“Glomerular” diseases - the past 60 years: what have we learned, and how?

J Stewart Cameron São Paulo

Nephrology Division - University of São Paulo22nd October 2013

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

What can we learn from the story of glomerulonephritis about how knowledge is

acquired and applied in medicine?

animals are not humans, science can mislead, and clinical observations often surprise, and contain vital clues

Clinic Laboratory

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.

“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

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….

• 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...

A time of rapid changeevery 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)

Homer SmithUSA

(1895-1962)

Jean Hamburger France

(1905-1989)

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

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

A world without renal journals

• The first nephrological journal was Minerva Nefrologica, in 1957 - in Italian

• The first English-language journal was Nephron from 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, sitting in 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

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.. “

Thomas Sprat. The history of the Royal Society of London, 1673, p. 74

1673

Renal biopsy- a turning point in study of human glomerular diseases

• thinner (3 - 5 μm) sections (Pirani 1956)• silver staining (Jones 1957)• electron microscopy (Folli, Farquhar etc 1957)• immunofluorescent staining (Kark 1959)

Poul Iversen & Claus Brun (1910- )

Robert Kark (1911-2003)

1951 1954

Then

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

- 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

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)

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!

At Cornell in New York, I studied glomerular permeability in nephrotics using multiple endogenous protein clearances

Cameron & Blandford Lancet 1966

Joachim ,Cameron & Becker JCI 1964

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.

A new clinico-pathological nosology of nephritis from biopsy

The idea of different levels of diagnosis emerged, with poor correspondence

Cameron 1968Cameron 1972

New descriptive and analytic technology (1970)

The Venn diagram allowed expression of complex relationshipsnephrotic

haematuric

peristent low complement

Cameron 1972

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.

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)

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

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

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%

Another success story – lupus nephritis

• In the 1950s, almost all lupus patients with III-IV nephritis were dead by 5 years

• Corticosteroids alone in the 1950s had little impact (Pollak, Kark et al.)

• We tried both azathioprine (1965) and oral cyclophosphamide (1968) + steroids which improved results. But at a price.

• Then, we tried different “induction” and “maintenance” therapies: cyclo + methylpred followed by aza + low-dose oral pred

Lupus treatment 1950-1990

• The result was a dramatic improvement in results

• But still in 2000 after 10 years, of 110 class IV patients 49% had side-effects or complications, and 30% died eventually (Bono, Cameron et al. QJM 2001)

Lupus in the 21st century

• Half a dozen other drugs have been used to treat lupus with some effect

• The major impact has been monoclonal antibodies acting on aspects of the immune response

• At the moment major interest lies in rituximab, with conflicting results.

• The latest paper, from Condon et al. in London, showed great benefit, without the use of any maintenance steroids. Two controlled trials, however, gave negative results, but may have asked the “wrong” questions

“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

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 ?

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.

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

Mediator explosion II: soluble factors

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)

1960 : only 2 2013: more than 100, and counting….

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)

• 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)

Cameron 1981

The interstitium as the field of 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 ?

Interstitial cells in renal injury

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)

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

• 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)

The renal parenchyma: loss of “innocence” II

protective, pathogenic, or both ?can transform into myofibroblasts (1994)

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...

Cameron 1979

“Therapies designed to reduce proteinuria per se may have a role in the treatment of glomerulonephritis”

Cameron 1989

Brewer 1982

Proteinuria: how does it happen ?

Electron microscopy of the filtration barrier (1950s/60s)

...we still don’t know

Molecular anatomy of the podocyte (1995- )

Proteinuria: how does it relate to oedema ?

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

1962

Proteinuria + reabsorption induces tubular sodium retention directly, distally via NaK-ATPase (Doucet) and proximally via activating amiloride sensitive ENaC channel

2004

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

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.g rituximab (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

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 proteinuria nephrotoxic ?• What drives interstitial damage? And how can we stop it

- or promote non-scarring healing ?

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 proteinuria nephrotoxic ?• What drives interstitial damage? And how can we stop it

- or promote non-scarring healing ? • Is Elvis still alive ?

“ 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

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