Watson - Renal Scarring Flashcards
What can cause renal scarring, and how?
- 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
How does blood flow through kidney?
- enters glomerulus through afferent arteriole
- thin wall capillaries allow filtration into Bowman’s capsule
- leaves through efferent arteriole
- enters tubules = delicate exchange system
What is the final product of the kidneys?
- urine
What is the tubulointerstitium and where is it?
- surrounds tubules
- 85-90% of kidney
- quite complex and susceptible to damage
What is the functional unit of the kidney?
- nephron
What parts of the kidney are sensitive to scarring?
- tubules and glomerulus
What is scarring?
- 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
What is the glomerulus and its structure?
- 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
What are mesangial cells?
- smooth muscle cells w/ contractile properties
What is the role of Bowman’s capsule?
- forms capsular space into which glomerular filtrate can drain prior to passage down capsule
In what order does filtration occur?
- sequentially across glomerular capillary through:
- -> endothelial cell pores (fenestrations)
- -> glomerular basement mem (GBM)
- -> podocytes (epithelial cells) form filtration slits between their pedicels
What is filtration limited by?
- size and charge of filtered mols w/ cut off size/radius around 65 kDa
Why does filtration favour +vely charged mols?
- GBM -vely charged
What happens to mols after they have been filtered?
- can be subsequently reabsorbed as travel down tubule, before reaching collecting duct
What is the gross structure of the kidney?
- 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
How much of cardiac input do the kidneys receive?
- 20%
How much urine do the kidneys prod?
- 1-2L a day
What are the 1° functions of the kidneys?
- 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
What is an important clinical measure of kidney function?
- creatinine clearance and glomerular filtration rate (GFR)
How is creatinine formed?
- 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
What is the role of phosphocreatine and when can it be used for energy?
- 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
What can we do to slow disease?
- 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
What does renal scarring mean to a patient?
- once function below 10% req dialysis
- small % may receive transplant –> will lead to better QoL
What are the 2 types of dialysis?
- haemodialysis
- peritoneal
What does haemodialysis involve?
- 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
Is haemodialysis an effective replacement of kidney function, why?
- at best achieves 10% of normal renal function → not effective replacement, but is essential
What does peritoneal dialysis involve?
- 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)
What are the disadv of peritoneal dialysis, comp to haemodialysis?
- less efficient
- fibrosis of peritoneal mem restricts LT use
- procedure also prone to infectious complications
What is the role of dialysis?
- 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
What are the additional kidney functions (which dialysis can’t perform)?
- prod of erythropoietin –> stims bone marrow prod of mature erythrocytes
- hydroxylation of vit D –> causes Ca absorption from gut
Why is it essential to treat renal failure w/ recomb erythropoietin and hydroxylated vit D?
- 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
So what does complete treatment for renal failure need to inc?
- dialysis
- nutritional supplements and vitamins to prevent wasting
- recomb erythropoietin
- active vit D
How many stages of renal failure are there?
- 4
- ranging from normal tissues to advanced scarring when patient reaches ESRF (end stage renal failure)
Where is progression of renal failure easiest to see?
- in tubulointerstitial compartment
What is seen as renal failure progresses from normal to stage 3?
- 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
What is the normal tubulointerstitium like?
- tight packing of tubules
- fine bore lumen (high SA:vol, so efficient reabsorption)
- small interstitial fibroblasts, w/ packing
- intricate structure
- good blood supply
What is the tubulointerstitium like in stage 3 ESRF?
- 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
What major changes occur in renal scarring?
- inflam response
- fibroblast prolif
- tubular cell death
- ECM accum
What causes platelet aggreg?
- results from damage to glomerular capillary endothelial lining favouring platelet adhesion and aggreg
What happens after platelets are aggreg?
- 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
How does endothelial cell damage contrib to scarring?
- inflam cytokines released by activated renal cells now act on lining of endothelial cells
- accelerate changes induced by initial insult
How does exp of CAMs contrib to scarring?
- triggered by stim of cellular damage and GFs
- changes properties of endothelial cells and allow immune cells to migrate into kidney tissues
What are chemokines?
- class of cytokines that drive migration of immune cells into sites of damage by chemotaxis
How do chemokines contrib to scarring?
- 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
What is the overall pathway to renal scarring?
- 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
What are myofibroblasts important in?
- wound healing
What is the role of myofibroblasts, and how do they differ?
- 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
How is ECM dep balanced?
- 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
How is ECM balance affected in fibrosis?
- offset, so dep outweighs breakdown
How could obesity be targeted for renal scarring?
- control blood pressure and reduce glomerular damage
- to reduce functional load on kidneys
What env factors could be targeted, and why?
- 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
How could hypertension be targeted?
- 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
In what diff ways could inflam be targeted?
- immunosuppression
- TGFβ antagonists
- other cytokines
- GFs
Could immunosuppression be used to target inflam?
- some evidence for effectiveness, but serious pot side effects, inc infection, lymphoproliferative disorders, malignancy
- so not good for LT, but do need before transplants
How could TGFβ antagonists be used to target inflam?
- 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
How could other cytokines be used to target inflam?
- 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
How could GFs be used to target inflam?
- knockdown eg. PDGF/VEGF (all pro-fibrotic)
- have other roles and neutralising them systematically will have side effects
Could the fibrosis pathway be blocked as a target?
- 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
What enzs degrade the ECM?
- MMPs, ser proteases, cys/asp proteases
How could the ECM be stab?
- tissue transglutaminase (TG2)
- exp in all tissues
- increasing its activity would expect to increase fibrosis
What is the role of transglutaminases?
- 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
What is the structure of TG2?
- 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)
How is TG2 activated?
- ec Ca levels
- causes large conformational shift, β barrels 1/2 move into extended position, exposing cat core and critical Cys277
What conservation is there between TG2 and other fam members, and how did this affect drug design?
- 4 dom structure fairly well conserved
- Cys277 in all members of fam and cat core is well conserved –> issue when designing drugs
What diseases has TG2 been implicated in?
- 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
What animal models are there for CKD?
- Streptozotocin rat
- UUO mouse (unilateral uretary obstruction)
- Subtotal nephrectomy (rat) (SNx)
How is the Streptozotocin rat a model for CKD?
- causes diabetes, leads to renal symptoms
How is the UUO mouse a model for CKD?
- 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)
How is the subtotal nephrectomy rat a model for CKD?
- 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
How is TG2 implicated in kidney scarring?
- immunohistochem shows increased TG2 prod
- immunofluorescence shows increased ec TG2
- specific Abs show TG2 crosslinking
- correl between TG2 and crosslinking in renal scarring
Could TG2 be a good therapeutic target, evidence?
- 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)
What success has there been w/ chemical inhibitors of TG2?
- 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?
Why is KO of TG1/3 and factor XIIIa when targeting TG2 a problem?
- 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)
Why may Abs be a better alt to target TG2?
- pot could target just TG2 and not cross react w/ other members of fam (despite seq conservation) –> no off target effects
How would Abs target TG2?
- could target cat site and block access of sub
- block conform change assoc w/ activation
- block Ca binding and prevent activation
What was the strategy for making an Ab to target TG2?
- 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)
What is ELISA?
- enz linked immunosorbent assay
What were the results after immunisation and screening w/ Ab against TG2?
- 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
What are conformational epitopes?
- composed of residues brought together in space as result of prot folding (so only in native form)
After hybridomas obtained what were the results after testing w/ ELISA? (for Ab targeting TG2)
- 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
Why did this experiment to find Ab to target TG2 fail?
- 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
What was a revised immunisation strategy to control epitope recognition in mice in the immunised mice?
- exp indiv doms in subsequent immunisations –> forces mice to respond to each dom in turn, hopefully stopping immunodom
What was the result after the revised immunisation strategy in finding Abs to target TG2?
- 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
Why is it important to find linear epitopes?
- 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
What 3 groups were epitopes mapped into (for Ab to target TG2)?
- 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
What is the next step for TG2 inhibitory Abs (and any other therapeutic Ab)?
- 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
What were the results of the 1st step in assessing TG2 antagonists in animal models?
- 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)
What was the revised strategy for testing TG2 antagonists on animal models?
- 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
If mice/rat models not available, what can be used, and what are the issues w/ this?
- can use primates, as homologous to humans
- but cost and ethical problems (so not used much)
What is phage display, and what is it used for?
- Abs displayed on surface of bacterial virus
- now a widespread methodology for rapid cloning of mAb
What is the conventional hybridoma tech (not phage display)?
- 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
What are the problems w/ humanisation of mAbs?
- when change framework can cause unforeseen interactions between res in framework and in CDR
- changing conf can change specificity
