Diabetic Nephropathy

 Diabetic nephropathy is the leading cause of chronic renal failure in the industrialised world.
It is also one of the most significant long-term complications in terms of morbidity and mortality for individual patients with diabetes. Diabetes is responsible for 30-40% of all end-stage renal disease (ESRD) cases in the United States. Although both type 1 diabetes mellitus (insulindependent diabetes mellitus [IDDM]) and type 2 diabetes mellitus (noninsulin-dependent diabetes mellitus [NIDDM]) lead to ESRD, the great majority of patients are those with NIDDM.
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 The glomeruli and kidneys are typically normal or increased in size initially, thus distinguishing diabetic nephropathy from most other forms of chronic renal insufficiency, wherein renal size is reduced (except renal amyloidosis and polycystic kidney disease).

Signs and Symptoms Approximately 25% to 40% of patients with DM 1 ultimately develop diabetic nephropathy (DN), which progresses through five predictable stages.

Stage 1 (very early diabetes)

Increased demand upon the kidneys is indicated by an above-normal glomerular filtration rate (GFR). Hyperglycemia leads to increased kidney filtration (see later) This is due to osmotic load and to toxic effects of high sugar levels on kidney cells Increased Glomerular Filtration Rate (GFR) with enlarged kidneys
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Stage 2 (developing diabetes)

Clinically silent phase with continued hyper filtration and hypertrophy The GFR remains elevated or has returned to normal, but glomerular damage has progressed to significant microalbuminuria (small but above-normal level of the protein albumin in the urine). Significant microalbuminuria will progress to endstage renal disease (ESRD). Therefore, all diabetes patients should be screened for microalbuminuria on a routine basis.
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Stage 3 (overt, or dipstick-positive diabetes)

Glomerular damage has progressed to clinical albuminuria. Basement membrane thickening due to AGEP

The urine is "dipstick positive," containing more than 300 mg of albumin in a 24-hour period.
Hypertension (high blood pressure) typically develops during stage 3.

Stage 4 (late-stage diabetes)

Glomerular damage continues, with increasing amounts of protein albumin in the urine. The kidneys filtering ability has begun to decline steadily, and blood urea nitrogen (BUN) and creatinine (Cr) has begun to increase. The glomerular filtration rate (GFR) decreases about 10% annually. Almost all patients have hypertension at stage 4.

Stage 5 (end-stage renal disease, ESRD)

GFR has fallen to <10 ml/min and renal replacement therapy (i.e., haemodialysis, peritoneal dialysis, kidney transplantation) is needed.

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CAPILLARY ENDOTHELIUM BASEMENT MEMBRANE FOOT PROCESSES OF PODOCYTES

FENESTRATION

FILTRATION SLIT

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NORMAL GBM. LEFT - a single glomerulus. There are one million of these in each kidney. RIGHT - a close up of the GBM (G) around part of one tiny blood vessel in a glomerulus (red circle in left hand diagram)
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Glomerular Histology:
The glomerular capillary wall is composed of an endothelial cell layer (blood side), a thick basement membrane, and epithelial cell layer (urine side). (i) Glomerular Endothelium

The glomerular endothelium is fenestrated. The fenestrae (0.07 to 0.1 mm-micrometers- in maximal diameter) allow the passage of electrolytes, proteins, and globulin.
However, platelets (3 mm), red cells (7 mm) and neutrophils (15 mm) can't pass through the endothelial layer.
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(ii) Glomerular Basement Membrane (GBM):

The GBM is a tri-laminar structure, 0.3 microns in thickness, composed of collagen, proteoglycans and laminin. It is product of the fusion of the endothelial and epithelial basement laminae. The dense central GBM area, or lamina densa, is due to the overlapping of the two laminae. Around 50% of the GBM is collagen IV.
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 The negative charge of the GBM has been attributed to the presence of the heparan sulphate proteoglycan (HSPG) called perlecan. These negatively charged molecules are geometrically arranged in clusters separated by about 0.003 m from each other. This anionic molecular sieve restricts the passage of molecules according to size and charge. Water, salts, glucose, amino acids and neutral, or cationic, molecules with radii less that 0.0035 m are filtered with relative ease. The albumin molecule measures 0.0035 m and is negatively charged. Therefore its filtration is restricted.
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 Presence of protein in the urine is a sign that either the charge or the distance between the anionic clusters, or both, are pathologically altered. The presence of red cells in the glomerular urine, is certain indication of GBM ruptures. Other classical constituents of the basement membrane are type IV collagen, laminin, and entactin.

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Glomerular mesangium:
The intra-capsular glomerular capillary network is kept together by the mesangium that is is composed of mesangial cells type I and II, and other tissue matrix. Mesangial type I cells are monocytes with phagocytic functions. These cells can extend cytoplasmic projections into the glomerular capillary. They also "clean" the mesangium of materials that leak from the capillary lumen into the matrix. These cells are stimulated by cytokines to produce free radicals and cytotoxic peptides.
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 Mesangial type II cells are myofibroblasts with the ability to contract upon ADH and angiotensin stimulation. Their contraction causes a reduction of the effective glomerular filtration area. Mesangial Matrix is a tissue mesh composed of different types of collagens (I, III, IV), laminin and proteoglycans.

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Three major histologic changes occur in the glomeruli of persons with diabetic nephropathy.
1. Mesangial expansion is directly induced by hyperglycemia, perhaps via increased matrix production or glycosylation of matrix proteins.

2.
3.

GBM thickening occurs.

Glomerular sclerosis is caused by intraglomerular hypertension (induced by renal vasodilatation or from ischemic injury induced by hyaline narrowing of the vessels supplying the glomeruli).
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Glomerular Hyper filtration

Glucose provides an osmotic diuretic effect Result is increased renal filtration, leading to glomerular hypertrophy Glomerular pressure increases Kidney responds with hypertrophy of epithelium and endothelium Accelerates glomerular cell failure Result is premature glomerulosclerosis

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Metabolic Perturbations
Oxidant Stress - related to glomerular hypertrophy and abnormal metabolism Non-enzymatic glycosylation of macromolecules particularly basement membrane (BM) Activation of glucose metabolizing enzymes

Cytokine and other humoral imbalances

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Non enzymatic Glycosylation Biochemical studies have shown that basement membranes in diabetes include excess amounts of type IV collagen, the main component of basement membranes, and decreased amounts of proteoglycans Both changes decrease the permeability of capillaries and disturb leukocyte diapedesis, oxygen diffusion, nutrition and metabolic waste removal.
Altered charge on BM may explain albuminuria Macrophage receptor activation leads to IL1, TNF production which stimulates matrix AGEP formation leads to abnormal collagen, increased toxic oxygen species
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Humoral Imbalances in DM Nephropathy

Insulin Deficiency Elevated Glucagon Concentrations Increased Transforming Growth Factor (TGF)- Increased angiotensin II Abnormally regulated thromboxanes and endothelins Abnormal insulin like growth factor (IGF)-1 Elevated platelet derived growth factor (PGDF)

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Role of TGF-
Stimulates extracellular matrix synthesis Inhibits extracelluular matrix degradation Up regulates protease inhibitors; down regulates matrix degrading enzymes Stimulates synthesis of integrins (matrix receptors) Key role in glomerular and tubuloepithelial hypertrophy, basement membrane thickening, and mesangial matrix expansion TGF- has been implicated in a number of chronic, scarring diseases

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 Angiotensin II and Thrombospondin (TSP1) can both stimulate the production of transforming growth factor- (TGF-) by tubuloepithelial cells and fibroblasts. TGF-, in turn, causes proliferation of fibroblasts and tubuloepithelial cells.
TGF- ultimately increases extracellular matrix proteins, likely by several mechanisms. TGF- stimulates production of several growth factors including basis fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF) that stimulate the formation of extracellular matrix (ECM) proteins.

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Ultrastructural changes of the glomerular basement membrane in diabetic nephropathy revealed by newly devised tissue negative staining method.
The normal human GBM showed a fine meshwork structure consisting of fibrils forming the small pores. The diameter of these pores was slightly smaller than that of human albumin molecules. The GBM in patients with diabetic nephropathy showed irregular thickening. At higher magnification, unknown cavities and tunnel structures, which were not seen in normal controls, were observed in the thickened GBM.
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 In some portions, these cavities presented a honeycomb-like appearance.

The diameters of the cavities and tunnels were far larger than the dimensions of albumin molecules. These enlarged structures are believed to allow serum protein molecules to pass through the GBM from the capillary lumen to the urinary space. These results suggest that the cause of massive proteinuria in diabetic nephropathy is the disruption of the size barrier of the GBM.

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Glomerular and vascular pathology is linked to hyperglycemia.

Changes in glomerular basement membrane structure occur very early in diabetic nephropathy, before even microalbuminuria is apparent. Collagen IV deposition is directly stimulated by hyperglycaemia and increased urinary levels indicate changes in the glomerular basement membrane. Contributing factors include the formation of advanced glycosylation end products (AGEs) due to nonenzymatic glycosylation of capillary basement membranes, as a consequence of long-term hyperglycaemia.
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 Non-enzymatic glycosylation has recently attracted increasing interest as a crucial pathophysiologic event behind all these hyperglycaemia-related alterations and in the pathophysiology of the development of diabetic complications. Proteins and lipids exposed to aldose sugars go through reactions which are not enzyme-dependent, and generation of reversible Schiff bases or Amadori products take place. Later, through further molecular rearrangements, irreversible advanced glycosylation end products (AGEs) are formed. This process also takes place during normal ageing, but in diabetes their formation is accelerated to an extent related to the level and duration of hyperglycaemia.
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 Hence large studies have shown a delay in onset or slowing of the progression of these complications if near normo-glycaemia can be maintained.
The glycated proteins cross-link, contributing to basement membrane (and mesangial) thickening, (culminating in the kidney in nodular glomerulosclerosis), as well as loss of the normal selective permeability (leading to proteinuria, retinal hard exudates and microhaemorrhages).

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 The potential pathophysiological significance of AGEs is associated with their accumulation in plasma, cells and tissues and their contribution to the formation of crosslinks, generation of reactive oxygen intermediates and interactions with particular receptors on cellular surfaces AGEs have direct effects on the host response by affecting tissue structures, e.g. by increasing collagen cross-links, which is followed by changes in collagen solubility and turnover. Thickening of basement membranes is partly due to glycosylation of membrane proteins or entrapment of glycosylated serum proteins into basement membrane It is evident that AGEs can interact with cell functions, tissue remodelling and inflammatory reactions in several different ways.
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 When Ang II is increased, greater AT1 receptormediated constriction of efferent than afferent arterioles increases single nephron glomerular filtration rate and raises intraglomerular pressure, causing glomerular hypertension. Sustained or severe increases in intraglomerular pressure can lead to GBM damage, glomerular endothelial dysfunction, and ultimately, extravasation of protein into Bowmans capsule. In addition to hypertension, conditions like diabetes that are associated with increased oxidative stress (increased formation of reactive oxygen species) independent of hypertension and glyco-oxidative modification of proteins (AGEs) comprising the glomerular basement membrane can lead to extravasation of protein.
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 Glomerular hypertension can lead to injury to the glomerular basement membrane causing it to leak plasma proteins into the urine. Attempts by the proximal tubules to reabsorb this filtered protein causes injury to the tubular cells, activates an inflammatory response, and is associated with the development of lipid metabolic abnormalities that create further oxidative stress on the already compromised glomerulus.
The resultant tubular inflammatory response and renal microvascular injury activate pathways that lead to fibrosis and scarring of both glomerular and tubular elements of the nephron.
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 An additional consequence of glomerular hypertension and resultant reduction in glomerular filtration rate (GFR) activates growth factors and cytokines that promote an influx of monocytes and macrophages into the vessel wall and into the renal interstitium, and also causes the differentiation of renal cells into fibroblasts.
Monocytes, macrophages and fibroblasts are capable of producing those growth factors and cytokines that activate pathways leading to expansion of extracellular matrix, fibrosis and loss of both tubular and glomerular structures.

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 Collagen IV is the principal component of the glomerular basement membrane and it is released into the urine during its turnover. Increased urinary levels of collagen IV are found in several conditions where glomerular injury is found, particularly in diabetic nephropathy. Collagen IV is too large to cross the glomerular membrane (MW 540 000) and so urinary collagen IV is a specific sensitive indicator of changes to the structure of extracellular matrix of the kidney. Unlike serum creatinine, that measures changes in glomerular function, increased levels of urinary collagen IV indicate that damage is occurring to the renal tissue. Urinary collagen IV is a very early and specific biomarker for pathological changes to the glomerular membrane, particularly in diabetic nephropathy.
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