diabetes is the most frequent cause of end - stage renal disease in industrialised countries [ 1 , 2 ] . clinically , diabetic nephropathy is characterized by the development of albuminuria and a subsequent decline in glomerular filtration rate . this severe complication significantly influences the risk of cardiovascular disease as well as mortality and quality of life [ 3 , 4 ] . at present the most important identified risk factors are diabetes duration , arterial blood pressure , and glycaemic regulation . as evident from an increasing incidence of affected patients , however , there is still a great need for new strategies in the treatment and prevention of diabetic nephropathy . clear evidence indicates that the pathogenesis of diabetic nephropathy is multifactorial and triggered by a complex series of pathophysiological events . the inflammatory response in diabetes is highly complex involving proinflammatory cytokines and chemokines , for example , il1 the impact of complement activation on the diabetic kidney may well , in part , be mediated through induction of cytokine response and inflammation [ 911 ] . several studies have linked diabetic late - complications to the complement system of the innate immune system [ 12 , 13 ] . the complement system plays a crucial role in recognition and clearance of infectious microbes and the system forms a link between innate and adaptive immunity . the activation of the complement system results in the release of multiple inflammatory signaling molecules . ultimately complement activation leads to the formation of pore - forming membrane attack complexes ( macs ) that are inserted in the cell membranes to mediate lyses of the cell through osmotic stress . however , in mammalian cells , it has been shown that sublytic amounts of mac can increase production of il-8 and monocyte chemoattractant protein 1 dependent on nfb nuclear translocation . furthermore , macs are shown to have a mitogenic effect and cause release of basic fibroblast growth factor and platelet - derived growth factor from endothelial cells leading to fibrosis in neighboring cells including glomerular mesangial cells [ 16 , 17 ] . the latter effects of mac may explain the link between complement and diabetic kidney damage . three activations pathways exist : the classical , the alternative , and the lectin pathway . the present paper focuses on the lectin pathway , in which at least five soluble pattern - recognition molecules are characterized that may activate the complement system , that is , mannan - binding lectin ( mbl ) , h - ficolin , l - ficolin , m - ficolin , and collectin - k1 . the three ficolins utilize a fibrinogen - like domain that binds , for example , n - acetylglucosamine , n - acetylgalactosamine , and n - acetyl - neuraminic acid , whereas mbl and cl - k1 have a carbohydrate recognition domain and through this bind specifically to patterns of monosaccharides . a very recent publication reports a close association between ficolin and diabetic nephropathy in patients with type 1 diabetes . the observational design of the study , however , limits its ability to study a cause - effect relationship . when ficolins bind they all initiate activation of associated serine proteases ( mbl associated serine proteases , masps ) , which subsequently cleave the complement factors , c2 and c4 , leading to further complement activation [ 20 , 21 ] . eventually , complement activation leads to the formation of macs causing cell lyses or induction of fibrosis . the balance between activation and inhibition of the complement cascade is tightly controlled by regulatory proteins in order to prevent damage of healthy host cells . in diabetes , inappropriate effects of the complement system may be present as glycation - induced dysfunction of the complement inhibitory mechanism and consequently overactivation of the system is indicated [ 2325 ] . it is speculated that diabetic patients are exposed to uncontrolled complement attack partly due to altered molecular patterns on the cell surfaces as a consequence of high blood glucose [ 24 , 25 ] . most significantly , an association is seen between diabetic nephropathy and the lectin pathway [ 2628 ] . we have previously demonstrated direct cause - effect relationship between presence of mbl and worsening of kidney injury in a mouse model of diabetic nephropathy [ 29 , 30 ] . we speculate that ficolins also exert detrimental effects in diabetes similar to mbl through activation of the lectin pathway as indicated in patients with type 1 diabetes . this study aimed to investigate the impact of ficolin b ( the orthologue to human m - ficolin ) on the development of diabetic nephropathy in a mouse model of type 1 diabetes . we used 11-week - old , female ficolin b knockout mice and age - matched , female c57bl/6j bomtac wild - type mice ( taconic , ry , denmark ) . the knockout ficolin b model was backcrossed more than 10 generations to a c57bl/6j bomtac genetic background ( own breeding ) . in each cage there were three to eight mice and they had free access to tap water and standard chow ( altromin number 1324 ; lage , germany ) . the environment was stable with a 12-hour light - dark cycle , temperature at 21  1c , and humidity of 55  5% . the ficolin b knockout mice and the wild - type mice were randomized into a diabetic and nondiabetic group ; thus four groups were made : ( 1 ) diabetic knockout mice ( n = 6 ) , ( 2 ) nondiabetic knockout mice ( n = 7 ) , ( 3 ) diabetic wild - type mice ( n = 11 ) , and ( 4 ) nondiabetic wild - type mice ( n = 11 ) . diabetes was induced by intraperitoneal injections of streptozotocin ( stz ) dissolved in a cold 10  mm citrate buffer ( doses of 55  mg / kg body weight , sigma aldrich , st louis , mo , usa ) on five consecutive days . the 18-week experiment was initiated when the mice were classified as diabetic ( blood glucose > 15  mm ) . animals with more than 15% sustained weight loss , signs of illness , or persistent ketonuria were excluded from the study . blood glucose was measured from tail vein by contour ( bayer diabetes care , kgs . lyngby , denmark ) . with combur test d strip ( roche diagnostics gmbh , mannheim , germany ) two mice from each diabetic group were excluded because of insufficient increase in blood glucose levels . furthermore , two mice from the diabetic knockout group were excluded because of weight loss > 15% of body weight . the excluded mice were not included in the number of animals per group indicated above . spot urine was collected in eppendorf tubes on five consecutive days prior to sacrifice of the animals . the blood samples were drawn from under the tongue at baseline and from the retroorbital venous plexus at study end and collected in potassium edta tubes ( sarstedt , nmbrecht , germany ) . the animals were anesthetized by an intraperitoneal dose of ketamine at 0.5  mg / g body weight and xylazine at 0.2  mg / g body weight ( ketaminol 4 vet and narcoxyl vet , resp . , urinary albumin excretion was determined by mouse albumin elisa quantification kit ( bethyl laboratories , inc . , urine creatinine was measured by isocratic high - performance liquid chromatography ( hplc ) on a zorbax scx300 column ( agilent , usa ) using a slight modification of a method first reported by yuen et al . . in brief , 5  l urine was added to 100  l acetonitrile containing 0.5% acetic acid and vortexed for 15 seconds to extract the creatinine . after 15  min of 20c storage and centrifugation the supernatants were evaporated and then reconstituted with 25  l 5  mm sodium acetate , ph 4.1 . duplicate samples ( 10  ul each ) were fractionated on a 50  mm  2.1  mm zorbax scx300 column with an in - front scx guard column . isocratic hplc was performed at a flow rate of 1  ml / min , and uv absorbance was monitored at 225  nm . a standard curve was created by including a 2-fold dilution series of creatinine anhydrous ( sigma aldrich ) . this study was designed with two independent factors ; diabetes / nondiabetes and knockout / wild - type and thus analysed by two - way anova for normal distributed variable with equal variance . the main focus of interest was the interaction between the diabetic factor and the knockout factor ; that is , does ficolin b modify the effects of diabetes on the effect parameters ? if no interaction was found , the independent effects of diabetes and ficolin b on the kidney were estimated . for pairwise comparison , normal distributed data was tested with student 's t - test , whereas otherwise the wilcoxon mann - whitney rank sum test was used . data are given as mean ( 95% confidence interval ( ci ) ) unless else is stated . at baseline , the knockout mice on average weighed 20.0  g , which was slightly less than the wild type mice , 20.8  g ( p = 0.04 ) . no difference was found between the two diabetic groups or between the two nondiabetic groups ( table 1 ) . after 18 weeks an expected difference in body weight was observed between the diabetic and the nondiabetic mice independently of knockout status ( p < 0.001 ) . the nondiabetic mice weighed 3.2  g ( ci : 2.0  g4.3  g ) more than the diabetic mice . furthermore the diabetic knockout mice were significantly smaller than the diabetic wild type ( p < 0.05 ) . as presented in table 1 , blood glucose , estimated as area under the curve ( auc ) , did not differ between the two diabetic groups ( p = 0.69 ) or between the two nondiabetic groups ( p = 0.13 ) . the kidney weight was equally increased in diabetic wild - type mice , 24% ( ci : 13%36% ) , and in the diabetic knockout mice , 29% ( ci : 12%47% ) , compared to the respective control groups ( figure 2(a ) ) . no interaction between knockout and diabetes was found ( p = 0.60 ) , indicating that wild - type and knockout mice develop the same degree of diabetes - induced renal hypertrophy . the considerable body weight difference between the two diabetic groups at study end indicated that the kidney weight was to be normalised to the body weight . ficolin b did not modify the diabetes - induced increase in kidney weight when testing for interaction ( p = 0.11 ) . furthermore , no significant statistical difference was found in kidney weight per body weight between the diabetic wild - type , 1.95  mg / g , and the diabetic knockout , 2.89  mg / g ( p = 0.09 ) . the albumin - to - creatinine ration ( acr ) was higher among the diabetic wild - type mice , 76  mg / g ( ci : 50103  mg / g ) , compared to the nondiabetic wild - type mice , 44  mg / g ( ci : 2563  mg / g ) , p = 0.07 . similarly , the acr of diabetic knockout mice was 96  mg / g ( ci : 71122  mg / g ) compared to the nondiabetic knockout group , 34  mg / g ( ci : 2344  mg / g ) , p < 0.001 . as depicted in figure 3 no interaction was observed between diabetes and ficolin b knockout , p = 0.21 . in the present study we found no association between diabetes - induced kidney changes and the presence of ficolin b. we conclude that ficolin b is not responsible for , or a crucial contributory factor in , the pathophysiology of diabetic nephropathy . in our study , the kidney weight and to some extent the acr were altered by diabetes as expected . the diabetes - induced increase in kidney weight , measured by comparing the diabetic mice with the nondiabetic mice , was not statistically different between the wild - type and ficolin b knockout mice . the diabetes - induced increase in kidney weight was 24% in the wild - type mice and 29% in the ficolin b knockout mice . taking the lower body weight of the knockout mice into account , the difference in renal hypertrophy was still insignificant when comparing the wild - type mice and the ficolin b knockout mice . similarly , the diabetic change seen in acr was not altered in the absence of ficolin b. the experimental setup including four groups matched on age , body weight , and genetical background was a strength to the study , as the diabetes factor and the knockout factor were the only modulators of the outcome . most importantly both diabetic groups did reach and sustain blood glucose levels of above 15  mm . at study end , the body weight differed among groups , which impeded the analyses of the diabetic kidney damage , because the knockout mice appear to be more vulnerable to type 1 diabetes mellitus . our study provides important new information on the association between the lectin pathway and diabetic kidney damage . we are the first to investigate the role of ficolin b ( which corresponds to ficolin m in human ) in the inflammatory response of diabetic nephropathy . in mice with deficiency of mbl , the classical functional and physical renal changes normally seen in this experimental model of type 1 diabetes were modified [ 29 , 30 ] . the fact that ficolin b does not appear to modulate diabetic effects on the kidney emphasizes the importance of mbl compared with ficolin b. both mbl and ficolin b activate the lectin pathway of the complement system , but only deficiency of mbl has been shown to protect against diabetic kidney damage . this indicates that the role of the lectin pathway in the development of diabetic nephropathy is complex and may depend on the specific carbohydrate - binding properties of mbl as previously described . the function of other complement factors in the first parts of the lectin pathway ( e.g. , ficolin a and masps ) in the pathology of diabetic kidney disease remains unknown and must be explored in further studies . one study indicates that ficolin a and ficolin b exert a cooperatively defensive role in destroying streptococcus pneumoniae , suggesting a synergetic immunological effect . this emphasises the need for further investigations involving both mouse ficolins . in order to fully understand the involvement of the lectin pathway in the development of diabetic nephropathy , an additional parallel experiment with masps is of particular interest given that they represent the limiting downward step in the complement activation in conclusion , this study demonstrates that ficolin b does not modify the kidney weight and acr in a type 1 diabetes mouse model . this indicates that the role of the lectin pathway in the development of diabetic nephropathy is specific and that hyperglycaemia - induced glycations on renal cells may be more prone to bind mbl than ficolin b.
background . 
 the innate immune system may have adverse effects in diabetes and cardiovascular disease . 
 the complement system seems to play a key role through erroneous complement activation via hyperglycaemia - induced neoepitopes . 
 recently mannan - binding lectin ( mbl ) was shown to worsen diabetic kidney changes . 
 we hypothesize that mouse ficolin b exerts detrimental effects in the diabetic kidney as seen for mbl . 
 methods . 
 we induced diabetes with streptozotocin in female wild - type mice and ficolin b knockout mice and included two similar nondiabetic groups . 
 renal hypertrophy and excretion of urinary albumin and creatinine were quantified to assess diabetic kidney damage . 
 results . in the wild - type groups , 
 the kidney weighed 24% more in the diabetic mice compared to the controls . the diabetes - induced increase in kidney weight was 29% in the ficolin b knockout mice , that is , equal to wild - type animals ( two - way anova , p = 0.60 ) . in the wild - type mice the albumin - to - creatinine ratio ( acr ) was 32.5 
 mg / g higher in the diabetic mice compared to the controls . 
 the difference was 62.5  mg / g in the ficolin b knockout mice , but this was not significantly different from the wild - type animals ( two - way anova , p = 0.21 ) . 
 conclusions . in conclusion , 
 the diabetes - induced effects on kidney weight and acr were not modified by the presence or absence of ficolin b.