Diabetic Nephropathy

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• Diabetic Nephropathy

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• 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
  (insulin-dependent 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).

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

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• 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
  end-stage 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.

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• 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.

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• Stage 5 (end-stage renal disease, ESRD)
• GFR has fallen to lt10 ml/min and renal
  replacement therapy (i.e., haemodialysis,
  peritoneal dialysis, kidney transplantation) is
  needed.

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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 µm 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 µm are filtered with relative ease.
• The albumin molecule measures 0.0035 µm 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.
• Mesangial expansion is directly induced by
  hyperglycemia, perhaps via increased matrix
  production or glycosylation of matrix proteins.
• 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)-ß
• 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-ß
• 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-ß 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-ß (TGF-ß) by tubuloepithelial cells and
  fibroblasts.
• TGF-ß, in turn, causes proliferation of
  fibroblasts and tubuloepithelial cells.
• TGF-ß ultimately increases extracellular matrix
  proteins, likely by several mechanisms.
• TGF-ß 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
  non-enzymatic 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 cross-links, 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
  receptor-mediated 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.