This document provides a review of renal amyloidosis. It begins by defining amyloidosis as a group of diseases caused by the misfolding and accumulation of various proteins. 27 human proteins are known to cause amyloidosis. The kidney is a common site of deposition for several types of amyloidosis. The document reviews the pathogenesis of amyloidosis, determinants of renal deposition, how it causes renal disease, classification, epidemiology including statistics from India, pathology findings including staining techniques, and methods to determine the type of amyloidosis involved.

1. Renal Amyloidosis - A Review
Dr Kiran Kumar M,
DM Senior Resident,
Dept of Nephrology

2. Introduction
• A generic term –
– group of diseases that are caused by the
misfolding and extracellular accumulation of
various proteins
• Misfolded proteins form fibrillar deposits
– Produce pathognomonic green birefringence
when stained with Congo red dye and viewed
under cross-polarized light

3. • Amyloidosis - remarkably diverse
•
– Hereditary or acquired,
– Localized or systemic
– Lethal or an incidental finding
• 27 human proteins with amyloidogenic
potential in vivo have been identified
– ~15 of these proteins cause systemic amyloidosis.

4. Pathogenesis of amyloidosis
• For each of these amyloidogenic “precursor proteins,”
– initial step in amyloid fibril formation is a misfolding event
• Misfolding can result from
– Proteolytic cleavage (e.g., amyloid β protein)
– An amino acid substitution (e.g., transthyretin [TTR])
– Intrinsic properties that become significant only at high
serum concentration or in the presence of specific local
factors (e.g., β2-microglobulin)

5. • Combination of these factors often determines the
amyloidogenic potential of a particular protein
– they are not sufficient to account for the occurrence,
timing, distribution or effects of amyloid deposition in vivo
• Amyloid fibrils
– avidly bind a normal circulating protein of uncertain
physiologic significance called serum amyloid P component
(SAP)
• This observation may be of diagnostic utility
– SAP scintigraphy -IV injection of radiolabeled SAP

6. • Regardless of the protein or the trigger for
misfolding, the misfolded variants are highly
prone to self-aggregation
• Self-aggregation generates protofilaments
that interact to form fibrils
• Amyloid fibrils have a characteristic - β pleated
sheet configuration
– Produces birefringence under polarized light when
stained with Congo red dye

8. Determinants of Renal Deposition of
Amyloid
• Factors that determine the organ distribution
of amyloid deposits - not well understood
– Kidney - frequent site of amyloid deposition in AL,
AA, fibrinogen, lysozyme, apoAII, and, to a lesser
extent, apoAI disease
– In contrast, TTR amyloidosis typically does not
involve the kidney

9. Variety of light chain sequences that are
amyloidogenic
In AL amyloidosis, disease can be restricted to a single
tissue type in one individual and involve as many as
five to six organ systems in another individual
Differences in amino acid sequence of an
amyloidogenic protein
– Familial TTR amyloidosis
• amino acid substitution of methionine for valine at position
30 (Val30Met) - predominant neuropathic involvement
• Val122Ile TTR variant - cardiomyopathy is the major
manifestation

10. Position of the mutation in the apoAI protein
a/w the distribution of organ involvement
– Mutations in the amino terminal portion are -
kidney, liver, heart involvement
– Mutations in the carboxy terminal portion -heart,
skin, laryngeal involvement

11. • Kidney tropism in AL disease
– Specific uptake by mesangial cells
– Does not seem to be a uniform requirement for amyloid
deposition in the kidney
• Other factors that might promote or retard amyloid
formation or deposition in the kidney
•
– Negative charge
– High glycosaminoglycan content of GBM
– Presence of certain proteases that could either render a
protein amyloidogenic or affect stability of amyloid
deposits
– Local PH

12. How Does Amyloidosis Cause Renal
Disease?
• Disruption of tissue architecture by amyloid deposits
• Amyloidogenic precursor proteins, folding intermediates,
and protofilaments have toxicities that are independent of
the amyloid deposits
– Lack of correlation between quantity of amyloid in tissue and
organ dysfunction
– in vitro demonstrations of direct toxicity of amyloidogenic
precursor proteins on cultured cells
– Detection of amyloidogenic precursor proteins in tissue in the
absence of amyloid
– Rapid improvement in markers of organ dysfunction after
treatment induced reductions in precursor protein production

14. Classification
• Based on
– the precursor protein that forms the amyloid
fibrils
– the distribution of amyloid deposition as either
systemic or localized

17. Epidemeology
• Predominantly a disease of mid-to-late life
• Renal amyloidosis is identified in ~4% of adult
renal biopsy samples
– accounts for ~1.6% of pts starting dialysis
• Age-adjusted incidence of AL amyloidosis in the
US and in the UK is estimated to be 5.1–12.8
cases per million persons per yr
• Disease develops in about 2% of individuals with
monoclonal B-cell dyscrasias

18. • AA amyloidosis - reported prevalence in
patients with chronic arthritides is 3–6%
• Duration of latency b/w onset of inflammation
and diagnosis of AA amyloidosis can vary from
less than a year to many decades
– median of approximately 17 yrs

19. Indian scenario
• Chugh et al – 1981
– 233 pts with renal amyloidosis
– Incidence of amyloidosis - 1.01% of 6431
postmortems and 8-4% of 1980 renal biopsies
– Secondary amyloidosis - 87.1 %
– Primary amyloid - 9.4 %
– Amyloidosis associated with multiple myeloma - 3.5 %
– Tuberculosis - commonest predisposing disease
accounting for secondary amyloidosis - 59%
– Chronic suppurative lung disease – 24.1 %.
– RA, chronic osteomyelitis and lepromatous leprosy -
small percentage of pts (2 to 8 %)

20. 1992-2002 2003-2010
No of biopsies 974 1524
Incidence of renal
amyloidosis
1.74% 1.9%
Mean age 38 ± 17.9 39.2 ± 19
Gender predominance Male female
Renal insufficiency in
patients with
renal amyloidosis
n = 2; 12.8% n = 14; 48.2%
Subnephrotic proteinuria 12.8% 48.82%
MC cause of secondary
amyloidosis
Infection (n = 10; 58.8%) Inflammatory disorders (n
= 14; 48.2%)
Prakash J1, Brojen T, Rathore SS, Choudhury TA, Gupta T. Ren Fail. 2012;34(10):1212-
6. doi: 10.3109/0886022X.2012.723514. Epub 2012 Sep 25.

21. Pathology
• Histologic Demonstration of Amyloid
– Usually by Congo red dye
– Congo red–stained amyloid
• Orange-red appearance under light microscopy
• Produces apple-green birefringence under polarized
light
– birefringence results from the ordered intercalation of Congo
red dye into the amyloid fibrils
– this optical property must be present to consider the staining
Congo red positive

26. • ThioflavinT staining - yellow-green fluorescence
– Congo red positivity greatest from sites with clinical
evidence of involvement
– If amyloidosis is suspected, abd fat aspiration rather than
an invasive Bx
– The sensitivity of Congo red staining of abd fat
• 80 to 90% - AL amyloidosis
• 65 to 75% - AA amyloidosis
• substantially lower in many of the familial amyloidoses
– Relatively noninvasive - Salivary gland and rectal biopsies

27. • Determination of the Type of Amyloidosis
– Types of amyloid - indistinguishable by LM or EM
– Most direct method for identifying the amyloidogenic
protein - Mass spectrometry or Amino acid
sequencing of proteins
• Not available routinely
• Usually not necessary unless other approaches are
unrevealing
– The most definitive method used in the clinical setting
• Immunofluorescence
• Immunohistochemical

28. Strong reactivity for λ light chains Weak reactivity for κ light chains

29. • Absence of immunoreactivity of tissue amyloid
for λ or κ light chain, evidence for AL disease –
– demonstration of monoclonal Ig protein in the blood
or urine or clonal plasma cells in the bone marrow
– Immunofixation electrophoresis vs protein
electrophoresis (monoclonal protein is less in AL than
in multiple myeloma)
– Nephelometric quantification of serum free light
chains
• presence of a monoclonal protein
• disease progression
• response to treatment

30. • Renal impairment-
– Ratio of the serum conc of two light-chain
isotypes rather than the absolute serum
concentrations
• Bone marrow biopsy – assessing
– plasma cell clonality
– plasma cell burden

31. • Renal Pathology
• Risk for procedure-related bleeding as a result
of vascular fragility- little evidence
• Glomerular deposition typically predominates
– appears as amorphous material in the mesangium
and capillary loops
• Substantial mesangial deposition produce nodules – vs
diabetic nephropathy or LCDD
– periodic acid-Schiff (PAS) and methanamine silver-
negative ( not extracellular tissue)

32. • Amyloid deposition in the tubulointerstitium
produces tubular atrophy and interstitial
fibrosis
• Small proportion of pts
– glomerular deposition is scant or absent
– amyloid is confined to the tubulointerstitium or
vasculature

33. • Immunofluorescence or immunohistochemical
studies arenegative for intact Ig, complement,
and fibrin
– AL disease – will reveal Ig light chain
• Reactivity - restricted to a single light chain
isotype
– Some degree of background staining for light
chains, albumin
• Absence of reactivity for either or light chain
does not rule out AL disease

34. • Commercially available reagents do not always
detect amyloidogenic light chains because of
– conformational change or fragmentation that
masks or eliminates the relevant epitopes
• AA amyloid - detected with available
antibodies against AA protein
• Loss of Congo red staining after treatment
with potassium permanganate is a property of
AA amyloid

35. • Electron microscopy
– Amyloid appears as nonbranching fibrils with a
diameter of 8 to 10 nm
– randomly arrayed without a specific orientation in the
mesangium, basement membranes, interstitium, and
vessels
– electron micrographic appearance of amyloid fibrils is
sufficiently characteristic that, if present, thediagnosis
of amyloidosis should continue to be considered even
when Congo red staining is negative

37. • D/D of monoclonal Ig light-chain disorders
• LCDD
– Congo red–negative deposits - granular pattern along the
glomerular and tubular basement membranes
– Immunoreactivity with anti-λ or anti- κ light-chain antibody
usually is positive
• light-chain epitopes are maintained to a greater extent in LCDD than
in AL amyloidosis
– In LCDD, the κ light-chain isotype is more common than the λ
isotype
– Compared with AL amyloidosis, there usually is less background
staining with nonpathologic lightchain isotypes or albumin
– PAS staining is much more intense than in amyloidosis
• deposition of light chains stimulates production of collagen and other
extracellular matrix components; as a result,

38. • Cast nephropathy or myeloma kidney –
– Light chains form intratubular casts
– light-chain casts are PAS negative
– they are highly refractile, and they often appear
lamellated and fractured
– Inflammatory cell infiltration and, in some cases,
granuloma formation often are present in the
interstitium that surrounds the affected tubules

42. Clinico-pathologic Correlates
• Proteinuria
– Present in the majority
– Ranges from subnephrotic to massive with urinary protein
excretion rates as high as 20 to 30 g/d
– Composed mostly of albumin
– Proteinuria usually is accompanied by other components of the
nephrotic syndrome
– Hypoalbuminemia can be profound
– Edema is severe and refractory to diuretics
• Cardiac and autonomic nervous system involvement can cause
hemodynamic fragility that limits the effectiveness or tolerability of
diuretics

43. • When amyloid is confined to the
tubulointerstitium or vasculature
– Proteinuria is minimal and reduced GFR is the
principal clinical manifestation.
– Renal impairment tends to progress less rapidly
• Normal or low BP
– Vascular involvement often is accompanied by
hypertension

44. • Unusual manifestations
– Nephrogenic diabetes insipidus caused by amyloid
deposition in the peri-collecting duct tissue
– Fanconi’s syndrome- injury to proximal tubular cells by
filtered light chains
• Amyloidosis can cause enlargement of the kidneys
– However, in most patients, the kidneys seem to be of
normal size by imaging studies
• Clear relationships b/w extent of amyloid deposition
evident by kidney Bx and severity of clinical
manifestations have not been demonstrated

46. Treatment
• The goal of current treatment approaches for
AL amyloidosis is to eradicate the clonal
plasma cells that produce the amyloidogenic
light chain
• Depends on whether pts are eligible to pursue
high dose melphalan followed by autologous
hematopoietic cell transplantation (HCT)

47. • Criteria for eligibility for HCT
– Physiologic age ≤70 years
– Troponin T <0.06 ng/mL
– NT-proBNP <5000 ng/L
– Creatinine clearance ≥30 mL/min (unless on chronic stable
dialysis)
– Eastern Cooperative Oncology Group (ECOG) performance
status ≤2
– New York Heart Association functional status Class I or II
– No more than two organs significantly involved (liver,
heart, kidney, or autonomic nerve)
– No large pleural effusions
– No dependency on oxygen therapy

50. • Achieving a haematologic response in AL
amyloidosis translates into improved overall
survival
• Complete haematologic responses are a/w
best clinical outcomes

52. • Complete hematologic response
– Improved survival
– Improvements in the function of affected organs
Complete hematological response Persistent hematological disease

53. Bortezomib-based regimens
• Newly diagnosed or relapsed/refractory AL
amyloidosis
– Proteosome inhibitors (eg bortezomib) in combination
with other agents
• Demonstrated promise with rapid responses seen
in a majority of pts
– 43 pts - cyclophosphamide, bortezomib,
and dexamethasone (CyBorD) - hematologic response
rate of 81 %
• Median follow-up of 14 mths, the estimated 2-year
progression-free survival was 67 percent and 41 percent for
newly diagnosed and relapsed patients, respectively

54. – 17 patients with AL amyloidosis treated with
another dosing variant of CyBorD, 16 patients
demonstrated a hematologic response (12
complete) with a median time to response of two
months
• Relapsed disease –
– the use of bortezomib-based or
immunomodulatory-based regimens is a
reasonable approach
– There are no good data to determine which of
these regimens will be of most benefit

56. Treatment of AA amyloidosis
• Current treatment approach for AA
amyloidosis is to treat the underlying
inflammatory disease
– Reduce the levels of SAA
• Suppression of inflammation can result in
reduction in the clinical manifestations of
amyloidosis and improved survival

57. • AA amyloidosis–associated kidney disease
– cytotoxic agents or TNF antagonists
• MOA:
– Suppression of SAA production and resultant
reduction in AA amyloid formation
– additional anti-amyloid effects through
• suppression of cytokine production or
• by altering the expression of specific mediatorsof
amyloid fibril–induced cellular toxicity

58. • Cytotoxic and immunosuppressive agents
– Azathioprine, chlorambucil, methotrexate,
and cyclophosphamide
– Only few reports
– 6 monthly cycles of cyclophosphamide
• Anticytokine therapy
• Much of experience – in RA pts and on first-generation
TNF-alpha antagonists (ie, etanercept and infliximab)
• Less information is available regarding the newer TNF-
alpha antagonists -adalimumab, certolizumab pegol,
and golimumab

59. • Effectiveness enhanced by pulse therapy with
glucocorticoids for induction
• Regression of tissue amyloid occurring as early
as three months
• Humanized anti-IL-6 receptor antibody –
tocilizumab
– IL-6 blockade may be more efficient than TNF
blockade in normalizing SAA levels in pts with
rheumatic disease

60. • Eprodisate - a negatively charged sulfonated
molecule with in vivo activity against
experimentally induced AA amyloidosis
– Interfere with interactions between GAGs and amyloid
proteins
• Tafamidis and Diflunisal
– stabilizes circulating transthyretin in its normal
conformation
– Used in ATTR amyloidosis (familial amyloid
polyneuropathy, hereditary amyloid cardiomyopathy
and senile systemic amyloidosis)

61. • Amyloidosis-Associated ESRD
• Large cohort of pts with amyloidosis-asstd
dialysis dependence
– Median survival after initiation of dialysis was 8.5
months
– Mortality – cardiac amyloidosis and malnutrition
–
• Dialysis dependence, in and of itself, should
not preclude aggressive treatment that aims
to reduce ongoing amyloid production

62. • Appropriateness of offering HDM/SCT to dialysis-
dependent patients with AL amyloidosis
– Hematologic response rate and treatment-asstd
mortality are similar in dialysis-dependent pts
compared with the overall population of pts who
undergo this treatment
• Kidney transplantation in amyloidosis-associated
ESRD in pts with AA amyloidosis
– Outcomes that were worse as well as outcomes that
were similar compared with the general renal
transplant population
– Recurrence of 71%

63. • AL amyloidosis
– kidney transplantation is a good option for
patients who attain a complete hematologic
response and do not have significant extrarenal
disease

64. Dialysis-related amyloidosis
• Levels of β2-microglobulin are increased in
patients on dialysis
• β2-microglobulin is preferentially deposited in
articular and periarticular structures
• Clinical manifestations are largely confined to
the locomotor system
– shoulders, knees, wrists and the small joints of the
hand, and results in swelling, chronic
tenosynovitis and occasionally haemarthroses

65. – Spondyloarthropathies and cervical cord
compression
– deposition of β2-microglobulin within the
periarticular bone results in the appearance of
subchondral erosions and cysts, which can
contribute to pathological fractures,
• Manifestations outside the musculoskeletal
systemic are rare
– congestive cardiac failure, gastrointestinal
bleeding, perforation and pseudo-obstruction,

66. • Treatment –
• Renal tx -only effective treatment
• Greater removal of β2-microglobulin is
attained in patients undergoing high-flux
haemodiafiltration