Introduction 1 2 3 4 Nearly 20 years on from the Steno hypothesis, the determinants of selective glomerular permeability to proteins at the cellular and molecular level are much better understood. In particular, the importance of podocyte-specific proteins in the regulation of selective permeability has been recognised. Similarly, much is now known about the biochemical derangements important in the pathogenesis of diabetic complications. We draw together these elements to consider the pathophysiological mechanisms through which diabetes exerts its effects on glomerular permeability in the initiating stages of diabetic nephropathy, i.e. at or before the appearance of microalbuminuria. These early changes establish the milieu in which the more advanced changes of overt diabetic nephropathy develop. Defining the mechanistic links from biochemical derangements to the appearance of increased urinary albumin highlights key elements in the pathophysiological pathway of the development of both diabetic nephropathy and micro- and macrovascular disease elsewhere. 5 In both general and diabetic populations, conditions associated with endothelial damage predispose to microalbuminuria 6 7 8 9 10 11 11 12 13 14 15 16 17 18  Microalbuminuria is a risk factor for macro- and microangiopathy, including advanced diabetic nephropathy 19 20 21 22 23 24  25 8 26 27 28 Generalised and glomerular endothelial dysfunction 27 29 30  1 Fig. 1 The relationship between hyperglycaemia, insulin resistance, endothelial dysfunction, macrovascular disease and microalbuminuria in type 1 and type 2 diabetes. Proposed major pathways are represented by red arrows; those of less certain significance by black arrows. The diagram illustrates, for the example, a possible mechanism for the increased risk of microalbuminuria in patients with type 1 diabetes and a susceptibility to insulin resistance. Particularly in type 2 diabetes, other pathways, not directly involving endothelial dysfunction, are likely in the pathogenesis of macrovascular disease and may also contribute to microalbuminuria (broken arrows)  The glomerular filtration barrier is a complex biological sieve 2 31 Fig. 2 Representation of a cross-section through the GFB showing the three-layer structure consisting of glomerular endothelium and glycocalyx, glomerular basement membrane (GBM) and podocyte foot processes. Albumin, represented by orange ellipses, does not pass through the normal GFB in significant amounts  Glomerular endothelial cells 31 32 33 34 35 36 37 31 32 38 39 40 41 42 43  The GBM 31 Podocytes 2 44 45 44 Passage of albumin across the normal GFB Filtration 46 Solute flux 31 47 48 47 31  Structural alterations in the GFB associated with microalbuminuria in diabetes 49 50 51 49 51 52 53 22 54 55  56 57 58 51 59  Functional alterations in GFB selective permeability associated with microalbuminuria in diabetes 60 61 61 62 63 58  We have already noted that physical alteration of the GFB is necessary to increase its permeability to albumin. Therefore, this confirmation of changes in GFB selective permeability in diabetic microalbuminuria in the absence of clearly identified structural correlates suggests that the key changes are yet to be elucidated. These considerations and the predominance of defects in charge selectivity point to alterations in the negatively charged glomerular endothelial glycocalyx as the missing link. We now move to consider what aspects of the diabetic milieu are responsible for these GFB changes and how they might cause endothelial, including glycocalyx, dysfunction. Metabolic pathways and effectors from hyperglycaemia to microalbuminuria 3 Fig. 3 Pathways to microalbuminuria in diabetes. Hyperglycaemia, through increased mitochondrial superoxide production, dysregulates key intracellular metabolic pathways. These in turn lead to the production of effectors that directly cause glomerular endothelial cell (GEnC) dysfunction (particularly of the glycocalyx) and disturb podocyte–endothelial cell communication. This results in microalbuminuria. Progression of these lesions and development of other glomerular changes, including podocyte damage, lead to overt diabetic nephropathy  64 65 Here we focus on selected intermediaries and effectors that have an identifiable role in microalbuminuria. In large part, dissection of these pathways and elucidation of their role in glomerular disease has necessarily relied on tissue culture or animal models. Such are described where human studies are lacking or where they provide additional significant insights. ROS 3 66 67 68 69 70 71 72 73  Vascular endothelial growth factor 74 75 76 77 78 79 80 53 81 52 82  The growth hormone/IGF system 83 84 85 86 87 88 Proinflammatory cytokines and adipokines 41 89 90 91 91 92 27 93 94 95 96 Conclusions 4 97 Fig. 4 Proposed mechanism of glomerular filtration barrier damage leading to diabetic microalbuminuria. High glucose causes dysregulation of mediators including TNFα and enhanced production of ROS, which directly damage the glomerular endothelial glycocalyx leading to microalbuminuria. Increased levels of pro-angiogenic molecules, including VEGF and inflammatory mediators, induce an activated and more permeable glomerular endothelial cell phenotype  4 4 98