Renal Unit, Guy’s Campus, King’s College, London UK

1. “Glomerular” diseases - the past 60 years: what have we learned, and how?J Stewart Cameron São PauloNephrology Division - University of São Paulo22nd October 2013 Renal Unit, Guy’s Campus, King’s College, London UK

2. What can we learn from the story of glomerulonephritis about how knowledge is acquired and applied in medicine? Clinic Laboratory animals are not humans, science can mislead, and clinical observations often surprise, and contain vital clues

3. Change or progress in Medicine depends upon many factors: • social milieu and beliefs • advances in other sciences • concepts and ideas • technology available • accident and luck Change is meandering, diffuse, intermittent and crabwise, rather than logical and linear. Blind alleys are easily entered and progress is NOT inevitable.

4. “In science the credit goes to the man who convinces the world, not the man to whom the idea first occurs” Sir William Osler (1849-1919): Aphorisms. Ed WB Bean, 1961

5. The importance of techniques: just imagine your lab, or your clinic, without: Lab Clinic • flame photometry ultrasound • radioisotopes CAT scans or MRI • immunoassay effective diuretics • electrophoresis effective BP agents • monoclonal antibodies long-term dialysis • immunofluorescence transplantation • electron microscopy renal biopsy • cell culture open-heart surgery • molecular biology - intensive care mRNA, knockouts etc. cardiac resuscitation etc. THIS was the early 1950s, 60 years ago….

6. When I was a medical student in the 1950s, I drained severely- oedematous patients’ legs using skin punctures, just as Frederick Dekkers had done, 3 centuries before - and there was an open coal fire in the ward...

7. A time of rapid change every item in these lists was introduced in a cataract of change in the 1950s or early 1960, almost all from outside nascent Nephrology Hugh de Wardener UK (1916-2013) Jean Hamburger France (1905-1989) Homer Smith USA (1895-1962)

8. A world almost without meetings • The world of science was very small, with few people involved • The first international meeting on Nephrology was held in London in 1953 • In Evian & Geneva in 1960, at which the ISN was formed

9. A world almost without meetings • The world of science was very small, with few people involved • The first international meeting on Nephrology was held in London in 1953 • In Evian & Geneva in 1960, at which the ISN was formed • the ASN was not founded until 1966, at the 3rd ISN meeting in Washington, DC

10. A world without renal journals • The first nephrological journal was Minerva Nefrologica, in 1957 - in Italian • The first English-language journal was Nephronfrom the ISN in 1963,followed by the Proceedings of the EDTA in 1964 (now NDT). • Searching for papers was by the volumes of Index Medicus, sittingin the library. No photocopiers, so you made notes on cards • Paper reprints of articles were common, and exchanged with colleagues and friends • Until 1963 there were just 2 books on the kidney

11. But people were avid to publish their work, as always: publish or perish “.. this Desire for Glory, and to be counted Authors, prevails upon all.. “ 1673 Thomas Sprat. The history of the Royal Society of London, 1673, p. 74

12. Renal biopsy- a turning point in study of human glomerular diseases 1951 1954 • thinner (3 - 5μm) sections (Pirani 1956) • silver staining (Jones 1957) • electron microscopy (Folli, Farquhar etc 1957) • immunofluorescent staining (Kark 1959) Robert Kark (1911-2003) Poul Iversen & Claus Brun (1910- ) Then

13. Renal biopsy opened up new horizons: • post mortem changes were avoided in tissue • sequential observations of renal lesions became possible • clinicopathological correlations were made more precise and simultaneous • thus a new nosology of glomerulonephritis could be put in place The CIBA Foundation symposium in 1961 marked the clinical arrival of biopsy

14. - and glomerular diseases could be classified by histology (1968-70) • Minimal change disease • Focal glomerulosclerosis • (Extra) membranous nephropathy • Acute endocapillary nephritis • Mesangial proliferative GN / segmental GN • Mesangiocapillary GN types I & II • Crescentic GN • IgA nephropathy (Cameron 1966) Looks familiar, doesn’t it ? Another of our failures has been to produce a more analytical and basic classification of GN during half a century

15. One thing we soon had - POISONS... • ACTH 1949 • nitrogen mustard 1949 • cortisone 1950 • prednis(ol)one 1955 • 6-mercaptopurine 1957 • azathioprine 1963 • cyclophosphamide 1964 What made the greatest difference to patients themselves: - effective diuretics(thiazides in 1958, furosemide in 1964) and - hypotensive agents(1954 onwards. ACE inhibitors only 1978)

16. Having worked on diabetes for 3 years, I trained as a nephrologist at Cornell (NY Hospital), on a Fulbright scholarship and an NIH grant of $ 5 000 p.a. awarded to physiologist Prof. Robert F Pitts. He seconded me for clinical research studies to Dr E Lovell Becker. Aboard US flagship “United States” September 1962, on the way to the New World - by boat!

17. Later we evolved a simple two-protein test of permselectivity to predict response to steroid treatment (in nephrotic patients). It was popular for a while. At Cornell in New York, I studied glomerular permeability in nephrotics using multiple endogenous protein clearances Joachim ,Cameron & Becker JCI 1964 Cameron & Blandford Lancet 1966

18. A new clinico-pathological nosology of nephritis from biopsy The idea of different levels of diagnosis emerged, with poor correspondence Cameron 1972 Cameron 1968

19. New descriptive and analytic technology (1970) peristent low complement The Venn diagram allowed expression of complex relationships nephrotic haematuric The life table permitted description of timing of survival and events - now we have relative risk, Mantel and Cox analyses, meta-analysis (1989) etc.etc. Cameron 1972

20. By 1968, a satisfying description of glomerulonephritis had emerged.. • a substantial minority of GN (15%) resulted from anti-GBM antibodies • However, most GN resulted from acute or chronic renal deposition of circulating pre-formed immune complexes, which could mimic most forms of human disease • the antigens in immune complex disease were not related to the kidney, which was an “innocent bystander” • C and polymorphs were the principal - perhaps unique - modes of injury • cell- mediated immunity played no role Frank J.Dixon (1968)

21. How well did human nephritis compare with the experimental models ? The task of aligning the variety of human nephritis with just two contrasting paradigms of experimental nephritis was, after years of effort from 1970, UNsuccessful… Several forms of human nephritis did not fit in: e.g. dense “deposit” disease, focal glomerulosclerosis, IgA nephropathy Many new different ways of experimental induction of glomerulonephritis were described

22. 1960-2010: The clinical paradigm • Today, we still classify human glomerulonephritis using optical microscopy appearances of glomerular injury on renal biopsy: a major failure to advance understanding to more fundamental levels • although great progress has been made in understanding anti-GBM nephritis, this forms only 1% at most of human glomerular disease, and details of the other 99% remain obscure

23. Anti-GBM disease 1960-2004 (1967) “linear” immunofluorescence described in humans with crescentic disease & lung haemorrhage (1967) human serum contains anti-GBM Ab & reproduces disease (1970s) cytotoxic agents lower antibody and improve prognosis (1978) plasma exchange added to treatment (1978) HLA linkage described (now *1501) (1980s) molecular structure of GBM (1994) antigen is in 3(IV)NC-1 collagen (2001) AA sequence of antigen defined 1960-2000: mortality falls from 96% to 6%

24. Anothersuccessstory – lupusnephritis • In the 1950s, almostalllupuspatientswith III-IV nephritisweredeadby 5 years • Corticosteroidsalone in the 1950s hadlittleimpact(Pollak, Karket al.) • Wetriedbothazathioprine (1965) andoral cyclophosphamide(1968) + steroidswhichimprovedresults. Butat a price. • Then, wetrieddifferent “induction” and “maintenance” therapies: cyclo + methylpredfollowedbyaza + low-dose oral pred

25. Lupustreatment 1950-1990 • Theresultwas a dramaticimprovement in results • But still in 2000 after 10 years, of 110 class IV patients 49% hadside-effectsorcomplications, and 30% diedeventually(Bono, Cameron et al. QJM 2001)

26. Lupus in the 21st century • Half a dozenotherdrugshavebeenused to treatlupuswith some effect • The major impacthasbeen monoclonal antibodiesactingonaspectsoftheimmuneresponse • Atthemoment major interestlies in rituximab, withconflictingresults. • Thelatestpaper, fromCondonet al. in London, showed great benefit, withoutthe use ofanymaintenancesteroids. Twocontrolledtrials, however, gavenegativeresults, butmayhaveaskedthe “wrong” questions

27. “Lumpy-bumpy” immune complex disease 1960-2004 • 1973: in situ immune complex formation was described (Mauer, McCluskey): totally ignored • 1978: rat membranous nephropathy deposits shown to form in situ(Couser, Hoedemaeker) • mesangial & sub-endothelial deposits continued to be considered from circulating immune complexes • a tiny number (~ 50) of human cases with demonstrable antigen and antibody in kidney were / are extrapolated to millions of others • VAST amounts of work on circulating complexes proved almost completely fruitless

28. Immune aggregate disease: a continuing mirage ? • Immune aggregates are present in most forms of nephritis in humans, both acute and progressive • Given the possibility that in situ combination of circulating antibody and antigen can take place within tissue, it has been almost impossible so far to prove that undissociated complexes may also localise into the kidney • The relevant antigens remain almost totally unknown • We must ask: do circulating immune complexes play ANY role in the pathogenesis of so-called “immune complex” nephritis ?

29. At last, two new ideas in the 21st century IgG4 disease/nephropathy the main glomerular antigen in the majority of membranous nephropathy patients was finally identified by Larry Beck in David Salant’s lab in 2009: PLA2M receptor. The Ab were Ig4 subclass - as are ADAMTS13 Ab in TTP. C3 nephropathy A re-examination of dense deposit disease, C3-only MCGN etc. has led to a new classification of these entities centering on the dysregulation of the complement cascade, often arising from inhibitor (H or I) mutations. Again, a link to HUS.

30. The mediator explosion I: cells • in 1960-70, only leukocytes and maybe platelets were in on the act • by 1978, macrophages were re-identified (EM in 1953, ignored) first in crescents (Atkins), then in glomeruli • THEN monoclonal antibodies arrived in 1981… bang! • activated macrophages and T cells identified in glomeruli and interstitium in models, and in humans • maybe mast cells involved ? (1990s) Parbtani & Cameron 1974 Nolasco, Cameron et al. 1983

31. Mediator explosion II: soluble factors 1960 : only 2 2013: more than 100, and counting…. first the “old guard” : complement & fibrin 1978: new ideas on complement: C3NeF as an antibody and C5b-9 as mediator in membranous 1975: angiotensin (1955) 1970: “lymphokines” - now cytokines IL1-27, TNF, interferons, etc. 1978: prostaglandins 1980: leukotrienes/lipoxins 1980: chemokines(named in 1993) 1985: PDGF, TGFβ … PAI-1, FGF, HGF, osteopontin 1988: endothelin 1991: nitric oxide 2004: hydrogen sulphideetc.etc. Platelets & PF4 (1979)

32. New perspectives in the 1980s • glomerular hypertension/perfusion is a major driver of proteinuria & glomerular damage (Brenner~1980) • interstitial inflammation and fibrogenesis/lysis determines prognosis (1980), thus cell-mediated injury mainly determines chronicity (1983) Cameron 1981 • loss of renal “innocence”: the kidney itself plays an active role in its immune injury (1985) • proteinuria is not just an indicator of severity, but a nephrotoxin (1988)

33. The interstitium as the field of injury? Interstitial cells in renal injury In the end, does it matter what the glomerular appearances may be, if outcome is dependent on interstitial changes and the degree of proteinuria ?

34. The interstitium as the field of injury? • In GN, tubulo-interstitial events correlate better with GFR than glomerular changes (Hutt & de Wardner 1968) and predict outcome better • In lupus nephritis, the number of monocytes predicts GFR up to 5 years later (Alexoupoulos, Cameron et al 1990)

35. The renal parenchyma: loss of “innocence” I: (1985-) The reaction of the resident renal cells to injury is now seen as crucial. Cell adhesion (1992) as well as cell attraction are important factors. 1988

36. The renal parenchyma: loss of “innocence” II • Renal tubular cells can synthesise and secrete: - complement components - eicosanoids - chemokines - growth factors, etc. • Renal tubular cells can express MHC class II - already known 1981 in Tx literature • Renal tubular cells can process antigen & induce an immune response/tolerance (1985) protective, pathogenic, or both ? can transform into myofibroblasts (1994)

37. Proteinuria emerges as a nephrotoxin • studies of overload proteinuria & podocyte damage were crucial but ignored(Brewer et al 1978-1982; Eddy 1988) • proteinuric urine induces many mediators from tubular cells (1990s) • we still do not know which component(s) of proteinuria are toxic... Brewer 1982 “Therapies designed to reduce proteinuria per se may have a role in the treatment of glomerulonephritis” Cameron 1989 Cameron 1979

38. Proteinuria: how does it happen ? Electron microscopy of the filtration barrier (1950s/60s) Molecular anatomy of the podocyte (1995- ) ...we still don’t know

39. Proteinuria: how does it relate to oedema ? 1962 The overthrow of this “classical” underfill explanation began 50 years ago and was completed in the early 1980s (Brown, Geers & Koomans etc. ). This came from studies in both animals and humans with renal disease 2004 Proteinuria+reabsorption induces tubular sodium retention directly, distally via NaK-ATPase(Doucet) and proximally via activating amiloride sensitive ENaC channel

40. Molecular biology and glomerulonephritis • genes of inherited glomerular disorders • genes which confer risk • sequence of relevant molecules • “knock-out” animals • mRNA analysis, RT-PCR etc. • arrays - a new nosology ? and so on ... Henger et al. KI 2004

41. New specific treatments for GN ? • Very little of the huge accumulation of knowledge about the immune system in the past 50 years has translated into clinical treatments. We still promote the same tired old anti-cancer poisons, plus: - 1976: plasma exchange - 1978: IV methyl prednisolone - 1983: cyclosporine - 1986: polyvalent IgG - 1995: mycophenolate - 1990: monoclonal antibodies, e.grituximab (1999) • But interference with antigen presentation, or the maturation of the immune response, has been explored only in lupus and vasculitis - and then, only to a limited extent…this needs to change

42. Glomerular disease: some “last great problems” from 40 years ago • What is the IgA doing in IgA nephropathy ? • What are the pathogenic antigens in aggregates of “immune complex” GN ? • What is/are the glomerular (auto)antigen(s) in human membranous nephropathy ? DONE  • What triggers minimal change nephropathy ? • What is dense deposit disease ? • What makes proteinurianephrotoxic ? • What drives interstitial damage? And how can we stop it - or promote non-scarring healing ?

43. Glomerular disease: some “last great problems” from 40 years ago • What is the IgA doing in IgA nephropathy ? • What are the pathogenic antigens in aggregates of “immune complex” GN ? • What is/are the glomerular (auto)antigen(s) in human membranous nephropathy ? DONE  • What triggers minimal change nephropathy ? • What is dense deposit disease ? • What makes proteinurianephrotoxic ? • What drives interstitial damage? And how can we stop it - or promote non-scarring healing ? • Is Elvis still alive ?

44. “ When problems in human medicine are being considered, the evidence from man in entitled to at least a little consideration” (Sir) George Pickering (1952) Clinic Laboratory

45. What will Nephrology be like in another 60 years, in 2073? • What is sure is that some of the questions of today will still be unanswered • Also that there will be new techniques and new questions of which we have as yet no idea at all… 1936