Chronic inflammation contributes to type 1 and type 2 diabetes mellitus and is involved in the development of cardiovascular complications 12. Microvascular complications, which are largely defined by the presence and degree of proteinuria, are associated with inflammation 3. In the case of diabetic nephropathy proteinuria and systemic inflammation define the cardio-vascular risk 4. It would be interesting to correlate these changes to the microvasculature.

Retinal vessels represent a direct non-invasive measurement of vascular lumen diameter on the level of resistance arteries, hence the microcirculatory level 5. In this respect an enlarged retinal arteriolar and venular caliber is related to diabetes and systemic inflammation 67. The dilation of the retinal vessels has been attributed to an increased production of nitric oxide as a result of up-regulated nitric oxide synthase messenger RNA, secondary to release of cytokines such as IL-1 and TNF-α 8.

Apart from these direct effects of inflammatory markers on the microcirculation in diabetic nephropathy, little is known about the contribution of inflammation-initiated transmigration of monocytes through the arteriolar wall. We hypothesize that the myeloid-related protein 8/14 complex (MRP8/14), a marker of monocyte and neutrophil activation 9, predicts microvascular changes at an early stage. MRP8/14, also termed calprotectin, leukocyte protein L1 complex or cystic fibrosis antigen, is a heterodimer of two calcium-binding proteins (S100A8 and S100A9, also referred to as MRP8 and MRP14, or calcranulin A and B involved in calcium-dependent signaling, cell differentiation, cell cycle progression, and cytoskeleton-membrane interactions 10. MRP8 and MRP14 are primarily expressed in cells of myeloid origin, particularly in monocytes and neutrophils 11. Upon phagocyte activation, MRP8 and MRP14 form the MRP8/14 complex, which translocates to the cytoskeleton and plasma membrane, where it is secreted 1213. This is an early event during transendothelial migration and thus represents an interaction of MRP-expressing neutrophils and monocytes with endothelium 14. Therefore we further hypothesize that systemic inflammation in diabetic nephropathy results in an increased transendothelial migratory activity of monocytes and neutrophils and hence microvascular changes in the glomerular and retinal vascular bed. It is the aim of this study to investigate in patients with type 2 diabetes and nephropathy whether the degree of proteinuria and retinal arteriolar lumen diameter are associated with markers of inflammation and endothelial transmigration. Furthermore we describe one patient demonstrating reduction of MRP8/14 and proteinuria under TNF-blockade, potentially mirroring the pathophysiological relevance of MRP8/14.

Forty-three patients with type 2 diabetes with nephropathy were recruited from the nephrological clinic Weissenburg. In the entire cohort, the simplified MDRD (Modification of Diet in Renal Disease) equation was used to estimate the glomerular filtration rate (eGFR (ml/min/1.73 m²) = 186 × (serum creatinine (mg/dl))^-1.154 × (age)^-0.203 × (0.742 if female) × (1.212 if black)). Proteinuria was quantified in spot urine by measurement of the protein-creatinine ratio (PCR). PCR was log-transformed to better approximate normal distributions. Additionally, cardiometabolic risk factors were evaluated and biological parameters measured, according to the recommendations of the American and European clinical practice guidelines. Anti-diabetic and anti-hypertensive medication was recorded including insulin-therapy, oral anti-diabetics, and the total number of antihypertensive drugs including differentiation of inhibitors of the renin-angiotensin system (RAS) or calcium channel blockers. The research has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association, and has been approved by the local Ethics Committee of the institution. Written informed consent was obtained from each patient after full explanation of the purpose, nature and risk of all procedures. One trained person measured anthropometric characteristics. For ≥3 hours before the examination, the participants refrained from heavy exercise, smoking, and alcohol or caffeine-containing beverages. The body mass index was weight in kilograms divided by height in meters squared. Venous blood samples, collected after overnight fasting, were analyzed by standard automated methods for lipids and blood glucose. Diabetes mellitus was defined as a fasting blood glucose level of at least 7.0 mmol/l or as the use of antidiabetic drugs.

IL-6, TNF-α (R&D Systems, Minneapolis, MN, USA) and MRP8/14 (Bühlmann Laboratories, Schönenbuch, Switzerland), were determined by commercially available ELISA using serum samples.

Our study demonstrates in patients with type 2 diabetes and nephropathy that MRP8/14, a marker for transendothelial migration of monocytes and neutrophils, may predict changes in the microvascular bed of glomeruli and retina beyond inflammation. These changes are for the glomerular bed the degree of proteinuria and in the retinal bed an increase of arteriolar lumen caliber. Thus, MRP8/14 is a potential biomarker for microvascular defects in patients with diabetic nephropathy which may represent a pathophysiological relevant feature. Furthermore, these results implicate a potential deleterious role of increased arteriolar lumen caliber if associated with inflammation and transmigration in patients with diabetic nephropathy.

Diabetic nephropathy is characterized by proteinuria and low-grade systemic inflammation 18. Both factors have been associated with microvascular complications 1 and hence predict cardiovascular outcome 2. In this context, MRP8/14 was in our study positively associated with Interleukin-6 as marker for systemic inflammation 19 and TNF-α as marker for metabolic driven inflammation 20 in patients with diabetic nephropathy. This is of relevance as sequential migration of neutrophils is stimulated by TNF-α induced inflammation and can be reduced by inhibition of Interleukin-6 secretion 21. Thus, a causal relationship with the option to modulate the degree of microvascular damage may be suggested. Besides, the degree of proteinuria and retinal arteriolar caliber as conventional and novel microvascular factors were positively associated with MRP8/14. Thus, systemic MRP8/14 values seem to reflect not only the grade of inflammation, but also may define the microcirculation as the place of action. The microcirculatory action is not restricted to one organ as glomerular and retinal effects are observed. Moreover, Alwegg et al. demonstrated the local coronary release of MRP8/14 in acute coronary syndrome 14. Thus we suggest that the MRP8/14 activity reflects a generalized microcirculatory effect of increased transendothelial migration. In contrast, there is a lack of association between arterial stiffness measured by the gold standard of aortic PWV and MRP8/14. This suggests that no dominant effect of transmigration is present in the context of the macrocirculation. This further emphasizes MRP8/14 as a novel and potentially selective marker for microcirculatory effects in diabetic nephropathy.

Studies in diabetic nephropathy are fraught with difficulty, given the recognized associations with hypertension and dyslipidemia, both of which are known to influence the microcirculation 22. It is important to notice that our cohort after stratification according to MRP8/14 remains comparable for the lipid profile and 24-hour blood pressure measurement. Therefore, it is unlikely that MRP8/14 as marker for transendothelial migration is largely influenced by these two factors. Hence, enhanced MRP8/14 may describe a pathomechanism independent of hypertension and dyslipidemia in patients with diabetic nephropathy.

Furthermore, we describe one patient in whom TNF-blockade was initiated due to an ankylosing spondylarthritis. In this patient MRP8/14 and proteinuria measurements were available before and during treatment covering a period of 18 month. The measurements showed a decrease both of transmigration and proteinuria as marker for microcirculatory damage. These observations may be interpreted as pathophysiological relevant link between inflammation, transmigration and microcirculatory alterations. However, in the future this clearly needs to be investigated in detail.

Is a larger retinal arteriolar caliber in patients with diabetic nephropathy deleterious? In patients with type 2 diabetes the myogenic responsiveness is impaired. Schofield et al. demonstrated in pressure myograph experiments using resistance arteries of type 2 diabetic subjects that above a luminal pressure of 50 mmHg no reactive contractility was present whereas control subjects gave a blood pressure dependent myogenic response 22. As a consequence wall stress may rise resulting in vascular hypertrophy 23.

In our cohort of patients with diabetic nephropathy high MRP8/14 values are associated with larger arteriolar lumen caliber, which may reflect a failure to autoregulate blood flow efficiently 24. This may increase high blood pressure flow to target organs, effecting downstream damage, as determined in our patients by increased proteinuria particularly in subjects with elevated MRP8/14 levels. The failure of blood flow autoregulation may result in enlarged preglomerular afferent arterioles. This increases intraglomerular pressure with the consequence of proteinuria 25. Secondly, capillary transmigration may directly damage podocytes thereby aggregating capillary leakage which corresponds with proteinuria 26. These options raise the new speculation whether the defective myogenic response observed in type 2 diabetes is not only regulated by metabolic factors 2728, but is also related to the degree of local inflammation and transmigration.

In summary, we demonstrate that MRP8/14 as marker for transendothelial migration describes not only the state of inflammation in diabetic nephropathy, but additionally the degree of microvascular alterations. Therefore, MRP8/14 is a potentially selective novel biomarker for microcirculatory defects, which may play a pathophysiological relevant role.

The authors declare that they have no competing interests.

KB designed, coordinated and wrote the manuscript. AB carried out the molecular genetic studies. SS performed retinal measurements. CRP performed macrocirculatory measurements.

FS carried out the MRP measurements. KK analyzed the retina measurements. ME performed statistical analysis. DS performed ELISAs. IL analyzed the retina measurements. UW designed, coordinated the manuscript. MB designed, coordinated and wrote the manuscript. All authors read and approved the final manuscript.