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Renal Function Tests
Notes on renal function tests… By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes

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Notes on Clinical Pathology Renal function tests By Dr. Ashish Jawarkar Consultant Pathologist Vadodara

OVERVIEW 1. Indications 2. Classification a. Tests for glomerular function i. Clearance tests 1. Inulin clearance 2. creatinine clearance 3. cystatin c clearance 4. urea clearance ii. Blood biochemistry 1. BUN 2. Sr. Creatinine 3. BUN/Sr. Creatinine ratio 4. Urine proteins b. Tests for tubular function i. Tests for proximal tubular function 1. Glycosuria, aminoaciduria, LMW proteinuria 2. Urinary concentration of Na+ 3. Functional excretion of Na+ ii. Tests for distal tubular function 1. Specific gravity 2. Urine osmolality 3. Water deprivation test 4. Water loading – ADH suppression test 5. Ammonium chloride loading test 3. Each test in detail

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* Indications for RFT 1. To identify early renal impairment in patients at risk, such as i. Diabetes mellitus ii. Hypertension iii. SLE iv. UTI v. UT obstruction vi. Older age To diagnose certain renal disorders to asses response to treatment in renal disorders to adjust dosage of chemotherapeutic drugs To plan renal replacement therapy in advanced renal diseases

2. 3. 4. 5.

* Classification

Tests for glomerular function 1. For GFR – clearance tests, indirect clearance 2. Blood biochemistry S. Creatinine, Bl Urea, BUN/S Creat ratio, Proteinuria (Albuminuria and microalbuminuria)

Tests for tubular function For Proximal Tubules For distal tubules i. Glycosuria, i. Specific gravity and Phosphaturia, osmolality Uricosuria, ii. water deprivation test aminoaciduria, LMW iii. water loading test Proteinuria iv. Ammonium chloride ii. Urinary excretion of test sodium iii. fractional sodium excretion

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*Tests to measure GFR GLOMERULAR FILTERATION RATE: Definition: Rate at which a substance is cleared from the plasma in unit time by the glomeruli (in ml/min) Rationale: i. ii. iii. iv. v. vi. vii. Best for assessing excretory renal function Varies according to age/sex/body surface area (BSA) Also depends on renal blood flow and pressure Normal GFR = 120ml/min/1.73m2 GFR declines with age after 40 @1ml/min/year due to progressive glomerular arteriosclerosis Fall in GFR leads to accumulation of waste products – GFR <15ml/min indicates uremia GFR <60ml/min/1.73m2 indicates >50% loss of renal function

Classification of chronic kidney diseases based on GFR: Stage Stage I Stage II Stage III Stage IV Stage V Disease Kidney disease with Kidney disease with Kidney disease with Kidney disease with Renal Failure GFR Normal GFR Mild decreased GFR Moderate dec GFR Severe dec GFR Value (ml/min/1.73m2) >90 60-89 30-59 15-29 <15

TESTS TO MEASURE GFR:

Direct assessment (Clearance Tests)

Indirect assessment from Sr. Creatinine

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(i) 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) Ideal agent for clearance studies: No ideal agent has been found, however the agent used should fulfill most of the following criteria: i. Should not bind to plasma proteins ii. should be freely filtered across glomeruli iii. should not be reabsorbed iv. should not be metabolized by kidney v. should be excreted only through the kidney Agents used: Exogenous i. ii. iii. iv. Inulin radiolabelled EDTA Radiolabelled 125I thiocynate 99 Tc-DTPA i. ii. iii. Endogenous Creatinine Urea Cystatin C

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(A) Inulin Clearance test:
Rationale: 1. Gold Standard for measurement of GFR 2. Neither secreted nor absorbed and is completely filtered by glomeruli Method: 1. Bolus dose is administered followed by constant i.v. infusion for maintaining constant plasma levels 2. Timed urine samples are collected and blood samples are obtained at mid points of urine collection Disadvantage: Rarely used in practice because 1. Time consuming 2. Expensive 3. Need to maintain steady plasma levels

Normal Values: Inulin clearance

Males : 125 ml/min/1.73 m2 Females: 110 ml/min/1.73 m2

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(B) Creatinine Clearance Test
Rationale: 1. Most commonly used for measuring GFR 2. Produced constantly from creatine in muscles 3. completely filtered by glomeruli, not reabsorbed, but is secreted in a small amount – there is overestimation of GFR by 10% 4. Can help in finding out the number of nephrons damaged by disease process Method: 1. 24 hour urine sample is preferred 2. First voided sample is discarded 3. Subsequently all urine passed is collected in containers 4. Next morning voided sample is collected and all containers are sent to laboratory 5. A blood sample is obtained at midpoint of urine collection 6. Cimetidine which blocks renal secretionocan be used to prevent overestimation 7. Final calculation is by the formula UV/P, with adjustment of 10% for secretion

As we can see from the graph, as the creatinine clearance decreases, the remaining nephrons in the kidney decrease Also the dotted line shows that the serum creatinine begins to rise only after 50% of the nephrons are damaged, i.e. serum creatinine though useful is a less sensitive indicator of renal function.

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Disadvantages: 1. small amounts of creatinine secreted by renal tubules can increase even further in advanced renal failure 2. Creatinine level is affected by intake of meat and muscle mass 3. collection of urine is incomplete often 4. Creatinine levels are affected by drugs such as cimetidine, probenecid and trimethoprim that block tubular secretion

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(C) Cystatin – C clearance test
Rationale: 1. It is a protease produced by all nucleated cells of the body at a constant rate 2. It is not bound to proteins, freely filtered by glomeruli and not absorbed Advantages over Creatinine: 1. More sensitive and specific 2. Not affected by sex/diet/muscle mass

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(D) Urea clearance test
Rationale: 1. Urea is freely filtered by the glomeruli but about 40% is reabsorbed 2. Thus it underestimates GFR and is not a sensitive marker

Importance of clearance tests:
As we saw in creatinine clearance graph, BUN and Sr. creatinine are not sensitive indicators of early renal impairment For serum creatinine to rise from 0.5mg/dl (normal) to 1.0 mg/dl, nearly 50% of the renal mass should have been damaged

Clearance tests are more helpful in this scenario of detection of early renal impairment

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(II) Indirect estimation of clearance from serum creatinine value

Creatinine clearance

=

(140 – age in years) x Body weight in kg ( 72 x serum creatinine in mg/dl)

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*Blood Biochemisty

(a) Blood Urea Nitrogen (BUN)
Earlier methods measured only nitrogen content of blood urea. 28 gms nitrogen is present in a gram mole of urea and molecular weight of urea is 60 . So urea: nitrogen = 60:28. ie BUN can be converted to urea by multiplying by 2.14 Newer methods directly measure blood urea. Production of Urea: Proteins

Amino acids

Synthesis of tissue proteins

Energy

Ammonia

Urea Cycle

Urea

Excretion in urine Rationale: 1. Completely filtered by glomeruli and 30-40 % is reabsorbed 2. State of hydration affects estimation 3. Affected by non renal factors such as - high protein diet - upper g.i. hemorrhage 4. Less sensitive – considerable destruction of renal parenchyma has to occur before urea is elevated

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Methods: 1. Direct method (Di acetyl monoxamine method) Urea + DAM High temp, strong acid, oxidizing agent Yellow diazine derivative

Intesity of color is measured

2. Indirect method (Urease Bertholet reaction) 37 C Urea Urease Ammonia Phenol Alkaline hypochlorite Iodophenol

Intensity of color is measured

Normal levels: Normal Adults – 7-18 mg/dl Adults > 60 years – 8-21 mg/dl

Causes of increased BUN: Azotemia – increase in level of BUN/urea Uremia – clinical syndrome resulting from azotemia Pre renal shock CHF dehydration high protein diet, trauma, burns, g.i. hemorrhage Renal Impairment of renal function Post renal Obstruction of urinary tract

1. 2. 3. 4.

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(b) Serum Creatinine
Production of Creatinine: Creatinine is a nitrogenous waste product formed in muscle from creatine phosphate. Rationale: 1. Creatinine is produced from muscles at a constant rate 2. Production is proportional to muscle mass and body weight 3. Its not reabsorbed, secreted in a small amount 4. It is not sensitive (see graph)

The dotted line shows that the serum creatinine begins to rise only after 50% of the nephrons are damaged, i.e. serum creatinine though useful is a less sensitive indicator of renal function.

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Methods: 1. Jaffe’s method Creatinine + Picric acid Alkaline reagent Colored product

Spectrophotometer Picric acid also reacts with glucose, protein and fructose, hence actual level is 0.2 to 0.4 mg /dl lower 2. Enzymetic method

Creatinine enzymes

H2O2 + phenol + dye

Colored product

spectrophotometer Normal Range: Serum Creatinine

Males 0.7 to 1.3 mg/dl Females 0.6 to 1.1 mg/dl

Causes of: Increased serum creatinine 1. Azotemia 2. dietary meat 3. Acromegaly, gigantism Decreased serum creatinine 1. Pregnancy (hemodilution) 2. Old age (decreased muscle mass)

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(c) BUN/Serum creatinine ratio
Normal: BUN:Sr. Creatinine Causes of 12:1 to 20:1

Ratio >20:1 INCREASED BUN WITH NORMAL CREATININE 1. 2. 3. 4. High protein diet Increased protein catabolism G.I. Hemorrhage Dehydration – decreased renal perfusion (Pre renal azotemia)

Ratio <12:1 INCREASED CREATININE WITH NORMAL BUN 1. Starvation 2. Low protein diet 3. severe liver disease In these three conditions, there is increased creatine breakdown in muscles to synthesize proteins – increased creatinine BUN is normal

In these conditions there is increased protein break down – increased BUN Muscle creatine is not broken down – hence no increase in serum creatinine INCREASED BUN AND INCREASED CREATININE BUT INCREASE IN BUN IS MORE 1. Post renal azotemia (obstruction) In this condition there is obstruction to urine flow which pushes urea back into circulation - increase in BUN is more than that of creatinine

INCREASED BUN AND CREATININE BUT INCREASE IN CREATININE IS MORE 1. Acute tubular necrosis

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(iv) Proteinuria
Rationale: 1. Normally a very small amount of albumin is excreted in urine. 2. Earlest evidence of glomerumlar damage in diabetes mellitus is occurrence of microalbuminuria (albuminuria in range of 30 to 300 mg/24 hrs) 3. Albuminuria >300mg/24 hour is termed clinical or overt proteinuria and indicates significant glomerular damage. For details see notes on urine analysis – Protein in urine

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*Tests to assess proximal tubular function:

(i) Glycosuria, aminoaciduria, LMW proteinuria
Rationale: 1. Proximal tubules reabsorb 99% of glomerular filterate. 2. Substances such as glucose, aminoacids and LMW proteins are reabsorbed by PCT. 3. Hence measurine these substances in urine gives us an idea about the function of PCT, if PCT are non functioning (or these substances are in excess) they will appear in urine. 1. Glycosuria – i. ii. in renal glycosuria, glucose is excreted in urine when blood levels are normal due to lesion in tubules Glycosuria can also occur in Fanconi syndrome

2. Generalised aminoaciduria i. many aminoacids are excreted in urine due to proximal tubular dysfunction 3. Tubular proteinuria (Low molecular weight proteinuria) i. substances such as beta 2 microglobulin, retinol binding protein, lysozyme and alpha 1 microglobulin are completely reabsorbed by tubules ii. Detected by urine protein electrophoresis.

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(ii) Urinary concentration of sodium:
Rationale: 1. Used to differentiate between pre renal azotemia and acute tubular necrosis 2. In pre renal azotemia, tubular function is preserved, i.e. reabsorption of sodium is preserved 3. In acute tubular necrosis, tubular function is not preserved, ie. Sodium is not reabsorbed. Values: 1. Pre renal azotemia: Urinary Na+ < 20 mEq/L 2. Acute tubular necrosis: Urinary Na+ > 20 mEq/L

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(iii) Functional excretion of Na (FNa )
Rationale: Measurement of urinary sodium is affected by urine volume (mEq/L)

+

+

Hence to avoid this we can measure the exact quantity of Na+ reabsorbed as a fraction of amount of Na+ filtered to amount excreted

As with above test, this test is used to differentiate between pre and renal azotemia Method: F Na+ = Urine Na+ Plasma Na+ x x Plasma Creatinine Urine Creatinine x 100

Values: 1. Pre renal azotemia - <1% 2. ATN - >3%

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*Tests that assess distal tubular function

(i) Specific Gravity
Rationale: 1. It is the ratio of density of substance to density of fresh water at 4˚C (39˚F) 2. At this temperature density of water is greatest and equals 1gm/dl 3. It means that a substance with specific gravity >1(@4˚C) will sink and <1(@4˚C) will float. Factors affecting specific gravity: 1. State of hydration 2. Tubule concentrating ability 3. Number and nature of dissolved particles – HMW solutes like proteins and glucose affect specific gravity Methods: See notes on urine examination Causes: Increased specific gravity 1. Proteinuria 2. Glycosuria (diabetes mellitus) 3. Nephrotic syndrome 4. urinary tract obstruction with preserved concentrating ability 5. decreased renal perfusion with preserved concentrating ability Normal Value: Specific gravity 1.003 to 1.030 Decreased specific gravity 1. Diabetes insipedus 2. CRF with decreased concentrating ability 3. increased water intake Fixed specific gravity (@1.010) Chronic renal failure

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(ii) Urine Osmolality
Rationale: 1. Osmolality measures the number of dissolved particles in a solution. 2. It is most sensitive and most commonly employed method to find out urinary concentrating ability Method: When solute dissolves in a solvent it leads to 1. Lowering of freezing point 2. increase in boiling point 3. decrease in vapour pressure 4. increase in oncotic pressure

These properties are used while measuring osmolality by a osmometer

Method:

0.1 M sucrose

Final level indicates osmolality

Semipermeable Membrane

as water enters The tube, its level rises

Water

Simple osmometer

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Factors affecting osmolality: 1. depends only on number of dissolved particles 2. it doesnot depend on nature or molecular weight of dissolved particles like specific gravity does Normal: Urine osmolality (24 hour)

500 - 800 mOsm/kg of water With restricted fluid intake - >800 mOsm/kg of water

Application: (Urine : plasma osmolality ratio is calculated, used to differentiate pre renal and renal azotemia) Decreased urine:plasma osmolality ratio (either urine osmolality is decreased or plasma osmolality is increased) Seen in Acute tubular necrosis (decreased concentrating ability) Increased urine:plasma osmolality ratio (either urine osmolality is increased or plasma osmolality is decreased) Pre renal azotemia – preserved concentrating ability

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(iii) Water deprivation test for urine osmolality and specific gravity
Rationale: Measures concentrating ability of kidney with fluid restriction Method: Measurement of urine osmolality and specific gravity

Restriction of water intake for a specified period of time

Measurement of urine osmolality and specific gravity and comparison with earlier values

Rise in specific gravity and urine osmolality (>800 mOsm/kg of water, >1.025)

No rise in specific gravity and osmolality

Urinary concentrating ability maintained Or false positive result*

Administer desmopressin

Rise in sp. Gravity

No rise

Central DI (diabetes insipedus)

Nephrogenic DI

* false positive result is obtained in case of low salt, low protein diet or major electrolyte disturbances

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(iv) Water loading – ADH suppression test
Rationale: Measures ability of kidney to dilute urine after water loading Method: Over night fast

Drink 20 ml/kg of water in 15-20 min

Collect urine at hourly interval for next 4 hours

1. 2. 3. 4.

Measure Specific gravity urine volume osmolality (serum and urine) plasma levels of ADH

Scenario 1 1. >90% of fluid load excreted in 4 hours 2. specific gravity <1.003 after 4 hours 3. Urine osmolality <100 mOsm/kg after 4 hrs 4. ADH level decreased with decreased osmolality

Scenario 2 1. <80% excreted 2. >1.003 3. >100 mOsm/kg 4. ADH fails to decrease

Normal diluting ability of kidney

Renal function impairment OR False negative*

* False negative seen in 1. dehydration 2. cirrhosis 3. Malabsorption 4. adrenocortical insufficiency 5. congestive heart failure

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(v) Ammonium chloride loading test
Rationale: After all the causes of metabolic acidosis have been ruled out

Renal tubular acidosis is the most likely diagnosis This test is done to confirm or rule out renal tubular acidosis

After overnight fast, urine pH should be <5.4

If results are inconclusive , we administer ammonium chloride which increases urinary pH and remeasure

Method: Measure baseline urinary pH and plasma HCO3- levels

Overnight fast and collect urine for next 6-8 hours

Scenario 1 1. Urine pH <5.4 2. plasma HCO3- Normal /high

Scenario 2 1. Urine pH > 5.4 2. Plasma HCO3- low

Scenario 3 Inconclusive results

Normal renal Acidifying ability

Type I renal tubular acidosis

Give NH4Cl orally

Collect urine samples Over next 6-8 hrs

If pH <5.4, acidifying Ability maintained
Notes on renal function tests… By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes