrosis (26, 44). We studied morphological damage and fibrosis following 14 days of 112 UUO and observed a shift from a largely fibrotic phenotype in young, to a more atrophic 113 phenotype of tubulointerstitial damage in old kidneys, although BMP7 and TGFβ were 114 not different. This phenotypic shift might derive from a synergistic effect of the observed 115 decrease of CTGF and increase of BMP6, even more so since we noted in vitro that CTGF 116 directly binds to BMP6 and inhibits its signaling activity. Together these findings 117 provide further understanding of the age associated response to injury, and might help 118 to identify better diagnostic methods and therapeutic interventions in the aging 119 population. 120