Proteinuria and paraproteinemias

1. Proteinuria and
Paraproteinemias
Apeksha Niraula
Junior Resident-3rd year
Department of Biochemistry
BPKIHS

2. Objectives
 Proteinuria
 Pathophysiology of Proteinuria
 Types of Proteinuria
 Paraproteinemias
 Types of Paraproteinemias

3. How well is protein conserved by Kidneys?
 In 24-hour, approximately 930 L of plasma containing 7
gm/dL of protein passes through kidneys(~65 kg of
protein), but less than 100 mg appear in urine(0.00015%)
 Filtration is dependent on adequate renal flow; maintained
by vasodilatation and vasoconstriction

4. Glomerular Membrane
 Modified capillary wall comprising
endothelium (50-100 nm pores)
 A cell-less basement membrane and
an outer specialized epithelial cell layer
(55 nm slit diaphragm)
 The whole of the glomerular membrane carries
a fixed net negative charge:
 Due to a Glycosialoprotein coat
 Charge increases in density from the Lamina
Interna towards the Lamina rara Externa with
the greatest density at the slit diaphragm of
the epithelium

5. Glomerular Filter
 Glomerular filter acts as a high capacity ultrafiltration
membrane
 Made up of highly modified vascular endothelial cells, with
only a thin cytoplasm and large pores allowing almost direct
access of filtrate to the basement membrane
 Thus the glomerular wall can be pictured as having two
filtration barriers in series:
 An Inner, charge : dependent membrane; and
 A more external, mainly size selective barrier in the
outer basement membrane
 Glomerular filter acts via two phenomenon size
selectivity and charge selectivity

6. The Glomerular Membrane
Adapted from: Proteinuria: Tubular handling of albumin—degradation or salvation?
Erik I. Christensen & Henrik Birn
Nature Reviews Nephrology 9, 700-702 (December 2013)

7. Adapted
from :
Notes For
USMLE

8. Normal Urinary Protein Content
 Normal Adult excretes < 140 mg/ 24 hour of protein
 Plasma Proteins represent only 25 mg/24 hr of total urinary
protein; of which about half is albumin; remaining protein are
of renal origin, Uromodulin being the major contributor (70
mg/ 24 hour)
 Low-level albuminuria (Microalbuminuria) has prognostic
value both for renal and non-renal diseases
 Proteins of Renal Origin- Tamm- Horsfall Protein, Urokinase,
Secretory IgA

9. Proteinuria
 Proteinuria means the presence of an excess of serum
proteins in the urine (> 500 mg/24 hour)
 Normal protein in urine <150 mg/day
 Approximately 30 mg is albumin
 Rest is secreted by tubules: Tamm Horsfall, IgA,
Haptoglobin, Transferrin, β2 microglobulin

10. 1. “Leaky” Glomerular capillary barrier”
 Allows albumin (and sometimes globulin) to
cross into Bowman’s space
 Seen in Glomerulonephritis
Proteinuria – Mechanisms

11. Glomerular barrier
Tubule
 Normally, the larger proteins are excluded
at the glomerular barrier
 Smaller proteins can pass, but are mostly
reabsorbed

12. Tubule
 Large proteins are able to pass by the
abnormal glomerular barrier
Leaky Glomerular barrier

13. 2. Malfunctioning tubular reabsorption of
smaller proteins
 Albumin excluded at (normal) glomerular barrier
 Sick tubules unable to reabsorb the normally-filtered
smaller proteins
 “Tubular” proteinuria
 eg:- Tubulointerstitial nephropathy

14. Tubule
 Malfunctioning tubules unable to reabsorb the smaller
proteins filtered at the glomerulus
Tubular Reabsorption Malfunctioning

15. 3. “Overflow” proteinuria
 Filtered load of proteins exceeds capacity of tubules
to reabsorb it all
“Filtered load” = plasma concentration X GFR
 Increased plasma concentration: ie:- excess light
chains
 Increased GFR: pregnancy, fever, hyperglycemia

16. Overflow Proteinuria
Glomerular barrier
Tubule
 Filtered load of proteins exceeds the tubular
reabsorption rate (similar to Glycosuria in
Hyperglycemia)

17. Determinants of urine protein excretion
Age, Sex and Diurnal variation
 In neonates, albumin excretion tends to be higher than in
older children and adults: this has been attributed to
greater permeability of the neonatal glomerulus;
Males
 Protein excretion in day > night time

19. Posture
 Ambulatory urine protein excretion is higher than it is
overnight or during recumbency
 Renal biopsies of patients with postural proteinuria
reveal that 8% have unequivocal evidence of well-defined
disease and 45% have subtle alterations in glomerular
structure
 >50% of patients has been shown to have there is reduced
blood flow to the left renal vein during standing owing to
entrapment of the left renal vein
 T/t: Conservative Management with annual assessment of
proteinuria and renal function

20. Exercise
 Exercise-induced proteinuria was discovered over a century
ago in soldiers after marches or drills
 Five –to 100-fold increases in the excretion of proteins such
albumin, transferrin and immunoglobulins have been
observed following 26-mile marathon runs
 Glomerular Pattern, although mixed glomerular and tubular
proteinuria has also been described, which persists for over 3
hours after exercise
 Reason: Some degree of Renal Ischemia owing to
redistribution of blood during exercise has been suggested
a possible mechanism

21. Pregnancy
 Small increase in albumin excretion during the third trimester
(Increased Glomerular permeability)
 Total urine protein excretion increases owing to decreased
renal tubular protein reabsorption
 Detection of significant proteinuria in new-onset
hypertension distinguishes between those pregnancies
with pre-eclampsia and those with gestational hypertension
 Reagent strip 1+ or greater, proteinuria should be
quantitated by a laboratory measure in a spot or 24 h urine
sample
 Significant Proteinuria > 500 mg of Protein excretion

22. Proteinuria in Kidney Disease
 Richard Bright (1836) Association between proteinuria
and kidney disease
 Total urine protein excretion is <150 mg/24 h in adults and
<140 mg/m2/24 h in children, normal concentrations are
often undetectable by chemical methods
 Albumin excretion rate < 20 µg/min

23. Type Pathophysiology Causes
Glomerular Increased glomerular capillary
permeability to protein
Primary and
Secondary
Glomerulo-nephritis
Tubular Decreased tubular reabsorption
of proteins in glomerular filtrate
Tubular or Interstitial
Disease
Nephrogenic
proteinuria
Increased excretion of protein
produced by the kidney (eg:-Tamm
Horsfall protein, N-Acetyl β-D
Glucosaminidase)
Acute Pyelonephritis
(Secretion of IgA)
Proteinuria of
Prerenal Origin
Increased production of low-
molecular-weight proteins
Light chain disease,
Myoglobinuria
Post-renal Proteinuria Obstruction of Urinary Tract or
Inflammation
UTI

24. Proteinuria in staging and prognosis of
chronic kidney disease
 Association of proteinuria with poorer prognosis in people
in the general population and across all stages of chronic
kidney disease (CKD)
 Quantitation of proteinuria (in the absence of a symptomatic
urinary tract infection and preferably using
the first morning urine) is an essential component of CKD
staging
 The decision limit is an Albumin/Creatinine ratio (ACR) >30
mg/mmol(~300 mg/24 h) or urine protein/creatinine ratio
(PCR) >50 mg/mmol (~0.5 g/24 h)

25. Glomerular proteinuria and Nephrotic
syndrome
 In the normal adult, the renal tubules reabsorb about
2–3 g of filtered albumin every 24 hour
 Nephrotic syndrome can be defined as proteinuria severe
enough to cause hypo-albuminemia and edema
 The degree of proteinuria varies but is generally >3.5 g/24
and is accompanied by a plasma albumin
<25 g/L
 However, it should be remembered that the amount of
protein in the urine may decrease as the plasma protein
concentration or the GFR falls.

26. Nephrotic Syndrome
 Massive Proteinuria (Loss of > 3.5 gm/day)
 Hypoalbuminemia
 Generalised Edema
 Hyperlipidemia and Lipiduria

30. Urine protein selectivity and classification of
glomerulonephritis
 Selectivity: based on assumption that there may be differentia
filtration of large molecular weight proteins in glomerular dise
 Protein selectivity is based on a comparison of the relative
clearance of IgG(150 000 Da) and transferrin (69 000 Da)
calculated as follows:
Clearance of IgG = [IgG]U x [Trans]P
Clearance of transferrin [IgG]P x [Trans]U
where:
[IgG ]u = Urine IgG concentration
[Trans]p = Plasma transferrin concentration
[IgG ]p = Plasma IgG concentration
[Trans]u = Urine transferrin concentration

31. Selectivity of proteinuria
 Excretion of relatively low M.W. protein (Albumin or
transferrin) is known as Selective proteinuria while if
excretion is predominately high M.W. protein (IgG, IgM or
α2 macroglobulin) it is Non-selective proteinuria
 Related to relative damage of Glomerular filter
 If there is predominantly loss of charge selectivity
Selective proteinuria
 If there is predominantly loss of size selectivity
Nonselective proteinuria

32. Interpretation of Selectivity Index
 >0.5 - “Non-Selective”
 0.3–0.5 - “Moderately Selective”
 <0.2 - “Highly Selective”
 Highly Selective - Minimal Change disease; Good
Prognosis
 Nonselective Protenuria - Membranous Nephropathy

33. Patho-Physiological Consequences of
Glomerular Proteinuria
 Hypoalbuminemia
 Urinary Albumin Loss
 Loss of Fixed Anionic charge on the glomerular
membrane
 Edema; Salt and Water Retention
 Hypoalbuminemia
 Decrease in Colloid Osmotic Pressure

34.  Abnormalities of Other Plasma Proteins
 Urinary loss of Antithrombin III
 Urinary Loss of Transferrin, Vitamin D binding
Protein
 Hyperlipidemia
 Increase in Total Cholesterol and TG ; Inverse
relationship with Albumin
 Decrease in HDL: Urinary Loss
 LCAT and LPL activity is decreased

35. Tubular Proteinuria
 Protein Excretion in tubular proteinuria is generally
<1–2 g/24 h
 Greater increase in proteins of <60 000 Da
 Majority of filtered proteins and other solutes are
reabsorbed by the proximal tubule
 In renal tubular disease, the reabsorption of protein
together with water, ions, glucose and amino acids is
impaired
 In tubular disease, the increase in excretion of low
molecular weight proteins is far greater than that
found for larger proteins such as albumin

36. Renal disorders associated with tubular
proteinuria
 Renal Interstitium and tubules are principally affected
Primary Tubular Proteinuria
 Large amounts of plasma proteins if being presented to the
proximal renal tubules, absorption of it causes Inflammation
and Secondary tubular damage
 Direct Tubular Toxic effects by Autoimmune or Infective
mechanisms, Drugs, Toxins, Metabolic disorders, pathology
leading to glomerular proteinuria
Tubular Dysfunction

38. Causes of Tubulointerstitial Nephritis
 Drugs
 Analgesics
 Cylcosporine
 NSAIDS
 Heavy Metals
 Lead
 Cadmium
 Metabolic Disease
 Hyperuricemia
 Hypercalcemia
 Tyrosinemia
 Infection
 Neoplastic Disease
 Multiple Myeloma

39. Methods of assessing tubular damage
 Tests of reabsorption of glucose, phosphate, bicarbonate
and amino acids
 High molecular weight protein are commonly used
markers of renal tubular damage
 N-Acetyl β-D Glucosaminidase (NAG) [1,50,000
Da]
 Lactate Dehydrogenase
 γ-glutamyl transferase
 Alkaline phosphatase

40. Low molecular weight protein markers of renal
tubular disease
Characteristics:
 It should have a constant plasma concentration
 Be freely filtered by the glomerulus
 Tubular reabsorption should be near saturation so that small
small reductions in tubular function result in significant
urinary excretion
 Tubular reabsorption should be unaffected by coexisting
glomerular proteinuria
 Should be stable in urine and easily measurable

41. Examples:
 Lysozyme (Muramidase): first low molecular
weight marker of tubular function
 β2- Microglobulin
 Retinol Binding Protein
 α1- Microglobulin

42. Proteinuria of Prerenal origin
 Occurrence in the urine of abnormal amounts of protein
filtered by the glomeruli in the absence of any glomerular
or tubular abnormality
 ‘Overflow’ proteinuria such as Bence Jones proteinuria,
Hemoglobinuria and Myoglobinuria
 Limitations: Abnormally high concentrations of protein at
the glomerulus and in the tubular lumen can cause
glomerular and tubular abnormalities

43. Bence Jones Proteinuria
 Presence of Bence Jones Proteins (22 kDa)
Important indication of the presence of Myeloma
 In approximately 20% of cases may occur in the absence of
a paraprotein band in the serum
 Detection Methods: Classic Heat Test
Bradshaw Test
Electrophoresis supplemented by
Immunofixation

44. Myoglobinuria
 Myoglobin (17.8 kDa)- Heme Containing protein catabolized
by endocytosis and proteolysis in proximal tubules following
glomerular filtration
 Typically, only 0.01 to 5% of filtered protein appears in urine
 Rhabdomyolysis: Causes rapid destruction of striated
muscle; release of Myoglobin to circulation
 Myoglobin Directly Toxic to renal tubules, cause
acute tubular necrosis with acute renal failure

45. Microalbuminuria as a marker of risk
 Microalbuminuria is defined as an albumin excretion rate of
20–200 µg/min (30–300 mg/24 hour or 3–30 mg/mmol
Creatinine)
 Microalbuminuria was coined to describe an increase in
urine albumin that is detectable by sensitive immunoassays
but is below the detection limit of chemical urine protein
methods and dye-binding stick tests
 Diabetic patients with microalbuminuria are at increased
risk of developing dipstick positive proteinuria and CKD

46.  Early identification allows aggressive treatment to improve
glycemic control, blood pressure and plasma lipids and
been shown to improve outcome
 Microalbuminuria in non-diabetic as well as diabetic
populations identifies patients at increased cardiovascular
risk
 Furthermore, microalbuminuria has been found to be a
predictor of outcome in critically ill patients following insults
such as major surgery, trauma or sepsis

48. Paraproteinemias
 A monoclonal immunoglobulin or immunoglobulin light
chain in the blood or urine resulting from a clonal
proliferation of plasma cells or B-lymphocytes
 Class of Ig produced by tumour gives clue to the site of B-
cell development where, malignant transformation has
occurred
eg:- Tumours of Early B-Cells secrete Monoclonal
IgM while tumours of end-differentiated B-cells
secrete IgG or IgA
 Elderly > Young

49. Paraprotein Structure
Heavy Chains
 IgG> IgA > IgM >
IgD
Light Chains
 Kappa
 Lambda

50. Monoclonal Proteins
 Appear as homogenous, compact bands on
electrophoretic separations
 Detectable in serum are intact immunoglobulins; if
there is renal damage, leaks into Urine
 Low Molecular weight fragments and monoclonal free
light chains [Bence Jones Proteins (BJPs)] can pass
through normal glomeruli and appear in urine [In the
absence of Renal Damage, may be detected in serum too]

51. Conditions associated with Paraproteinemias
Category Conditions
Malignant Multiple Myeloma
Plasmacytoma
Waldenstrom’s Macroglobulinemia
Lymphoma
Chronic Lymphoblastic leukemia
Non-Malignant (Asymptomatic) Benign
Transient
MGUS
Non-Malignant (Symptomatic) Amyloidosis
Cryoglobulinemia and Immune Complex
Disease

52. Symptoms Radiological findings
Backache Osteolytic lesions
Lassitude Pathological fractures
Recurrent infection Hypogammaglobulinemia
Clinical syndromes Biochemical Findings
Renal Failure Raised serum total protein
Nephrotic syndrome Raised serum globulin
Peripheral neuropathy Proteinuria
Hyperviscosity syndrome Subnormal Immunoglobulins
Carpal tunnel syndrome Hypercalcemia
Malabsorption Abnormal RFT
Indications for Investigation of Paraproteins

53. Laboratory Investigations of Paraproteins
 Serum Paraprotein and/or Bence Jones proteins (BJP)
 Highly sensitive marker for B-Cell malignancies;
particularly for Myeloma > 95%;
Waldenstorm’s madroglobulinemia (> 10%)
Identification of Paraproteins
 Serum Protein Electrophoresis (SPE) : Detection of
Monoclonal proteins is one of two clear indications for the
measurement of Serum IgG, IgA, IgM concentrations

54. Consensus Recommendation of the International
Myeloma Workshop Consensus Panel
 Protein Electrophoresis Only reliable method for the
detection of Paraproteins in serum and urine
 Abnormal bands in SPE- quantitated by Scanning
densitometry
 Immunofixation should be used to type the monoclonal
band Confirms the Clonality and identify the
heavy and light chains
 Paraproteins are seen in SPE If Concentration is
> 5gm/dl
 May be missed if < 5gm/dl

55.  Suspicion of B-Cell Malignancy with apparently
normal Electrophoresis Immunofixation
 Raised concentration of IgG or IgA, but no clear
staining in βγ region indicates a Polyclonal Increase
 IgD paraproteins and free heavy chains are susceptible to
post-synthetic degradation results in diffuse
paraprotein bands on electrophoresis, may be missed if
present at low concentrations

56. Mistaken Protein Variants for Paraproteins
 Allotypic variants, e.g. α1-antitrypsin, may result in two
bands in a normally homogeneous region
 Haptoglobin–haemoglobin complex as a result of invitro
haemolysis, causing splitting of the normally homogeneous
α2 zone
 Acute phase proteins, e.g. C-reactive protein, increases,
which may result in additional bands being present
 Fibrinogen in plasma or inadequately clotted samples
additional band in the γ region
 Lipoproteins, which may give distinct bands in the β region
in agarose systems

57. Urine Examination
 Urine should be examined in every patient suspected of B-Cell
Malignancy
 Should be done even when there is no serum monoclonal
component detected
 Also important to monitor the amount of BJP during a
patient’s follow-up, even when BJP is not detected at
presentation, it can appear later in the course of the disease
(BENCE JONES ESCAPE)
 Presence of BJP Strong evidence of Malignancy

58. Several proteins can be mistaken for monoclonal proteins in
urine electrophoresis (Particularly in case of Tubular
Proteinuria:
 α1- Macroglobulin
 Lysozyme (γ region)
 Degraded fragments of protein of glomerular origin
 β2- Microglobulin
 Combination of SPE and Free light chain measurement has
has been suggested as a suitable screening protocol for B-
Cell malignancy

59. Immunofixation
 Helps to identify heavy and light chain components
Indications
 Confirms Clonality of a band detected by Electrophoresis
 Tests for ɑ, γ or µ heavy chains and ƙ and ƛ light chains
 Tests for δ and ε heavy chains when a serum shows
monoclonal light chains without a corresponding ɑ, γ or µ
heavy chains
 Presence of Amyloid: Where SPE shows no band
 Investigate the possibility of an apparent paraprotein in
serum or urine caused by a high concentration of another
protein (eg:- Fibrinogen, Complement components, β2-
macroglobulin)

60.  Detect Minimal Residual Disease or complete remission
following hematopoietic stem cell transplantation for
Myeloma when no monoclonal component is seen on
electrophoretic separation
 Immunofixation techniques enhances the sensitivity of gel
electrophoresis both by removing background staining
selectively increasing the amount of protein (by adding
antibodies) in the band of interest

64. Multiple Myeloma
 Commonest of the malignant causes of
paraproteinemia
 Plasma cell tumour; increased
Immunoglobulin production (IgG)
 Disease of Elderly ; 70-80 years, Male>Female
Clinical Features:
 Bone Pain (70%)
 Hypercalcemia (30%)
 Fever (15%)
 Renal Insufficiency (10%)
 Infection (10%)

65. Three Major diagnostic features
 Identification of monoclonal protein in serum or urine
 Presence of neoplastic cells in bone marrow
 Destruction of Bone
 Laboratory Parameters:
 IgG, IgA and BJP: Account for majority of Paraproteins
in Myeloma
 IgD paraproteins occur usually with ƛ Light Chains
 Hyperviscosity Seen with IgA or IgG3 paraprotein, have
tendency to aggregate
 Monoclonal protein concentration > 30g/L

66. Staging System for Multiple Myeloma
 Durie Salmon Staging- previously used
 International Staging System currently used;
Concentration of β2- Microglobulin and Albumin is
used for staging

67. β2-Microglobulin
 Seen in Increased cell turnover, or example malignancy
(particularly lymphoid), acquired immune deficiency syndromes
and inflammatory conditions
 Cleared from the plasma by glomerular filtration followed by
proximal tubular reabsorption and catabolism
 Important prognostic indicator in Myeloma

68. Investigations in Myeloma
 Diagnosis ( 2 out of 3 are required for Diagnosis)
1. Serum and Urine for presence of Paraprotein
2. Bone Marrow Biopsy
3. Skeletal X-ray Survey – Punched Out Lesions

69. Management and Prognosis
 Serum Calcium
 Serum Creatinine and Urea
 Serum Albumin
 Full Blood Count and Film
 Erythrocyte Sedimentation Rate
 Quantitative immunoglobulins
 24 hour urine for UPEP, UIFE
 Serum β2 Microglobulin
 Serum Paraprotein Concentration
 SPE, Serum Free Light Chains
 Skeletal Survey, MRI Spine
 Bone marrow biopsy: H+E, Flow Cytometry, Cytogenetics
 Molecular Studies: FISH, Gene Expression Profiling (GEP), PCR
 Optional: PET, bone
density examination
 Future: Markers for
Apoptosis

70. Monoclonal Gammopathy of Undetermined
Significance
 Benign
 Monoclonal proteins < 30g/L
 Clonal bone marrow plasma cells < 10%
 No End organ damage (No Hypercalcemia, Renal Damage,
Anemia, Bone Lesions)
 No Bence Jones Proteins

71. Waldenstrom’s Macroglobulinemia
 IgM Myeloma is rare
 Waldenstrom Macroglobulinemia is characterized by the
presence of an IgM paraprotein and pleiotropic lymphoid
proliferation in the bone marrow
 Disease of elderly men
 Investigations
 Serum and Urine for presence of paraprotein
 Bone marrow Biopsy
 Lymph Node Biopsy
 Hematologic Investigations

73.  The heavy chain diseases are rare lympho-plasmacytic
malignancies
 Patients have absence of light chain and secrete a defective
heavy chain that usually has an intact Fc fragment and a
deletion in the Fd region
 Gamma, alpha, and mu heavy chain diseases have been
described
Heavy Chain Disease

74. Gamma Heavy Chain Disease (Franklin's Disease)
 Frequently associated with Autoimmune diseases, especially Rheumatoid
Arthritis
 Diagnosis: Anomalous serum M component [Often <20 g/L (<2 g/dL)]
Alpha Heavy Chain Disease (Seligmann's Disease)
 Most common of the heavy chain diseases
 Closely related to a malignancy known as Mediterranean lymphoma
 Alpha heavy chains assesment is difficult
Mu Heavy Chain Disease
 Seen in patients with Chronic Lymphocytic Leukemia
 Presence of vacuoles in the malignant lymphocytes and the excretion of
kappa light chains in the urine

75. Summary
 Proteinuria is a potent risk marker for progression of renal
disease in both non-diabetic and diabetic kidney disease
 Various type of proteinuria has been classified according to
the site of origin (Glomerular, Tubular) or incase of prerenal
origin (Overflow proteinuria)
 Microalbuminuria has been attributed to the assessment of
cardiovascular risk, Diabetic Nephropathy
 Paraproteinemias are most commonly diagnosed by
Electrophoresis scanned by Densitometry, Immunofixation

76. Acknowledgement
 To my Moderator Mr. Binod
Kumar Lal Das Sir for his
support and guidance during
the preparation of the
presentation

77. Thank You