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MBB334 - Biochemical Basis of Human Disease > Watson - Renal Scarring > Flashcards

Watson - Renal Scarring Flashcards

1
Q

What can cause renal scarring, and how?

A
  • glomerulonephritis (= inflam) –> immune or autoimmune
  • Diabetes Mellitus –> TI or any that lead to increased blood sugar (chemical insult to kidney, affects structure and cellular lining)
  • hypertension –> physical insult, changes structure of kidney
  • pyelonephritis (= recurrent kidney infections) –> causes inflam
  • polycystic kidney disease (inherited) –> changes structure
  • tubulointerstitial nephropathies –> urinary blockage (chemical insult), infection, drug/chemical toxicity
  • unknown causes –> some people just get gradual scarring process
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2
Q

How does blood flow through kidney?

A
  • enters glomerulus through afferent arteriole
  • thin wall capillaries allow filtration into Bowman’s capsule
  • leaves through efferent arteriole
  • enters tubules = delicate exchange system
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3
Q

What is the final product of the kidneys?

A
  • urine
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4
Q

What is the tubulointerstitium and where is it?

A
  • surrounds tubules
  • 85-90% of kidney
  • quite complex and susceptible to damage
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5
Q

What is the functional unit of the kidney?

A
  • nephron
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6
Q

What parts of the kidney are sensitive to scarring?

A
  • tubules and glomerulus
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7
Q

What is scarring?

A
  • accum of collagen and other components of ec matrix
  • normal to have some around tissues to support and give structure, but when excessive and replaces normal cellular structure causes loss of function of kidneys
  • ie. scarring is imbalance between deposition and removal
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8
Q

What is the glomerulus and its structure?

A
  • large capillary network supported by specialised podocytes
  • spatial architecture further maintained by mesenchymal cell type –> mesangial cells
  • encapsulated by epithelial cell forming Bowman’s capsule
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9
Q

What are mesangial cells?

A
  • smooth muscle cells w/ contractile properties
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10
Q

What is the role of Bowman’s capsule?

A
  • forms capsular space into which glomerular filtrate can drain prior to passage down capsule
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11
Q

In what order does filtration occur?

A
  • sequentially across glomerular capillary through:
  • -> endothelial cell pores (fenestrations)
  • -> glomerular basement mem (GBM)
  • -> podocytes (epithelial cells) form filtration slits between their pedicels
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12
Q

What is filtration limited by?

A
  • size and charge of filtered mols w/ cut off size/radius around 65 kDa
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13
Q

Why does filtration favour +vely charged mols?

A
  • GBM -vely charged
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14
Q

What happens to mols after they have been filtered?

A
  • can be subsequently reabsorbed as travel down tubule, before reaching collecting duct
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15
Q

What is the gross structure of the kidney?

A
  • glomeruli in cortex and tubules in medulla
  • millions of glomeruli filtering urine, collects through tubules in medulla
  • join together in bigger and bigger collection ducts until enters ureter
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16
Q

How much of cardiac input do the kidneys receive?

A
  • 20%
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17
Q

How much urine do the kidneys prod?

A
  • 1-2L a day
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18
Q

What are the 1° functions of the kidneys?

A
  • clear body of many toxins gen from various biochem events, inc nitrogenous waste products
  • reg body pH
  • maintain water balance
  • control Na/K and other electrolyte levels –> needs to be w/in v narrow range
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19
Q

What is an important clinical measure of kidney function?

A
  • creatinine clearance and glomerular filtration rate (GFR)
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20
Q

How is creatinine formed?

A
  • creatine synthesised in liver from meth of glycocyamine
  • transp through blood to other organs, eg. muscle and brain
  • forms high energy compound phosphocreatine
  • in periods of rest, excess ATP can be used to hydrolyse phosphocreatine
  • during synthesis, cat by creatine kinase, spont conversion to creatinine can occur (waste product)
  • -> prod mainly by energy consump in muscles
  • -> filtered out of blood by kidneys
  • -> levels in blood and urine can be used to calc creatinine clearance, which reflects GFR
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21
Q

What is the role of phosphocreatine and when can it be used for energy?

A
  • acts as transient ST store of high energy phosphate and can be used for energy
  • eg. used in 1st secs of muscle action or intense neuronal activity it donates P to gen ATP and energy
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22
Q

What can we do to slow disease?

A
  • treat underlying disease if poss
  • -> hypertension = angiotensin converting enz inhibitors
  • -> diabetes = insulin
  • -> inflam = steroids
  • -> blockage = surgery
  • -> infection = antibiotics
  • -> genetics = gene therapy?
  • low prot diets to reduce prot in urine, reduce urea/toxin levels
  • but controlling initial insult may not halt scarring process
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23
Q

What does renal scarring mean to a patient?

A
  • once function below 10% req dialysis

- small % may receive transplant –> will lead to better QoL

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24
Q

What are the 2 types of dialysis?

A
  • haemodialysis

- peritoneal

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25
Q

What does haemodialysis involve?

A
  • fistula permanently inserted into major vein/artery
  • blood pumped continuously to artificial kidney –> large dialysis mem sep blood from dialysis fluid (characteristics of fluid can alt dep what is dialysed from blood)
  • typical session 3-6 hrs, and 3x week
  • most feel unwell for around 24 hrs after
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26
Q

Is haemodialysis an effective replacement of kidney function, why?

A
  • at best achieves 10% of normal renal function → not effective replacement, but is essential
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27
Q

What does peritoneal dialysis involve?

A
  • permanent catheter inserted into peritoneal cavity
  • dialysis fluid runs through catheter to peritoneum, where contacts w/ blood capillaries that line peritoneum, which act as mem
  • after given dwell time, fluid drained, having equilibrated w/ blood and allowed removal of waste products that move from blood to fluid
  • each exchange around 6 hrs and typically 4 per 24 hrs = continuous ambulatory peritoneal dialysis (CAPD)
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28
Q

What are the disadv of peritoneal dialysis, comp to haemodialysis?

A
  • less efficient
  • fibrosis of peritoneal mem restricts LT use
  • procedure also prone to infectious complications
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29
Q

What is the role of dialysis?

A
  • removal of various small MW toxins (up to 5 kDa) that would normally be excreted by kidneys –> many toxins are nitrogen waste products, ie. from urea cycle, breakdown of peptides, AAs etc
  • removal of excess body water
  • balance Na/K blood levels
  • control blood pH
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30
Q

What are the additional kidney functions (which dialysis can’t perform)?

A
  • prod of erythropoietin –> stims bone marrow prod of mature erythrocytes
  • hydroxylation of vit D –> causes Ca absorption from gut
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31
Q

Why is it essential to treat renal failure w/ recomb erythropoietin and hydroxylated vit D?

A
  • lack of erythropoietin leads to low erythrocyte count, lethargy and anemia
  • lack of vit D results in brittle and bendy bones, dental problems and if severe, biochem problem
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32
Q

So what does complete treatment for renal failure need to inc?

A
  • dialysis
  • nutritional supplements and vitamins to prevent wasting
  • recomb erythropoietin
  • active vit D
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33
Q

How many stages of renal failure are there?

A
  • 4

- ranging from normal tissues to advanced scarring when patient reaches ESRF (end stage renal failure)

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34
Q

Where is progression of renal failure easiest to see?

A
  • in tubulointerstitial compartment
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35
Q

What is seen as renal failure progresses from normal to stage 3?

A
  • distension and deformation of tubules –> shift from many to few large ones and large increase in inter tubule gaps that are filled w/ ECM and fibroblasts
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36
Q

What is the normal tubulointerstitium like?

A
  • tight packing of tubules
  • fine bore lumen (high SA:vol, so efficient reabsorption)
  • small interstitial fibroblasts, w/ packing
  • intricate structure
  • good blood supply
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37
Q

What is the tubulointerstitium like in stage 3 ESRF?

A
  • high level tubular atrophy
  • majority of tubules collapse and lost
  • remaining blocked w/ prot (filtered in urine)
  • major interstitial cell type now fibroblasts
  • failure to pass urine or reabsorb from filtrate
38
Q

What major changes occur in renal scarring?

A
  • inflam response
  • fibroblast prolif
  • tubular cell death
  • ECM accum
39
Q

What causes platelet aggreg?

A
  • results from damage to glomerular capillary endothelial lining favouring platelet adhesion and aggreg
40
Q

What happens after platelets are aggreg?

A
  • results in initiation of many causes of renal scarring
  • once begin to aggreg, they become self activating –> secrete GFs (eg. PDGF, VEGF) and cytokines (eg. IL-1), which act in paracrine manner on local renal cells
41
Q

How does endothelial cell damage contrib to scarring?

A
  • inflam cytokines released by activated renal cells now act on lining of endothelial cells
  • accelerate changes induced by initial insult
42
Q

How does exp of CAMs contrib to scarring?

A
  • triggered by stim of cellular damage and GFs

- changes properties of endothelial cells and allow immune cells to migrate into kidney tissues

43
Q

What are chemokines?

A
  • class of cytokines that drive migration of immune cells into sites of damage by chemotaxis
44
Q

How do chemokines contrib to scarring?

A
  • released by renal cells
  • attach to cells and ECM to form grad away from prod cells
  • grad acts as trail to attract, then guide infiltrating cells to site of chemokine release = chemoattraction
45
Q

What is the overall pathway to renal scarring?

A
  • exp of GFs
  • induction of cytokines and chemokines
  • invasion of tissue by cells of IS (lymphocytes and macrophages)
  • prolif of renal mesangial cells, increases in activity of fibroblasts (ECM exp) and increases no. myofibroblasts (high ECM exp)
  • scarring is result of:
  • -> changes in nature of cells in tissue
  • -> pathological increase in dep of ECM
46
Q

What are myofibroblasts important in?

A
  • wound healing
47
Q

What is the role of myofibroblasts, and how do they differ?

A
  • v good at prod ECM
  • some may be normal mesangial cells and/or resident fibroblasts that have undergone changes in phenotype due to influence of GFs
  • others may be infiltrating kidney
48
Q

How is ECM dep balanced?

A
  • ECM exists in state of eq
  • dep balanced by turnover and removal
  • normally dep and breakdown finely balanced to provide optimum levels of ECM for cell viability and function
49
Q

How is ECM balance affected in fibrosis?

A
  • offset, so dep outweighs breakdown
50
Q

How could obesity be targeted for renal scarring?

A
  • control blood pressure and reduce glomerular damage

- to reduce functional load on kidneys

51
Q

What env factors could be targeted, and why?

A
  • smoking: effects on blood pressure etc. seem to be pro-fibrotic
  • stress: increases blood pressure
  • infections: chronic nephritis leads to scarring, antibiotic treatment and monitor to prevent reoccurrence
52
Q

How could hypertension be targeted?

A
  • ACE inhibitors –> reduce glomerular pressure and proteinuria
    » increased cardiovascular risk, os even though benefit of lowering blood pressure to treat renal scarring, the risk prob don’t outweigh these risks
  • renin-aldosterone-system
    –> controls NA+ filtration and blood pressure, reduce glomerular pressure
53
Q

In what diff ways could inflam be targeted?

A
  • immunosuppression
  • TGFβ antagonists
  • other cytokines
  • GFs
54
Q

Could immunosuppression be used to target inflam?

A
  • some evidence for effectiveness, but serious pot side effects, inc infection, lymphoproliferative disorders, malignancy
  • so not good for LT, but do need before transplants
55
Q

How could TGFβ antagonists be used to target inflam?

A
  • central mediator of fibrosis, so knockdown
  • but also important in immunosuppression t/o body (serious side effects if KO/down)
  • deficient mice die of multifocal inflam
56
Q

How could other cytokines be used to target inflam?

A
  • IL-1β antagonist (as pro-inflam), but no real data
  • IL-10 = key inflam cytokines, but local delivery could be issue as not always effective when used systemically
  • TNFα = key inflam cytokine, anti TNF therapy well estab in rheumatoid arthritis, but side effects inc infection and malignancies
57
Q

How could GFs be used to target inflam?

A
  • knockdown eg. PDGF/VEGF (all pro-fibrotic)

- have other roles and neutralising them systematically will have side effects

58
Q

Could the fibrosis pathway be blocked as a target?

A
  • MMP local delivery been shown to reduce collagen content in rat model of neuropathy
  • but overexp caused more fibrosis in mice
  • may be harder to influence equilib than we thought –> controls prob more complex than we know
  • selective pharmacological inhib caused increased fibrosis
59
Q

What enzs degrade the ECM?

A
  • MMPs, ser proteases, cys/asp proteases
60
Q

How could the ECM be stab?

A
  • tissue transglutaminase (TG2)
  • exp in all tissues
  • increasing its activity would expect to increase fibrosis
61
Q

What is the role of transglutaminases?

A
  • cat formation of covalent bond between glu in prots/peptides and lys in donor prots/peptides
  • glutamyl-lysyl dipeptide bond covalent and unbreakable under phys conditions
  • so stab ECM by crosslinking collagen mols and other components
62
Q

What is the structure of TG2?

A
  • 4 doms
  • -> sandwich dom = important to anchor to ECM and has binding site for fibronectin
  • -> catalytic core
  • -> β barrel 1 and 2 (cover active site when inactive, this is folded form its in when secreted)
63
Q

How is TG2 activated?

A
  • ec Ca levels

- causes large conformational shift, β barrels 1/2 move into extended position, exposing cat core and critical Cys277

64
Q

What conservation is there between TG2 and other fam members, and how did this affect drug design?

A
  • 4 dom structure fairly well conserved

- Cys277 in all members of fam and cat core is well conserved –> issue when designing drugs

65
Q

What diseases has TG2 been implicated in?

A
  • tissue scarring and fibrosis: in kidney, liver, lung, heart –> ECM accum
  • Coeliac disease –> deamidation of Gliadin
  • neurodegen disease: AD, PD, HD –> form of neurofibrillary plaques
  • cancer and chemo resistance –> stab cells against apoptosis, angiogenesis and metastasis
66
Q

What animal models are there for CKD?

A
  • Streptozotocin rat
  • UUO mouse (unilateral uretary obstruction)
  • Subtotal nephrectomy (rat) (SNx)
67
Q

How is the Streptozotocin rat a model for CKD?

A
  • causes diabetes, leads to renal symptoms
68
Q

How is the UUO mouse a model for CKD?

A
  • tie off 1 ureter, so other unobstructed and can act as control
  • causes accum of waste products, inc urea etc. in kidney leading to renal failure
  • shorter time course than human CKD (so less reflective of chronic scarring)
69
Q

How is the subtotal nephrectomy rat a model for CKD?

A
  • most widely used model and accepted by FDA
  • 1 kidney and 2/3 other removed (so 5/6 total nephrectomy)
  • overburdened kidney so prog fails
  • after 8 wks ESRF
  • resembles human disease and prog can be measured by similar physiological indicators (creatine clearance, GFR)
  • can take tissue samples
70
Q

How is TG2 implicated in kidney scarring?

A
  • immunohistochem shows increased TG2 prod
  • immunofluorescence shows increased ec TG2
  • specific Abs show TG2 crosslinking
  • correl between TG2 and crosslinking in renal scarring
71
Q

Could TG2 be a good therapeutic target, evidence?

A
  • chem inhibitors available that target cat core of TG2 and block enz activity
  • all target cat Cys277, so called pan transglutaminase inhibitors as target all members
  • drug delivered locally by implanted catheter and minipump
  • result: TG inhibition prevents renal scarring and failure, and prolongs kidney function in subtotal nephrectomy rat model of kidney disease –> suggests good target
  • this is ds approach, treating result, ie. fibrosis
  • further evidence from UUO model:
  • -> KO TG2 and get much reduced collagen dep comp to WT
  • -> shows not deleterious, as viable and not much diff in phenotype, so can block w/o serious physiological side effects (although don’t know true in humans)
72
Q

What success has there been w/ chemical inhibitors of TG2?

A
  • SNx rat treated w/ small mol inhibitor –> tubular structure preserved and scarring burden reduced
  • but TG inhibitors broad spectrum and KO TG1, TG3 and factor XIIIa
  • almost imposs to prod small mol inhibitor w/ no other targets, so do we need an alt?
73
Q

Why is KO of TG1/3 and factor XIIIa when targeting TG2 a problem?

A
  • TG1/3 effects epidermal layer = parakeratosis, disintegration of keratin layer, complete loss of keratin
  • factor XIIIa = symptoms similar to haemophilia as important in blood clotting (ie. bleeding, haemorrhage, weak clots)
74
Q

Why may Abs be a better alt to target TG2?

A
  • pot could target just TG2 and not cross react w/ other members of fam (despite seq conservation) –> no off target effects
75
Q

How would Abs target TG2?

A
  • could target cat site and block access of sub
  • block conform change assoc w/ activation
  • block Ca binding and prevent activation
76
Q

What was the strategy for making an Ab to target TG2?

A
  • immunise mice w/ recomb human TG2 (available commercially)
  • screen hybridomas for TG2 binding activity
  • screen for cross reactivity w/ other human TGs
  • all by ELISA → high throughput, need to screen lots hybridomas etc.
  • screen for inhib activity by assay (this is subset, so don’t need high throughput)
77
Q

What is ELISA?

A
  • enz linked immunosorbent assay
78
Q

What were the results after immunisation and screening w/ Ab against TG2?

A
  • approx 40 hybridomas obtained, all +ve for human TG2
  • WB analysis
  • -> see if epitope linear or conformational
  • -> if denature conf epitopes abolishes Ab binding (as in WB), difficult to map
  • -> epitope info critical to understanding function
  • only 2 reacted w/ TG2 (= linear), so majority conformational
79
Q

What are conformational epitopes?

A
  • composed of residues brought together in space as result of prot folding (so only in native form)
80
Q

After hybridomas obtained what were the results after testing w/ ELISA? (for Ab targeting TG2)

A
  • all showed cross reactivity w/ multiple members of fam (inc TG1/3 and factor XIIIa)
  • surprising, but perhaps as all conformational, so when enzs folded the residues exposed on the surface were more conserved than overall seq of enz
81
Q

Why did this experiment to find Ab to target TG2 fail?

A
  • could not control epitope recognition
  • TGs highly conserved and like all prots will possess “immunodom” epitopes (don’t gen Abs to all of prot)
  • there are epitopes that are particularly favourable for gen Ab response
82
Q

What was a revised immunisation strategy to control epitope recognition in mice in the immunised mice?

A
  • exp indiv doms in subsequent immunisations –> forces mice to respond to each dom in turn, hopefully stopping immunodom
83
Q

What was the result after the revised immunisation strategy in finding Abs to target TG2?

A
  • found lots of hybridomas for sandwich, barrel-1 and barrel-2 doms, all TG2 specific, but none inhibitory
  • did achieve specificity and all linear
  • for cat core: AB1 was the most effective and had IC50 superior to that of best chem inhibitor
84
Q

Why is it important to find linear epitopes?

A
  • as makes mapping easy
  • have to map conf epitopes by crystallisation which is a long process, but can do linear by deletions
  • so can patent seq of target as well as epitope
  • and could reveal mech of action
85
Q

What 3 groups were epitopes mapped into (for Ab to target TG2)?

A
  • Ab-1 binding site –> binds at access site for substrate, so sterically block access
  • DF-4 binding site –> targets cat core, near to Cys227
  • DD-9 binding site –> remote from active site, but think on Ca binding site, so may inhib enz activation
86
Q

What is the next step for TG2 inhibitory Abs (and any other therapeutic Ab)?

A
  • conversion to human IgG (from mouse)
  • mouse Abs would cause signif IR in patients as non self
  • before starting dev must demonstrate therapeutic benefit –> chemical data and KO mouse results promising, but need animal models to show efficacy
87
Q

What were the results of the 1st step in assessing TG2 antagonists in animal models?

A
  • none of the Abs had any effect on TG2 (only works on humans)
  • makes sense as so highly specific
  • MSA of human-mouse-rat TG2 = v similar, even target epitope of AB1 is almost identical (suggests res where there is a diff are critical to Ab binding)
88
Q

What was the revised strategy for testing TG2 antagonists on animal models?

A
  • can’t just repeat immunisation using purified mouse/rat catalytic core, due to tolerance –> wont respond to own cat core
  • get mice to respond to mice cat core by immunising TG2 KO mice –> as never exp TG2, so is foreign antigen
  • used phage display as more rapid way of cloning Abs than conventional hybridomas
89
Q

If mice/rat models not available, what can be used, and what are the issues w/ this?

A
  • can use primates, as homologous to humans

- but cost and ethical problems (so not used much)

90
Q

What is phage display, and what is it used for?

A
  • Abs displayed on surface of bacterial virus

- now a widespread methodology for rapid cloning of mAb

91
Q

What is the conventional hybridoma tech (not phage display)?

A
  • immunise mice w/ antigen
  • create immortal hybrid cells
  • screen IgG by ELISA for specific binding
  • use plastic well to try and contain prot product w/ gene that made it –> this is why 1 cell per well
92
Q

What are the problems w/ humanisation of mAbs?

A
  • when change framework can cause unforeseen interactions between res in framework and in CDR
  • changing conf can change specificity

MBB334 - Biochemical Basis of Human Disease (7 decks)

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