Origen, formulación y difusión de la doctrina de la hispanidad:

In DN the structure of the glomerulus undergoes a number of changes so that the kidney is unable to function and subsequently fails, leading to ESRD; requiring the need for dialysis or renal

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transplantation (Thrailkill, Nimmo et al. 2009). Changes include glomerular hyperfiltration, hyperperfusion and subsequently the development of histological features: thickening of the glomerular basement membrane (GBM), glomerular hypertrophy, mesangial expansion and glomerulosclerosis (GS). These histological features are currently used to diagnose DN on renal biopsy tissue. There has been a recent change in classification, in order to account for interstitial and vascular changes seen in DN, see Table 1.0 (Tervaert, Mooyaart et al. 2010).

Table 1.0 Histological classification of DN adapted from Tervaert et al 2010 Renal biopsy Lesion

Glomerular Class I II III IV

Glomerular basement thickening a - Mild mesangial expansion b - Severe mesangial expansion

Nodular sclerosis (Kimmelstiel-Wilson lesion) Advanced glomerulosclerosis

Interstitial 0-3 Fibrosis 0-2 Inflammation Vascular 0-2 Arteriolar hyalinosis

0-2 Large vessel arteriosclerosis

Mesangial cells maintain glomerular capillary structure and modulate glomerular filtration via smooth muscle activity (Dronavalli, Duka et al. 2008). Hyperglycaemia is known to result in mesangial cell proliferation, increased matrix production and GBM thickening (Dronavalli, Duka et al. 2008). Hyperglycaemia has also been shown to increase extracellular matrix production from mesangial cells in vitro. Increased intracellular glucose is thought to be a critical step.

Overexpression of upregulated glucose transporters (GLUT1/GLUT4) in normal glucose concentration increases glucose entry to cells and also upregulates the synthesis of extracellular matrix by mesangial cells in vitro (Heilig, Concepcion et al. 1995). There may also be an upregulation of vascular endothelial growth factor (VEGF) expression on podocytes thus causing increased vascular permeability (Wolf and Ziyadeh 2007). These all contribute to the histological changes seen.

No difference has been seen in the degree of mesangial expansion between normoalbuminuric and microalbuminuric T1DM/T2DM (Doi, Mima et al. 2008). The glomerular filtration rate (GFR),

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however, is seen to decrease and correlate with mesangial expansion rather than GBM thickening with GS occurring at the glomerular capillary wall (Abbate, Zoja et al. 2006).

Recently there has been an increase in understanding of the role of the glomerular endothelium in the development and progression of DN. The glomerular endothelium glycocalyx is thought to be lost and this affects the fenestrations in the glomerular endothelium that have high water permeability.

These changes contribute to thickening of the GBM and widening of the podocyte foot processes leading to podocyte loss with disruption of the slit diaphragm (Satchell 2012). The state of the glomerular endothelium correlates more strongly with decreasing GFR and increasing albumin/creatinine ratio (ACR) than seen with a decline in podocytes. It is being increasingly reported that the decline in renal function seen in DM may be independent of proteinuria (Kramer, Nguyen et al. 2003).

A study into Pima Indians with T2DM showed significantly higher levels of podocyte detachment in macroalbuminurics compared with normoalbuminurics and microalbuminurics (Weil, Lemley et al.

2012). Podocyte detachment correlated with albuminuria however, the percentage of endothelial cell fenestration correlated significantly with GBM thickness and GFR, indicating a relationship between the histopathological findings and renal function decline in DN.

α1 and α2 chains of type IV collagen (Col4) are the main components of GS and are affected by a transcription factor (Smad1) critical for its development (Doi, Mima et al. 2008). Col4 is upregulated by advanced glycation end-products (AGEPs) and Smad1. In mesangial cells, Angiotensin II (Ang2) induces Col4 synthesis and increases the expression of Smad 1 and the phosphorylated Src gene that encodes tyrosine kinases involved in cell growth, movement and proliferation. Angiotensin 2 receptor blockers (ARB) are effective at blocking this pathway in the streptozotocin (STZ) rat model and hence may induce these beneficial effects in DN if treatment is commenced early (Doi, Mima et al. 2008).

Tubulointerstitial fibrosis occurs with glomerular changes and with proteinuria (Mauer, Steffes et al.

1984). Fibroblasts, macrophages and lymphocytes are found within the tubulointerstitium during the

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process of fibrosis however, the effects of their individual roles remain uncertain, whether causative or beneficial in the phagocytosis of cellular components (Iwano and Neilson 2004). Different theories have been proposed for the origin of fibroblasts in the kidney. Fibroblasts may be derived by epithelial mesenchymal transition (EMT) in their activation following tissue injury via cytokines and cell signalling mechanisms. Thus, fibroblasts may be produced by tubuloepithelial cells (Wada, Sakai et al. 2007). Alternatively, bone marrow derived cells have been implicated in the ureteric obstructive mouse model to be the source of fibroblasts resulting in fibrosis (Jang, Kim et al. 2013).

EMT may arise from the loss of epithelial cells’ ability to adhere, thereby allowing cells to infiltrate into the interstitium, or be washed into the tubular fluid. Their effects are then exerted by downstream, orchestrating signals to induce renal fibrosis (Conway and Hughes 2012).

Transforming growth factor-beta (TGF-β) together with activation of transcription factors such as Smad3 contribute to the disruption of the tubular epithelial membrane and inhibit proteases that maintain the function of cell adhesion molecules such as matrix metalloproteinases (MMPs). Bone morphogenic protein-7 (BMP7) inhibits TGF-β induced EMT and when administered systemically has led to the repair of severely damaged renal tubular epithelial cells and reversed renal fibrosis (Iwano and Neilson 2004). A decrease in E-Cadherin has been associated with a rise in fibronectin and MMP2. Once fibroblasts are present they proliferate rapidly and produce a number of collagens and fibronectin exacerbating fibrosis. Downregulation of BMP7 in STZ mice was seen to be related to an increase in Tamm-Horsfall protein, that has been described as an early event in DN (Qu, Du et al. 2012).

Production of Ang2 and inhibition of nitric oxide (NO) occur following haemodynamic changes in DN that have been shown in diabetic mouse models (Zhang, Wang et al. 2012). This leads to longstanding vasoconstriction with chronic tissue ischaemia and hypoxia that may contribute to the formation of tubulointerstitial fibrosis (Nakagawa, Sato et al. 2007).

1.4 Proteinuria

Proteinuria has been established as a strong independent predictor of renal outcome in both diabetics and non-diabetics (Breyer, Bain et al. 1996). The Ramipril Efficacy In Nephropathy (REIN) trial showed the baseline urinary protein excretion correlated significantly with GFR decline and progression of non-diabetic proteinuric renal diseases to ESRD (1997, GISEN).

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The significance of the development of proteinuria is amplified by the toxic effects of protein on the renal tubular epithelial cells (Navarro-Gonzalez, Mora-Fernandez et al. 2011). This interaction induces chemokines, adhesion molecules and pro-inflammatory cytokines that lead to interstitial infiltration with monocytes, macrophages and lymphocytes, resulting in renal cell and tubulointerstitial damage and fibrosis (Abbate, Zoja et al. 2006; Galkina and Ley 2006; Navarro-Gonzalez, Mora-Fernandez et al. 2011).

Albumin forms the greatest proportion of proteinuria in DM (McIntyre and Taal 2008). Urinary ACR is the preferred method of monitoring in DN (McIntyre and Taal 2008). Albuminuria is not a marker of the earliest phase of DN, even with GS formation therefore; earlier phase sensitive markers are required. GS may occur in normoalbuminuric and microalbuminuric DN and can occur following hyperfiltration, hyperperfusion and thickening of the GBM. Previous reports show there may be no significant structural changes of the glomerulus in diabetics, with or without microalbuminuria, unless this was combined with a raised blood pressure or a change in serum creatinine (Chavers, Bilous et al. 1989; Fioretto, Steffes et al. 1994). The process of loss of renal function therefore can occur before the initiation of proteinuria (Rosolowsky, Niewczas et al. 2008).

Normoalbuminurics have been found to have established changes of DM that would normally be seen in proteinuric DM who go onto progress. In contrast, microalbuminuric DM have been reported thus ACR does not predict progression (Chavers, Bilous et al. 1989; Fioretto, Steffes et al. 1994). A decline in GFR with glomerular changes may occur without proteinuria (Najafian, Alpers et al.

2011). The presence of microalbuminuria may be predictive of decline; however, microalbuminurics do not always progress as previously reported. It is plausible that different genotypes of diabetic renal disease may exist that will influence or predetermine who will progress, thereby emphasising the need for more sensitive and specific markers. Alternatively proteomics may be a more sensitive technique (Zurbig, Jerums et al. 2012).

There is no established step progression from no nephropathy to microalbuminuria to macroalbuminuria and ESRD, as patients may go from any stage to ESRD at any point (Adler, Stevens et al. 2003; Zoppini, Targher et al. 2012). In addition, Perkins showed there may be regression of microalbuminuria that may reflect the introduction of angiotensin converting enzyme

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inhibitors (ACEi), (Perkins, Ficociello et al. 2003). The use of albuminuria as a marker of progression of DN is not robustly associated with progression and does not reflect underlying pathological stages associated with progression. Better markers are required that are more sensitive and reflective of disease stage and progression (Perkins, Ficociello et al. 2003; MacIsaac, Tsalamandris et al. 2004).