Lab diagnosis in renal diseases

1. LABORATORY DIAGNOSIS
IN RENAL DISEASES
- Dr. Meghana P

2. INTRODUCTION
Kidney is a highly specialized organ.
Functions:
• ECF volume and composition
• Metabolic waste products
• Blood pressure
• Erythropoietin
• Vit D3

3. FACTORS AFFECTING RENAL
FUNCTION
• Diffuse renal disease
• Pre renal conditions – dehydration, CCF, shock
• Post renal conditions – obstruction

4. INVESTIGATIONS
• Renal function tests
• Urine analysis
• Renal biopsy

5. RENAL FUNCTION TESTS

6. INDICATIONS
• Early identification of renal impairment in patients at risk.
• Diagnosis of renal disease.
• Assess response to treatment.
• Adjust dosage of certain drugs.
• Plan renal replacement therapy in advanced renal diseases.

7. CLASSIFICATION
GLOMERULUS PROXIMAL
TUBULES
DISTAL
TUBULES
Clearance tests Glycosuria Specific gravity
S. Creatinine Aminoaciduria Osmolality
BUN Tubular proteinuria Water deprivation
test
BUN/S.Creatinine Urinary excretion of
sodium
Water loading test
Proteinuria Fractional sodium
excretion
Ammonium
chloride loading test

8. GLOMERULAR FILTRATION
RATE
• Rate at which a substance is cleared from the plasma in unit
time by the glomeruli.
• ml/min

9. GFR - RATIONALE
• Varies according to age, sex, BSA.
• Normal GFR is 120 – 130 ml/min/1.73m2.
• Declines with age.
• Fall in GFR leads to accumulation of waste products.
• GFR < 60ml/min/1.73m2
- >50% loss of renal function.

10. CHRONIC KIDNEY DISEASES
STAGE DISEASE GFR VALUE
(ml/min/1.73m2
)
Stage I Kidney disease
with
Normal GFR >90
Stage II Kidney disease
with
Mildly reduced
GFR
60-89
Stage III Kidney disease
with
Moderately
reduced GFR
30-59
Stage IV Kidney disease
with
Severely reduced
GFR
12-29
Stage V Kidney failure <15

11. METHODS TO MEASURE GFR
• Direct assessment - Clearance tests
• Indirect assessment from S. Creatinine

12. CLEARANCE TESTS
• Volume of plasma that is completely cleared of that substance
per minute.
• C = UV/ P
• C - Clearance (ml/min)
U - Concentration of substance in urine (mg/dl)
V - Volume of urine per min (ml/min)
P - Concentration of substance in plasma (mg/dl)

13. AGENT FOR CLEARANCE
STUDIES
• Not bind to plasma proteins.
• Freely filtered across glomeruli.
• Not reabsorbed
• Not secreted.
• Not metabolized by kidney.
• Excreted only through the kidney.

14. AGENTS USED
EXOGENOUS ENDOGENOUS
Inulin Creatinine
Radiolabelled EDTA Urea
Radiolabelled 125I-iothalamate Cystatin C

15. INULIN CLEARANCE TEST
• Gold standard for measurement of GFR.
Method:
• Bolus dose – 25 ml of 10 % solution.
• 500 ml of 1.5 % solution – 4 ml/ min.
• Timed urine and blood samples are collected.

16. INULIN CLEARANCE TEST
Disadvantage:
• Time consuming
• Expensive
• Need to maintain steady plasma levels
Normal values:
• Males – 125 ml/min/1.73 m2
• Females – 110 ml/min/1.73 m2

17. CREATININE CLEARANCE
TEST
Rationale:
• Most commonly used.
• Produced from creatine in muscle.
• Secreted in small amount – overestimation of GFR.

18. CREATININE CLEARANCE
TEST
Method:
• 24 hour urine sample.
• A blood sample is obtained at midpoint of urine collection.
• Final calculation is by the formula UV/P.
• Cimetidine can be used to prevent overestimation.

20. CREATININE CLEARANCE
TEST
Disadvantages:
• Small amounts of creatinine secreted by renal tubules can
increase even further in advanced renal failure.
• Collection of urine is often incomplete.
• Creatinine levels are affected by -
- meat and muscle mass.
- certain drugs (cimetidine, trimethoprim)

21. CYSTATIN-C CLEARANCE TEST
Rationale:
• Protease inhibitor produced by all nucleated cells.
Advantages over creatinine:
• More sensitive and specific.
• Not affected by sex, diet and muscle mass.

22. INDIRECT ASSESSMENT OF
GFR
Convenient but insensitive.
Creatinine clearance
(140 – age in years) x Body weight in kg
( 72 x serum creatinine in mg/dl)
=

23. BLOOD UREA NITROGEN (BUN)
• Biochemical parameter.
• Adults: 7-18 mg/dl

24. PRODUCTION OF UREA
Proteins
Amino acids
Synthesis of tissue
proteins
Energy Ammonia
Urea Cycle
Excretion in urine
Urea

25. DISADVANTAGES - BUN
• Less sensitive.
• Completely filtered by glomeruli and 30-40 % is
reabsorbed.
• Affected by -
- Protein diet
- Upper G.I haemorrhage
- Liver function
• Considerable destruction of renal parenchyma - blood urea
elevation.

26. METHODS
1. Diacetyl monoxamine method
+ DAM
Yellow diazine derivative
High temp, strong acid, oxidizing
agent
Intensity of color is measured
Urea

27. METHODS
2. Urease Berthelot reaction
Iodophenol
Intensity of color
is measured
Urea
370 C
Urease
Ammonia
Alkaline hypochlorite
Phenol

28. CAUSES OF INCREASED BUN
PRE RENAL RENAL POST RENAL
• Shock
• CHF
• Dehydration
• High protein diet
• Trauma
• Burns
• GI haemorrhage
• Impairment of
renal function
• Obstruction of
urinary tract

29. SERUM CREATININE
Rationale:
• Creatinine is produced in muscles at a constant rate.
• Production is proportional to muscle mass and body weight.
Normal range: 0.7 to 1.3 mg/dl

30. METHODS
1. Jaffe’s method
Creatinine Colored product
Alkaline reagent
+ Picric acid
Spectrophotometer

31. METHODS
2. Enzymatic method
Creatinine H2O2 + phenol + dye
Colored
product
enzymes
Spectrophotometer

32. SERUM CREATININE
INCREASED DECREASED
• Azotemia
• Dietary meat
• Acromegaly
• Gigantism
• Pregnancy
• Old age

33. BUN/SERUM CREATININE
RATIO
• Discriminate different types of azotemia.
• Normal value - 12:1 to 20:1

34. BUN/SERUM CREATININE
RATIO
RATIO >20:1 RATIO <10:1
↑BUN WITH NORMAL
CREATININE
↑CREATININE WITH NORMAL
BUN
High protein diet
Increased protein catabolism
GI haemorrhage
Dehydration
Starvation
Low protein diet
Severe liver disease
↑BUN & CREATININE, BUT ↑IN
BUN IS MORE
↑BUN & CREATININE, BUT ↑ IN
CREATININE IS MORE
Post renal obstruction Acute tubular necrosis

35. PROTEINURIA
Rationale:
• A very small amount of albumin is excreted in urine.
• Microalbuminuria (30 to 300 mg/24 hrs)
• Clinical or overt albuminuria ( >300mg/24 hr)

36. TESTS TO ASSESS PROXIMAL
TUBULAR FUNCTION

37. GLYCOSURIA, AMINOACIDURIA,
TUBULAR PROTEINURIA
Rationale:
• Proximal tubules reabsorb 99% of glomerular filtrate.
• If PCT are non functioning (or these substances are in excess)
they will appear in urine.

38. URINARY CONCENTRATION OF
SODIUM
Rationale:
• Pre renal azotemia – normal sodium absorption.
• Acute tubular necrosis – decreased sodium absorption.
Values:
• Pre renal azotemia: < 20 mEq/L
• Acute tubular necrosis: > 20 mEq/L

39. FUNCTIONAL EXCRETION OF
SODIUM (FENa)
Rationale:
• Better indicator.
• Distinction between pre renal failure and renal failure.

40. Values:
• Pre renal failure: <1%
• ATN: >1%
FENa
Urine Na+
x
Plasma Creatininex
100
Urine CreatininePlasma Na+
= x
FUNCTIONAL EXCRETION OF
SODIUM (FENa)

41. TESTS TO ASSESS DISTAL
TUBULAR FUNCTION

42. SPECIFIC GRAVITY
• Normal value: 1.003 to 1.030
• Depends on – hydration and fluid intake.

43. SPECIFIC GRAVITY
INCREASED DECREASED FIXED
(1.010)
• Proteinuria
• Glycosuria
• Glomerulonephritis
• Urinary tract obstruction
• Decreased renal perfusion
• Diabetes insipidus
• Increased water intake
• Diseases of tubules
• CRF

44. URINE OSMOLALITY
Rationale:
• Most sensitive and most commonly employed method.
• Depends on number of dissolved particles.
• Normal value: 500 - 800 mOsm/kg of water.

45. URINE OSMOLALITY
Application:
• ↓ urine : plasma osmolality - Acute renal failure
• ↑ urine : plasma osmolality - Pre renal azotemia

46. WATER DEPRIVATION TEST
Rationale:
• Measures concentrating ability of kidney with fluid restriction.
• Differentiate between central DI and nephrogenic DI.

47. WATER DEPRIVATION TEST
Method:
Rise in specific gravity and
urine osmolality
Urinary concentrating ability
maintained
No rise in specific gravity and
osmolality
Administer desmopressin
Rise No rise
Nephrogenic DICentral DI
Restriction of water intake

48. WATER LOADING – ADH
SUPPRESSION TEST
Rationale:
• Measures ability of kidney to dilute urine after water loading.

49. Method:
Over night fast
Drink 20 ml/kg of water in 15-20 min
• <80% excreted
• >1.003
• >100 mOsm/kg
• ADH fails to decrease
• >90% excreted
• Specific gravity <1.003
• Urine osmolality <100 mOsm/kg
• Decreased plasma osmolality and
ADH levels
Normal diluting ability
of kidney
Renal function
impairment
Collect urine for next 4 hours

50. AMMONIUM CHLORIDE
LOADING TEST
Rationale:
• Diagnose type 1 renal tubular acidosis.

51. • Urine pH<5.4
• Plasma HCO3
-
normal/ high
• Urine pH > 5.4
• ↓ Plasma HCO3
-
Inconclusive results
Give NH4Cl (0.1 gm/kg)
Normal renal
acidifying ability
RTA
Collect urine samples for
next 6-8 hrs
If pH <5.4 - Acidifying
ability maintained
Urine pH and plasma HCO3
-
levels
Overnight fast
Method:

52. URINE ANALYSIS

53. URINE ANALYSIS
Physical Examination
Volume
Color
Turbidity
Odor
pH
Specific gravity
Biochemical Examination
Proteins
Sugars
Ketone bodies
Bile salts
Bile Pigments
Blood
Microscopy
Cells
Crystals
Casts
Microorganism
Parasites

54. RENAL BIOPSY

55. INDICATIONS
• Nephrotic syndrome
• Acute renal failure
• Systemic diseases
• Sub-nephrotic proteinuria
• Hematuria
• Post transplant

56. CONTRAINDICATIONS
• Solitary kidney
• Multiple cysts
• Acute infection
• Renal neoplasm
• Bleeding diathesis
• Severe hypertension

57. COMPLICATIONS
• Haemorrhage
• AV fistula
• Infection
• Accidental biopsy of another organ or perforation of viscus
• Death

58. NEEDLE BIOPSY
OPEN BIOPSY

59. ADEQUACY
• Two biopsy cylinders
• Length - 1 cm
• Diameter - 1.2 mm
• 10–15 glomeruli

60. • Light microscopy - neutral buffered formaldehyde.
• Electron microscopy - 2-3% glutaraldehyde.
• Immunofluorescence - do not need any fixative.

61. 1. H&E - General
2. PAS - Basement membrane and matrix
3. Trichrome - Fibrosis
4. Silver - Basement membrane and matrix
5. Congo red - Amyloid
STAINING

62. EVALUATION
• Glomeruli
• Tubules
• Interstitium
• Vessels

63. NORMAL GLOMERULUS

64. GLOMERULAR FILTER

65. GLOMERULUS: NO / MINIMAL
HYPERCELLULARITY
DISEASE LM IF EM
MCD Normal glomeruli,
lipid vacuolation in
tubules
Negative Loss of foot
processes, no
deposits
FSGS Focal and segmental
sclerosis and
hyalinosis
IgM, C3 Loss of foot
processes, electron
dense deposits
MGN Diffuse thickening
of capillary wall
Granular
IgG, C3
Subepithelial
deposits (spikes)

66. MINIMAL CHANGE DISEASE

67. MINIMAL CHANGE DISEASE

68. FSGS

69. MEMBRANOUS
GLOMERULONEPHRITIS

70. MEMBRANOUS
GLOMERULONEPHRITIS
SPIKES GRANULAR DEPOSITS

71. GLOMERULUS: WITH
HYPERCELLULARITY
DISEASE LM IF EM
MPGN Double
contour of
GBM
I – IgG, C3
II – C3,
properdin
III – C3, IgG,
Ig M
I - Sub endothelial deposits
II – Dense intra membranous
deposits
III – Sub endothelial and sub
epithelial deposits.
Acute GN Diffuse
proliferation,
leukocytic
infiltration
Irregular IgG, C3 Subepithelial deposits (humps)
Crescentic
GN
Proliferation,
crescents
I- Linear Ig G, C3
II- Granular Ig G,
C3
III - Negative
I - Linear deposits along GBM
II - Sub epithelial deposits
III - No deposits

72. MPGN
TRAM - TRACK

73. MPGN
I - Subendothelial II - Intramembranous

74. ACUTE GLOMERULONEPHRITIS

75. ACUTE GLOMERULONEPHRITIS
IRREGULAR DEPOSITS HUMPS

76. RPGN

77. RPGN

78. TUBULO-INTERSTITIAL
DISEASE LIGHT MICROSCOPY
ATN Dilated, necrosed tubules
Acute
pyelonephritis
Extensive acute inflammation, destruction
of the tubules
Chronic
pyelonephritis
Thyroidization, chronic inflammation,
periglomerular fibrosis

79. ACUTE PYELONEPHRITIS

80. CHRONIC PYELONEPHRITIS

81. VASCULAR CONDITIONS
DISEASE LIGHT MICROSCOPY
Benign nephrosclerosis Hyaline arteriosclerosis, intimal
thickening
Malignant nephrosclerosis Necrotising arteriolitis,
hyperplastic intimal sclerosis
Thrombotic
microangiopathy
Fibrinoid necrosis, thrombi in
renal microvasculature

82. BENIGN NEPHROSCLEROSIS

83. MALIGNANT
NEPHROSCLEROSIS

84. THROMBOTIC
MICROANGIOPATHY

85. RECENT ADVANCES
• Genomics and proteomics.
• Laser-capture micro dissection - selection of glomeruli or a
specific tubular segment for study.
• ISH
Advantages:
• Identification of specific mRNA expression pattern and their
correlation with diagnosis, prognosis and response.

86. SUMMARY
• Renal diseases causes no symptoms until 50-75% of kidney
function is destroyed.
• Lab tests often provide the initial recognition of renal
impairment and can be used to direct further therapy.
• The simplest diagnostic test is the examination of the urine.
• Renal biopsy is performed to establish the specific diagnosis
of renal disease.

87. REFERENCES
• U.Satyanarayana, U.Chakrapani, Biochemistry, Arunabha Sen
Books Pvt. Ltd., 2010.
• Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart MJ,
Shepherd J. Clinical Biochemistry: An illustrated colour text
(3rd Ed). Edinburgh: Churchill Livingstone, 2004.
• Stevens LA, Levey AS. Measurement of kidney function. Med
Clin N Am 2005;89:457-73.
• Pathological basis of disease : Robbins and Cotran, 9th edition.

88. THANK YOU