Biochemistry and Renal Function

Kidney

1. Human urinary system: 2. Kidney, 3. Renal pelvis, 4. Ureter, 5. Urinary bladder, 6. Urethra. (Left side with frontal section)7. Adrenal glandVessels: 8. Renal artery and vein, 9. Inferior vena cava, 10. Abdominal aorta, 11. Common iliac artery and vein12. Liver, 13. Large intestine, 14. Pelvis

Functions of the Kidney:Maintaining balance

• Regulation of body fluid volume and osmolality• Regulation of electrolyte balance• Regulation of acid-base balance• Excretion of waste products (urea, ammonia, drugs, toxins)• Production and secretion of hormones• Regulation of blood pressure

The organization of the kidney

Cortex Medulla

Nephron

The first step in the production of urine is called glomerular filtration.

Filtration: the forcing of fluids and dissolved substances through a membrane by pressure occurs in the renal corpuscle of the kidneys across the endothelial capsular membrane (Bowman's) capsule.

- The resulting fluid is called the filtrate.

- Filtration is a passive process.

- The total filtration rate of the kidneys is mainly determined by the difference between the blood pressure in the glomerular capillaries and the hydrostatic pressure in the lumen of the nephron

Glomerular Filtration

% ReabsorbedWater 99.2Sodium 99.6Potassium 92.9Chloride 99.5Bicarbonate 99.9Glucose 100Albumin 95-99Urea 50-60Creatinine 0 (or negative)

Proximal convoluted tubule

• Carries out most of the reabsorption of electrolytes from the filtrate back into circulation

• 75% of sodium and chloride• Water (follows sodium and chloride

passively by osmolality)• Almost all bicarbonate, calcium,

potassium, glucose and amino acids

Loop of Henle• Has a descending limb and ascending limb• Not all nephrons have a loop of Henle

Extends from the cortex down into the medulla and back up again

• It is responsible for creating a hyperosmolar medulla by a mechanism called countercurrent multiplication

•This is necessary for the production of a concentrated urine

Distal Convoluted Tubule• Carrries out the ‘fine-tuning’ of

electrolyte reabsorption or excretion

• Specifically Na+, K+, H+

• Affected by concentration of these in plasma to carry out homeostasis

• Under hormonal control (Aldosterone)

Collecting duct• Carries out the reabsorption of water• Naturally impermeable to water

Passive diffusion under the control of osmolar difference between tubular cells and lumen (created by counter-current system of Loop of Henle)

ADH

If there is a need to conserve water: ADH is stimulated

Causes aquaporins ‘water transporters’ to move to the impermeable membrane to allow water to pass through

Summary of Nephron

• Glomerulus – filters blood

• Proximal tubule – bulk reabsorption

• Loop of Henle – production of osmostic gradient for control of water reabsorption

• Distal tuble – fine tuning of reabsorption

• Collecting duct – water reabsorption (or excretion)

Assessing renal function

• The evaluation of kidney function focuses on assessing the filtering capabilities of the kidney and examining the urine produced for the presence of kidney disease.

• Examining filtering capabilities mainly relies on blood tests to check that waste products are being excreted. The two most widely used markers are urea and most importantly creatinine.

• Examination of the urine relies on testing for substances which should not escape through the kidneys. The most widely used marker is urine protein.

Clearance

• The of the volume of plasma from which a substance is removed by glomerular filtration during its passage through the kidney.

• This is the basis for establishing Glomerular Filtration Rate (GFR)

Glomerular Filtration Rate (GFR)

• Kidneys usually filter ~170L of water each day (120 mL/min)

• Affected by:– Number of nephrons– Blood supply to the nephron– Integrity of the glomerulus

These can be altered in disease and affect the GFR

The volume of plasma that is filtered by the kidneys and from which a substance is completely cleared

per unit of time

Properties of a suitable Marker for assessing GFR

• Endogenous (produced in the body)• Inert (not metabolised)• Continuously produced• Cleared (not reabsorbed or secreted in the kidney)

• Increased levels in blood• Measurable

Creatinine• Creatinine is formed by the

metabolism of phosphocreatine, a high-energy molecule which provides a rapid supply of ATP to muscles. Phosphocreatine is converted spontaneously to creatinine on a regular basis (shown below). Consequently, creatinine is released into the blood and excreted by the kidney as a metabolic waste.

• In a steady state of release and excretion, creatinine is at constant concentration in the blood.

• Normal levels vary according to gender:

– Men: 62-121 uMoles/L– Women: 44-110 uMoles/L

HN NH2

C

H3C CH2

C

O O-

HN NH

C

H3C CH2

C

O O-

P

O

O-

O

CH3

N

NH

NHO

Creatine kinase

ATP ADP

Creatine Phosphocreatine

Spontaneous

Spontan

eous

PiH2O

Creatinine

HN NH2

C

H3C CH2

C

O O-

HN NH2

C

H3C CH2

C

O O-O-

HN NH

C

H3C CH2

C

O O-

P

O

O-

O

HN NH

C

H3C CH2

C

O O-O-

P

O

O-O-

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N

NH

NHO

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NH

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Creatine kinase

ATP ADP

Creatine Phosphocreatine

Spontaneous

Spontan

eous

PiH2O

Creatinine

Renal handling of creatinine

• Glomerulus- Freely filtered• Proximal tubule- Actively secreted (10-20%)• Loop of Henle- • Impermeable to creatinine; not reabsorbed, secreted, or

metabolized• Distal tubule- Impermeable to creatinine; not reabsorbed,

secreted, or metabolized• Collecting ducts- Impermeable to creatinine; not

reabsorbed, secreted, or metabolized

Assessment of Renal Function• Serum Creatinine

– 1 blood sample – convenient, cheap, quick– BUT - not sensitive

Serum creatinine only starts to increase above normal when the kidney ia at about half function with a GFR of ~60mL/min

A small person with a low muscle mass will has a much lower serum creatinine

A body builder will have a much higher serum creatinine

AND affected by muscle mass

Remember, creatinine levels are inversely related to GFR.

A high creatinine meand a low GFR.

Used to calculate Glomerular Filtration Rate

– More sensitive than creatinineat picking up small changes in renal function

– BUT 24 hr urine collection plus serum required– Inconvenient, lots of measurements and calculation = lots

of room for error!

Clearance = U x V

P

[Urine Creatinine (umol/L)] x Urine Volume (mL) = mL/min [Plasma Creatinine (umol/L)] x Collection period (min)

U = Urine creat’concentrationV = Urine VolumeP = Plasma creat’ concentration

•Creatinine Clearance

eGFR

• eGFR (estimated GFR)• Uses serum creatinine to ‘estimate’ GFR

– More sensitive than creatinine– Uses only serum creatinine measurement and a

calculation, therefore only 1 blood sample required – more convenient

– BUT not to be used in certain situations such as acute illness, pregnancy, young and elderly

– As it uses serum creatinine in its formula, still affects by variations in muscle mass

eGFR• Various equations exist• Based on large scale studies that measured GFR or Cr Cl by

gold standard methods and serum creatinine and worked out a formula to calculate these

• Built in several other factors that affect serum creatinine measurement – age, sex, ethnicity, weight

Cockcroft-Gault

MDRD – widely accepted and used as the best formula for estimating GFR

GFR (mL/min/1.72m2) = 175 x [serum creatinine (umol/L) x 0.011312] -1.154 x (age)-0.203

x 0.742 if female

x 1.210 if African American4 variables – serum creatinine, age, sex, ethnicity

Cr Cl (mL/min) = [(140 – age) x weight/0.814 x serum creatinine (umol/L)] x 0.85 if female

Limitations of eGFR

Serum Creatinine = 40 umol/L Serum Creatinine = 180 umol/LSerum Creatinine = 110 umol/L

Calculated eGFR = >90 mL/min/1.73m2

Calculated eGFR = 66 mL/min/1.73m2

Calculated eGFR = 37 mL/min/1.73m2

Actual GFR = 45 mL/min/1.73m2

Actual GFR = 66 mL/min/1.73m2

Actual GFR = >90 mL/min/1.73m2

Specialist tests for GFR • In some situations, a very accurate GFR may be needed• Or in children, where MDRD equation is not validated

• Exogenous markersInulin clearance = Gold standard measure of GFR– Infusion of inulin and urinary clearance– Collect blood and urine samples– Gold Standard measure of GFR

51Cr-EDTA = standard clinical measure of GFR– Injection bolus of 51Cr-EDTA– Collect blood samples– Calculate eGFR from known amount injected and the decrease

in activity over time

• Endogenous markersCystatin C– Protein produced by all nucleated cells – More specific than creatinine– But assay = expensive

Many renal diseases with various glomerular, tubular, interstitial or vascular damage can

cause an increase in plasma urea concentration.

The reference interval for serum urea of healthy adults is 2-7 mmol/l. Plasma

concentrations also tend to be slightly higher in males than females. High protein diet causes

significant increases in plasma urea concentrations and urinary excretion.

Measurement of plasma creatinine provides a more accurate assessment than urea

because there are many factors that affect urea level.

Nonrenal factors can affect the urea level (normal adults is level 5-39 mg/dl) like:

Mild dehydration,

high protein diet,

increased protein catabolism, muscle wasting as in starvation,

reabsorption of blood proteins after a GIT haemorrhage,

treatment with cortisol or its synthetic analogous

Plasma Urea

Urea is the major nitrogen-containing metabolic product of protein

catabolism in humans,

Its elimination in the urine represents the major route for nitrogen

excretion.

More than 90% of urea is excreted through the kidneys, with small

losses through the GIT and skin

Urea is filtered freely by the glomeruli

Urea production is increased by a high protein intake and it is

decreased in patients with a low protein intake or in patients with

liver disease.

Plasma Urea

Clinical Significance

• States associated with elevated levels of urea in blood are referred to as uremia or azotemia.

• Causes of urea plasma elevations:Prerenal: renal hypoperfusionRenal: acute tubular necrosisPostrenal: obstruction of urinary flow

The glomerular basement membrane does not usually allow passage of

albumin and large proteins. A small amount of albumin, usually less than 25

mg/24 hours, is found in urine.

Urinary protein excretion in the normal adult should be less than 150

mg/day.

When larger amounts, in excess of 250 mg/24 hours, are detected,

significant damage to the glomerular membrane has occurred.

Albumin excretion in the range 25-300 mg/24 hours is termed

microalbuminuria

Proteinuria

– Normal < 150 mg/24h. • TYPES OF PROTEINURIA 

– Glomerular proteinuria – Tubular proteinuria – Overflow proteinuria 

Proteinuria

Glomerular proteinuria

• Glomerular proteinuria — Glomerular proteinuria is due to increased filtration of macromolecules (such as albumin) across the glomerular capillary wall. The proteinuria associated with diabetic nephropathy and other glomerular diseases, as well as more benign causes such as orthostatic or exercise-induced proteinuria fall into this category. Most patients with benign causes of isolated proteinuria excrete less than 1 to 2 g/day

Tubular proteinuria

•  Low molecular weight proteins — such as ß2-microglobulin, immunoglobulin light chains, retinol-binding protein, and amino acids — have a molecular weight that is generally under 25,000 in comparison to the 69,000 molecular weight of albumin. These smaller proteins can be filtered across the glomerulus and are then almost completely reabsorbed in the proximal tubule. Interference with proximal tubular reabsorption, due to a variety of tubulointerstitial diseases or even some primary glomerular diseases, can lead to increased excretion of these smaller proteins

Overflow proteinuria 

•  Increased excretion of low molecular weight proteins can occur with marked overproduction of a particular protein, leading to increased glomerular filtration and excretion. This is almost always due to immunoglobulin light chains in multiple myeloma, but may also be due to lysozyme (in acute myelomonocytic leukemia), myoglobin (in rhabdomyolysis), or hemoglobin (in intravascular hemolysis

Microalbuminuria

• Loss of small amounts of albumin in the urine• Measured as a ratio with urine creatinine( Should be less

than ~3 mg/mmol)• Requires a sensitive urine albumin method.• Performed as part of monitoring diabetes.

An increased ratio enables treatment to reduce the risk of progressive kidney damage (Diabetic nephropathy)

Biochemical testing of urine involves the use of commercially available disposable strips When the strip is manually immersed in the urine specimen, the reagents react with a specific component of urine in such a way that to form color

Colour change produced is proportional to the concentration of the component being tested for.

To test a urine sample:

fresh urine is collected into a clean dry container

the sample is not centrifuged

the disposable strip is briefly immersed in the urine specimen;

The colour of the test areas are compared with those provided on a colour chart

Urinalysis using disposable strips

Urine Dip stix

Renal Failure

• Kidneys no longer function properly

• Kidneys unable to excrete waste

• kidneys cannot concentrate urine

• Kidneys cannot conserve electrolytes

• PRERENAL or factors external to the kidney which interferes with renal perfusion (55% cases of ARF) – E.g Dehydration

• INTRARENAL: conditions that cause direct damage to renal tissue (35-40% cases of ARF) – E.g Autoimmune disease (SLE)

• POSTRENAL: mechanical obstruction in the urinary tract (5% cases of ARF) – E.g Bladder outflow obstruction / renal calculi

The excretory function of the kidneys declines over hours or days

Acute Kidney Injury (AKI)

AKI

Main Biochemical signs• Increased creatinine , Hyperkalaemia,

Acidosis, fluid imbalance.

• Pattern of events• Anuria / Oliguria

• Diuresis

• Recovery (not always!)

Chronic Kidney Disease (CKD)

• Kidney damage or GFR <60mL/min for at least 3 months.

Various stages (1-5)

Stages of CKD

Text Book

• Ahmed N (2012) Clinical Biochemistry Fundamentals of

Biomedical Science (ISBN978-0-19-953393-0)

• Marshall et al (2012) Clinical Chemistry accessible via e-book