VET343 Urinary Lecture Notes 2014

VET343 Lecture notes to the Urinary System Clinical Pathology

VET 343 - 2014 (Mary McConnell and Tibor Gaal) 1.

VET343 Urinary Lecture Notes 2014

LEARNING OBJECTIVES

Given the information provided in the study guide, at the end of this section you should be able to:

1. Use your knowledge of the normal structure and physiology of the urinary system to select and interpret appropriate laboratory tests for urinary tract disease and dysfunction.

2. List the major extrarenal factors which influence renal function and describe the effect of each of these factors on renal function.

3. List the consequences of renal failure and explain their pathophysiological bases.

4. Name the two major components of laboratory evaluation of the urinary system.

5. Describe the different means of collecting a urine sample and explain the advantages and disadvantages of each of these.

6. Describe the major physical characteristics of urine.

7. Explain how specific gravity is measured and how the measurement of urine specific gravity is related to the hydration state of a healthy animal.

8. Describe the different types of inappropriate urine concentration.

9. Explain how the biochemical tests that can be done on urine using urine dipsticks are interpreted in the diagnosis of urinary system disease.

10. List the cells, microorganisms and other objects that may be found on the microscopic examination of a urine sediment and explain the significance of the presence or absence of each

of these.

11. List the tests of renal function.

12. Explain the metabolism and excretion of urea and creatinine.

13. Define prerenal, renal and postrenal azotemia and explain how laboratory tests can be used to distinguish between them. Describe the physiological basis of the water deprivation and

exogenous AVP (ADH) tests.

14. List the indications for and advantages of measurement of the urine protein:creatinine ratio.

15. Explain the role that laboratory testing can play in the early detection of chronic kidney disease.

16. Explain what a urine fractional clearance ratio is.

17. Explain why it would be advantageous to have access to a practical means of measuring GFR.

18. Define the nephrotic syndrome.

Note: The background review information provided in pages 2-9 of these notes is for review purposes

only. It will not be specifically covered in the lectures and is not directly examinable. However, a good

working knowledge of renal physiology is essential for understanding the diagnostic tests used in the

investigation of urinary system disease.

The Urinary System Clinical Pathology

2. VET343 2014 (Mary McConnell and Tibor Gaal)

THE URINARY SYSTEM

Odds & Ends

Albumin mg/dL x 10 = g/L

ARF = acute renal failure

AVP = arginine vasopressin = antidiuretic hormone = ADH

BTA = Bladder tumour antigen

BUN = urea nitrogen; mg/dL x 0.357 = mmol/L urea

CDI = central diabetes insipidus

CKD = chronic kidney disease

Creatinine mg/dL x 88.4 = mol/L

CRF = chronic renal failure

EPO = erythropoietin

FCR (urine) fractional clearance ratio = FER

FER = (urine) fractional excretion ratio = FCR

ECF = extracellular fluid

GFR = glomerular filtration rate

hpf = high power field (usually x 40)

lpf = low power field (usually x 10)

NDI = nephrogenic diabetes insipidus

Phosphorus mg/dL x 0.3329 = mmol/L

PTH = parathyroid hormone

PU-PD = polyuria and polydipsia

TCC = transitional cell carcinoma

Urea mg/dL x 0.1665 = mmol/L

1,25 vitamin D = 1,25-dihydroxycholecalciferol, 1,25-(0H)2 vitamin D, calcitriol

The urinary system is central to the investigation of almost all other body systems and it is essential to

have a good understanding of renal function and the assessment of renal function. The kidney is an

important effector organ through which many hormones exert their effects. For example, fluid balance

is regulated in part by the actions of arginine vasopressin (AVP, previously called antidiuretic hormone,

ADH) and the glucocorticoids on the kidney; plasma osmolality and electrolyte balance are partly

regulated by the effect of aldosterone; and calcium and phosphorus homeostasis is regulated by

parathyroid hormone (PTH) and activated vitamin D (1,25-dihydroxycholecalciferol, 1,25-(0H)2

vitamin D, calcitriol). The urinary system is defined as consisting of the kidneys and the lower urinary tract. The lower urinary

tract includes those structures that are not involved in the active formation of urine; the renal pelvices,

ureters, bladder and urethra. Functions of the Urinary System

1. Excretion of waste products and toxins from the body through glomerular filtration and the formation of urine.

2. Maintenance of the plasma extracellular fluid (ECF) volume (isovolaemia) and composition; i.e. regulation of plasma and ECF osmolality (isoosmosis).

3. Recovery and recycling of potentially valuable metabolites such as glucose and amino acids. 4. Regulation of acid-base and electrolyte balance (isohydria and isoionia). 5. Calcium (Ca) and phosphate (P) homeostasis. 6. Hormone synthesis. 7. The function of the lower urinary tract is to store and voluntarily discharge the urine formed by the

kidneys.



Renal Structure and Perfusion

The functional unit of the kidney is the nephron and most renal functions are the sum of the activities of

thousands of nephrons. The healthy kidney has a remarkable ability to retain a constant blood flow (and

therefore constant glomerular filtration rate, GFR) over a wide range of arterial blood pressures; this is

autoregulation. The number of nephrons varies between species, with larger species having more

nephrons than smaller species. For example, the cat has 190,000/kidney and the dog has 430,000/kidney (regardless of breed).

The kidneys have a rich blood supply and normally receive 15-20% of the cardiac output: i.e.

the rate of vascular perfusion is higher than most other tissues.

Glomerular Filtration

In summary, perfusion of glomerular capillaries with blood under pressure results in the passage of

material (filtrate) through the capillary walls into Bowman's space. This glomerular filtrate is then

reduced in volume and modified to form urine as it passes down the renal tubule by:

Reabsorption of components from the tubular lumen into the tubular cells and then back into the body.

Secretion of components from the tubular cells into the filtrate in the tubular lumen.

Glomerular Filtrate

The glomerular filtrate is an ultrafiltrate of plasma; i.e. it has a similar composition to plasma except that it is almost free of proteins.

Proteins with a molecular weight lower than albumin (68,000 Da) are filterable.

Negatively charged proteins are less easily filtered than those with a positive charge.

The capillary endothelium provides a barrier to red and white blood cells and the basement membrane is impermeable to macromolecules.

Formation of the filtrate is a passive process with the filtration rate of the kidneys being mainly determined by the difference between the blood pressure and oncotic pressure in the

glomerular capillaries and the hydrostatic pressure in the lumen of the nephron, as well as the

nature of the glomerular basement membrane and the number of nephrons.

Afferent

arteriole

70 mm

BP

Hydraulic

pressure

28 mm 36 mm

plasma oncotic pressure

(increases as fluid leaves)

18 mm pressure in

Bowmans space

Oncotic pressure

Efferent

arteriole



Tubular Modification of the Glomerular Filtrate

The renal tubules modify the glomerular filtrate to achieve body homeostasis for water and electrolytes

(death from hypovolemia and electrolyte depletion would occur in less than one hour if all the glomerular

filtrate formed were lost from the body). A volume equivalent to the extracellular fluid volume is filtered

every two hours ......... anything which interferes with this filtration has enormous potential for affecting

water, salt and H+ balance as well as the excretion of waste products. In humans, failure to absorb 1%

of glomerular filtrate would cause loss of 2 litres of fluid loss in a day.

Proximal Tubule (PT)

The PT is lined by thick cuboidal cells with a prominent brush border on the luminal side

There is active reabsorption of sodium, potassium, glucose, amino acids, phosphate, -hydroxybutyrate, acetoacetate, pyruvate, and other cations and anions.

Water passively follows the active transport of solute and the filtrate remains isoosmotic with plasma (the volume of filtrate is decreased by 60% in this segment).

85% of filtered HCO3- is reabsorbed in the PT by a complex system of ion dissociations and

exchanges.

Loop of Henle

The loop of Henle is responsible for the countercurrent concentrating mechanism which is

dependent upon:

selective segmental permeability or impermeability to solute and water

an energy-dependent active transport mechanism

medullary hypertonicity

The following sequence occurs as fluid passes through the loop of Henle:

When tubule fluid enters the loop of Henle from the PT, it is isotonic to plasma.

The descending loop of Henle is freely permeable to water and, to a lesser extent, solute; therefore water moves out of the tubule lumen into the interstitium down a concentration

gradient and the tubular fluid becomes hypertonic.

The ascending thick limb of the loop of Henle is impermeable to water and actively pumps chloride out of the tubule to generate hypotonic tubular fluid by the time it enters the

collecting duct.

Sodium follows chloride to maintain electrical neutrality; it is estimated that 25% of the filtered load of sodium is absorbed by this chloride transport mechanism.

The active transport of chloride represents the primary source of energy expenditure for both the concentration and dilution of urine.

Distal Tubule (DT) & Collecting Duct (CD)

The DT is the site of fine control over the composition of the tubular fluid.

Several DT's coalesce to form the CD which discharges urine into the renal pelvis.

Urine entering the DT is hypotonic relative to plasma, 90% of the filtered Na+ and 80% of the filtered water has been reabsorbed by this point.

Depending on the hydration state of the animal, urine in the DT may remain hypotonic or be returned toward hypertonicity.



The permeability of the distal segment of the DT and the CD to water is controlled by arginine vasopressin (AVP, antidiuretic hormone, ADH) via V2 receptors. In the presence of

AVP the permeability of the membrane is greatly increased and water moves passively into

the hypertonic interstitium of the medulla resulting in increased concentration of the urine.

In the absence of AVP the membranes are impermeable to water and dilute urine is excreted.

AVP also enhances the diffusion of urea from the CD into the medulla (this is important in the establishment and maintenance of medullary hypertonicity).

In addition to its role in the concentration and dilution of urine, the DT is also very important in the regulation of K

+ and H

+ excretion by the kidney; K

+ and H

+ ions are secreted by the

DT.

Na+ reabsorption in the DT and CD is controlled by both aldosterone and aldosterone-independent mechanisms.

Ammonia is secreted and it also buffers H+ ions as NH4+.

Conservation and Excretion of Analytes

Net Result of Normal Renal Function on Plasma Analytes

Conserved Excreted

H2O

Glucose

Amino acids

Proteins

Na+

Cl-

HCO3-

Ca2+

(except in horses on Ca2+

-rich diet)

Mg2+

Urea

Creatinine

PO4

K+

H+

(mostly bound to PO4 or in NH4+)

NH4+

Lactate

Acetoacetate

-Hydroxybutyrate Bilirubin Hemoglobin dimers

Myoglobin

From: Fundamentals of Veterinary Clinical Pathology (2008), Stockam, SL & Scott, MA. Page 417

Glucose

Glucose passes freely through the glomerular filtration barrier.

At normal blood glucose levels, reabsorption of glucose is nearly complete within the first 20% of the PT; glucose absorption at the luminal surface involves secondary active transport

that is coupled to sodium reabsorption. This is active transport because in healthy animals this

is against a concentration gradient.

The kidney has definite limits for glucose reabsorption and glucosuria will occur when the concentration of glucose in the filtrate exceeds the tubular maxima (Tm) for the glucose

carrier proteins. (Tubular maxima: the concentrations of solutes at which the renal tubules are

working at full capacity and further increases in concentration will not increase the function.)

Renal glucosuria occurs in animals in which the glucose transport mechanism is absent or defective (e.g. Fanconi syndrome in the Basenji, renal glucosuria in the Norwegian elkhound

and Scottish terriers). True renal glucosuria is not common.



Amino Acids and Proteins

In nearly all species, over 99% of filtered amino acids are reabsorbed by specific mechanisms in the tubules (mostly in the early PT); the exception is the domestic cat in which the

sulphur-containing amino acid felinine (precursor of the putative cat pheromone) is excreted

in the urine.

Several distinct transport mechanisms for amino acids exist and genetic absences of specific transport systems have been reported (e.g. Fanconi syndrome in the Basenji, cystinuria in the

dachshund, basset hound, bull dog, Chihuahua, Yorkshire and Irish terriers).

Small peptides are hydrolysed at the brush border and reabsorbed as amino acids.

Larger proteins (including albumin) enter the tubular cells through endocytosis and then broken down to amino acids.

Hemoglobin and myoglobin are also reabsorbed and can cause tubulopathy

Sodium (Na+ )

Na+ is the major extracellular cation and a large amount of sodium is filtered daily

Approximately 75% of the Na+ is reabsorbed in the PT.

In the PT, Na+ movement into the cell is passive while sodium extrusion out of the cell across the basolateral membrane involves Na

+-K

+ ATPase active transport.

Water passively follows the reabsorption of Na+ according to osmotic gradients.

Additional Na+ is removed from the tubular fluid in the ascending loop of Henle.

The reabsorption of sodium in the PT and loop of Henle occurs regardless of body needs.

The diuretic furosemide acts on the thick portion of the loop of Henle to prevent chloride, sodium and water reabsorption.

Further active reabsorption under the control of aldosterone occurs in the DT and CD and it is in these areas that control of body Na

+ balance occurs.

There is also aldosterone-independent reabsorption in the DT and CD via a Na+-Cl- cotransporter (thiazide diuretics block this mechanism).

Na+ resorption in the distal nephron is reduced during volume expansion through the action of atrial natriuretic peptide (ANP).

However, diseases of the PT or loop of Henle may result in flooding of the distal segments such that their capacity to absorb Na

+ is exceeded.

Chloride (Cl- )

Cl- appears in the glomerular filtrate in the same concentration as in the blood and is reabsorbed according to the body's needs.

Reabsorption (75%) occurs passively in the PT down a concentration gradient.

Cl- is reabsorbed by active transport in the loop of Henle in conjunction with the transport of Na

+.

Potassium (K+ )

K+ is the major intracellular cation, high intracellular concentrations are maintained by the activity of the Na

+-K

+ ATPase which pumps Na

+ out of cells and K

+ into cells.

K+ appears in the glomerular filtrate in the same concentration as in blood and 70% is reabsorbed in the first two thirds of the PT.

K+ moves passively from tubular cells into the lumen of the DT down a concentration and electrochemical gradient through K

+-channels that are opened by aldosterone (release of

aldosterone is stimulated by hyperkalemia).

Movement down this concentration gradient is enhanced when urinary flow rate through the tubules is high; this rapidly removes the K

+ and the concentration gradient is maintained.



Conversely, lower flow rate reduce the gradient and decreases the excretion of K+. This is partly compensated for by the promotion of K

+ excretion by ADH (AVP).

Adaptation to chronic increase in potassium intake correlates with an increase in Na+-K+ ATPase activity in cells of the distal nephron; this increases the ability of the DT to excrete

K+.

In chronic renal failure the kidney adapts to excrete a much larger percentage of the filtered load of K

+; this allows increased K

+ excretion per remaining amount of nephron mass (it is

important to avoid hyperkalemia).

The amount of K+ that can be secreted by DT cells is dependent upon the cellular [K+ ] rather than the serum K

+ concentration, both of which are affected by factors such as K

+ loading,

acidosis, alkalosis.

Inorganic Phosphorus (Phosphate)

Blood phosphorus exists in the HPO42-

and H2PO4- forms at a 4:1 ratio.

The kidney is the major site of control of phosphate concentrations and almost all phosphate reabsorption occurs in the PT which is inhibited by PTH

Reabsorption occurs via a Na+-PO4 cotransporter.

Activity of the cotransporter is increased by hypophosphatemia and insulin.

Activity of the cotransporter is decreased by hyperphosphatemia and increased PTH activity, resulting in an increase in the fractional clearance of phosphate (FCR-P).

Vitamin D metabolites and calcium also influence renal phosphate reabsorption.

Calcium (Ca2+

)

Protein-bound calcium remains in the blood, only ionised and complexed calcium enters the filtrate.

In carnivores and omnivores, the intestinal absorption of calcium is controlled and therefore the kidney plays a less important role in calcium homeostasis.

`80-85% of filtered Ca2+ is reabsorbed in the PT and loops of Henle via a passive process.

Both PTH and activated vitamin D promote Ca2+ reabsorption.

Magnesium (Mg2+

)

80% of plasma magnesium appears in the glomerular filtrate (the rest is protein-bound).

Only 20-30% is reabsorbed in the PT and 10% in the distal nephron.

Most is reabsorbed in the thick ascending loop of Henle which is the major site of magnesium homeostasis.

Bicarbonate (HCO3-)

HCO3- is an important buffer that is available to neutralise H

+ ingested or produced during

metabolism.

HCO3- freely passes through the glomerular filter and must be reabsorbed.

The PT normally reabsorbs 80-90%of the filtered HCO3- by a process that requires H

+

secretion and the enzyme carbonic anhydrase.

Filtered HCO3- combines with H

+ in the filtrate to form H2CO3 and is then converted to

CO2 and H2O.

This CO2 and H2O then diffuse into the PT cells and are reconverted to HCO3- by the

reversal of these steps. The HCO3- is then transported to the peritubular fluid.

The H+ is recycled and there is no net secretion of H+ in association with this HCO3-

reabsorption.



If the [HCO3- ] is tending to decrease, then all filtered HCO3

- is reabsorbed and new HCO3

-

can be synthesised by renal tubular cells using CO2 produced in renal cell metabolism (this

process requires net secretion of H+ ).

If the [HCO3-] is tending to increase, then less of the filtered HCO3

- is reabsorbed and the

excess is excreted in the urine.

Protons (H+ )

The kidneys secrete H+ as a defence against acidosis (remembering that H+ is continuously being produced in cellular metabolic processes).

Fluid leaving the PT has a pH of 6, even in acidosis; therefore H+ has to be secreted against a concentration gradient.

The PT has a limited capacity to secrete against a concentration gradient, but in the DT and CD H

+ can be secreted against a cell:lumen concentration gradient of 1:1000 .

Buffers that accept H+ efficiently remove protons from solution and negate their pH effect. The phosphate system works constantly to remove protons.

HPO42-

+ H+ H2PO4

-

The ammonia system is induced in response to the need to excrete an increased acid load with ammonia being synthesised in the tubular cells primarily from the amino acid

glutamine

NH3 + H+ NH4

+

The pK of NH4+ is 9.15; it is a weak acid which tends to hold onto its H+.

Urea

Urea is recycled between the interstitial fluid and fluid in the loop of Henle, and then between the medullary CD and medullary interstitial fluid.

60-65% of filtrate urea is reabsorbed in the PT down a concentration gradient created by the movement of H2O into the cells.

As a result of this, the medullary interstitial concentration of urea is high and some urea will move down a concentration gradient back into the ascending loop of Henle which

further increases the concentration of urea in filtrate in the medullary CD fluid.

Urea reabsorption in the distal nephron is greatly enhanced by AVP. Urea contributes nearly 50% of the interstitial solute necessary for establishing medullary

hypertonicity and renal concentrating ability.

It has been known since 1913 that the urine concentrating ability of healthy dogs is increased on a high protein diet.

Creatinine

In some dogs, there is some secretion of creatinine by the PT, but this does not appear to occur in cats and dogs.

Overall, renal processing of creatinine is minimal.

Water Homeostasis

All mammals and birds can concentrate urine above the osmolality of plasma to provide a means by

which body water can be conserved when necessary; conversely water can be excreted when necessary.

Because of the extremely high volume of water filtered through the glomeruli, water is reabsorbed in the

PT, descending loop of Henle and proximal DT without regard to the state of water balance.

In the PT, 60-80% of water is reabsorbed secondary to reabsorption of solutes.



Further reabsorption occurs in the loop of Henle and the proximal part of the DT .

Only 9% of filtrate remains as it enters the distal section the DT; urine volume is determined by the

amount of this 9% that is reabsorbed.

In water excess; water reabsorption is decreased and a high volume of low solute concentration urine is

excreted.

In water deficit; water reabsorption is increased and a low volume of high solute concentration urine is

excreted.

Reabsorption of water occurs secondary to osmotic gradients; i.e. the reabsorption of solute in the PT is

associated with water reabsorption.

Medullary interstitial hypertonicity provides an osmotic gradient for water reabsorption from the lumen

of the DT and CD when the cells of the DT and CD are made water permeable by the action of AVP.

AVP is released in response to increased plasma osmolality.

Extrarenal Influences on Renal Function

1. Age In puppies tubular function matures more slowly than glomerular function and concentrating ability

increases throughout the first 2.5 months of life; therefore puppies are less able to excrete a water

load than adults.

Calves are also less able to excrete a water load than adults; they can quite easily be overloaded.

Calves have a limited ability to concentrate urine (important in calf diarrhoea as they have a limited

ability to conserve water).

Neonatal foals often produce dilute urine (SG



PRIMARY RENAL DYSFUNCTION

Kidney/Renal Disease: Any functional or morphological abnormality of the

nephrons.

Renal Failure: Decompensated renal disease characterised by

inappropriately concentrated urine with renal azotemia.

The distinction between these two categories has become more important in recent

years with increased detection of chronic kidney disease (CKD) at earlier stages before

there are typical changes indicative of significantly decreased renal function. Not all

renal dysfunction results in renal failure as defined above (although this may develop

in the long-term).

1. Acute Renal Failure (ARF)

Acute renal failure is defined as abrupt deterioration of renal function with resultant

retention of nitrogenous waste products and loss of the ability to adequately regulate solute

and water balance. It may be reversible or irreversible and occurs within hours or days of

the renal insult that markedly decreases GFR.

The magnitude of the azotemia can be mild to severe (as for chronic disease), but develops within hours to days (rather than months in chronic disease).

Acute, Intrarenal Renal Failure is an abrupt deterioration of renal function due to events occurring in the kidney itself. All/most of the nephron population is abnormal and there

is less scope for compensation. Nephrotoxins or infectious agents may be the cause, but

the pathogenesis of acute intrarenal failure is often not clear.

Prerenal ARF is a sudden decline in renal function provoked by inadequate perfusion (eg severe dehydration, cardiac insufficiency, hypovolemia). The nephron population is

normal and remains so unless the state of inadequate perfusion is protracted.

Postrenal Renal Failure is a sudden decline in renal function except that postrenal ARF is provoked by urinary obstruction or bladder rupture

2. Chronic Renal Failure (CRF)

CRF is often the result of slow destruction of the renal parenchyma resulting in destruction

of the nephron population. The precise pathogenesis is rarely clear; compensation by the

surviving nephrons usually prevents clinical signs appearing until the damage is irreversible.

There are usually no clinical and few detectable biochemical abnormalities until most tissue

have been destroyed.

The causes are generally poorly defined, but include infectious, immune-mediated and possibly dietary.

Adaptation occurs in CRF e.g. Single nephron GFR increases as the renal mass decreases

e.g. Residual nephrons increase their excretion of K+ to compensate

New nephrons cannot be generated in mature animals, but hypertrophy and hyperplasia may occur.

There are no fixed rules that allow CRF to be staged, but the following functional categories are quite useful:



Diminished renal reserve: The GFR is 50% of normal and the animal is clinically healthy and non-azotemic. However, the kidneys are less able to cope with any insult

such as poor perfusion or dehydration.

Chronic renal insufficiency: The GFR is 20-50% of normal and azotemia and impaired concentrating ability (polyuria) appear. Anemia may appear.

Chronic renal failure: The GFR is



Consequences of Renal Failure (Acute or Chronic)

1. Azotemia and the clinical signs of Renal Failure

Azotemia = increased serum urea and/or creatinine (increased

non-protein nitrogenous compounds in the blood)

Uremia = azotemia plus clinical signs of renal failure

The clinical signs associated with renal failure have multiple causes which include accumulation of compounds normally filtered by the kidneys: e.g. urea, creatinine,

ammonia, uric acid, leucine, tyrosine, sulphates, phosphates, chloride, potassium,

organic acids (acidosis and increased anion gap are often associated with RF).

Polyuria/polydipsia (see below).

The gastrointestinal tract is severely affected; vomiting occurs in carnivores, oral and GI ulceration, diarrhea, constipation, "uremic" or ammoniacal breath.

Gastrin is a digestive hormone that causes the stomach to secrete acid. It is excreted by the kidneys and gastrin levels rise with renal insufficiency resulting in increased gastric

acidity, nausea and other gastrointestinal signs.

The central and peripheral nervous systems are affected causing depression (most species) and peripheral neuropathy.

Anemia often occurs in chronic renal failure (see below).

Osteopathy (renal osteodystrophy) may also develop

2. Altered Water Homeostasis

Anuria = failure of urine production

Oliguria = decreased urine production

Dysuria = difficulty in urination

Stranguria = frequent, difficult and painful discharge of urine

Pollakiuria = unduly frequent urination of small volumes of urine

Polyuria = increased urine production

Changes in urine volume are associated with renal failure.

A marked decrease in GFR is associated with oliguria or anuria and may occur in acute renal failure as a terminal event or in chronic renal failure.

Polyuria is a common sign seen in chronic renal failure.

Polyuria is most commonly associated with chronic renal failure, but may also occur in less severe acute renal failure; the inability to regulate water excretion predisposes

animals with polyuric renal failure to dehydration and prerenal azotemia.

Polyuria/polydipsia (PU/PD) is a very common change that is associated with many problems, both renal and nonrenal; the table below lists some of the more common

causes of PU/PD).

Polyuria occurs in renal disease because:

As nephrons are destroyed or become nonfunctional, each of the remaining functional nephrons takes on an increased solute load resulting solute (osmotic) diuresis.

Decreased reabsorption of Na+ and Cl

- in the ascending loop of Henle which results in

osmotic retention of water within the tubule.



Decreased medullary hypertonicity occurs as renal tissue and blood flow are damaged, less Na

+ and Cl

- are reabsorbed in the ascending loop of Henle and the damaged cells

in the distal nephron become less responsive to AVP/ADH.

Water intake in dogs is very variable, depending on activity level, environment and diet.

Water intake in cats is more predictable.

Measurement of water intake is often very difficult for clients, especially in multi-pet households.

Cause of PU/PD Classification Mechanism(s)

Central diabetes

insipidus (CDI)

Primary polyuria Lack of AVP (ADH) production

(partial or complete)

Chronic renal failure Primary polyuria

(2NDI)

Congenital lack of renal response to

ADH

Diabetes mellitus Primary polyuria

(2NDI)

Osmotic diuresis

Hyperadrenocorticism Primary polyuria (2&

1 NDI)

Primary polydipsia

Impaired release of AVP

Impaired tubule response to AVP

Psychogenic polydipsia

Hypercalcemia Primary polyuria

(2NDI)

Interferes with action of AVP on

renal tubules

Hyperthyroidism Primary polyuria

(2NDI)

Primary polydipsia?

Loss of medullary hypertonicity

Psychogenic?

Hypokalemia Primary polyuria

(2NDI)

Down-regulation of aquaporin-2

Loss of medullary hypertonicity

Polyuric acute renal

failure

Primary polyuria

(2NDI)

Osmotic diuresis

Post-obstructive

diuresis

Primary polyuria

(2NDI)

Osmotic diuresis

Down-regulation of aquaporin-2

Primary NDI Primary polydipsia (1

NDI)

Congenital inability of the nephron

to response to AVP (ADH)

Primary polydipsia Primary polydipsia Psychogenic polydipsia

Hepatic encephalopathy

Hyperthyroidism

Gastrointestinal disease

Pyelonephritis Primary polyuria

(2NDI)

Bacterial endotoxin reduces tubular

sensitivity to AVP/ADH

Damaged countercurrent

mechanism

Pyometra Primary polyuria

(2NDI)

Bacterial endotoxin reduces tubular

sensitivity to AVP/ADH

Renal medullary solute

washout

Primary polyuria

(2NDI)

Decreased renal medullary

hypertonicity with loss of osmotic

gradient

Source: Comp Cont Vet Ed (October 2007). Pages 612-624 Normal and Abnormal Water

Balance: Polyuria and Polydipsia. This article contains a more comprehensive list than above.



3. Hematological Abnormalities

A nonregenerative, normochromic, normocytic anemia is often seen in CRF because erythropoietin which is the hormone required for normal erythropoiesis is produced by

the kidney; erythropoietin production is decreased in CRF and this is at least partially

responsible for the anemia.

Bleeding abnormalities due to defective platelet function are common in people, but have only been reported in low numbers of uremic dogs.

4. Electrolyte Abnormalities

Sodium

In dogs, plasma Na+ concentration remains normal until the terminal stages. Hyponatremia has been seen in ARF in cattle and CRF in horses.

Potassium

In most species the plasma K+ remains normal in the polyuric stage of renal failure, but hyperkalemia develops with oliguria or anuria (most common in ARF).

In cats, hypokalemia is a relatively common occurrence in chronic renal failure and is increasingly being recognised as a contributor to the progression of kidney failure.

Hypokalemia is common in cattle with ARF because of reduced food intake, decreased absorption and increased salivation.

Calcium and Phosphorus

The metabolism of these is markedly altered during uremia and renal secondary hyperparathyroidism occurs in monogastrics.

The kidneys are responsible for the final activation step in vitamin D synthesis and decreased functional kidney mass decreases activation of vitamin D (calcitriol).

Early in renal failure there is a slight hyperphosphatemia due to decreased GFR which is accompanied by a slight hypocalcemia (inorganic phosphorus x ionized calcium

constant); these changes are not detectable on routine tests).

The hypocalcemia stimulates PTH release which induces phosphaturia and normalises plasma phosphate and calcium values, but to maintain this state PTH secretion has to

keep increasing.

Increased PTH can compensate only up to a point, after which hyperphosphatemia occurs (at about the time that azotemia occurs).

Although this increased PTH production is physiologically appropriate, excessive amounts of PTH are toxic to the kidneys and other organs, including the brain.

Hyperphosphatemia also contributes to decreased activation of vitamin D. Decreased levels of activated vitamin D contribute to decreased dietary calcium

absorption, decreasing serum calcium levels and further stimulating PTH release.

Plasma P concentrations in cattle and horses in renal failure are variable because of the relationship between ingestion, renal excretion and extrarenal excretion. Either

hypocalcemia or hypercalcemia can occur in advanced renal failure; hypercalcemia is

more likely to occur in horses because of their high dietary intake of calcium.



LABORATORY EVALUATION OF THE URINARY SYSTEM

Note: Collect samples of urine and blood (as close together time-wise as possible) as well as any other

appropriate samples before initiating treatment as, more than in any other body system, treatment may

affect the laboratory findings and make it difficult to interpret the results.

Indications for Urinary System Evaluation

1. Urinary problems

These include oliguria, polyuria, polydipsia, pollakiuria, dysuria, abnormal urine, inappropriately concentrated urine and azotemia.

Involvement of the urinary system may by primary or secondary; e.g. Prostatic enlargement may account for dysuria and abnormal urine in the male dog.

2. Genitourinary problems

Vaginal or urethral discharges may relate to either system.

3. Systemic problems

Systemic problems may occur as the result of primary renal disease; e.g. dehydration, ascites, edema, vomiting, oral ulceration, osteodystrophy, depression, coma.

Objectives of Urinary System Evaluation

1. Confirm the problems and, as far as possible, reach as specific a diagnosis as possible.

e.g. PU/PD, dysuria and incontinence are all problems reported by owners and should be verified before laboratory investigation if possible.

e.g. The problem of red urine can be refined as hematuria (due to blood), hemoglobinuria (due to Hb) or myoglobinuria due to release of myoglobin in severe

myonecrosis).

2. Localise the problems as primarily urinary or extraurinary;.

Urinary tract disease may be localised as either lower urinary tract, glomerular, tubular

Azotemia may be prerenal, renal or postrenal.

PU-PD may be related to renal disease or endocrine diseases such as diabetes mellitus, diabetes insipidus and hyper- or hypoadrenocorticism (see the list above).

Inflammatory cells or bacteria in urine may relate to either the urinary or genital systems.

3. Use the information obtained 1 & 2 to plan an appropriate treatment and management plan.

URINALYSIS (physical, chemical, and microscopic examination of urine)

Note: A very reference available on this topic for dogs and cats is the 1999 publication put out by Bayer

titled Urinalysis: A Clinical Guide to Compassionate Patient Care written by Carl A Osborne, and Jerry

B Stevens. It is much more detailed than these notes and has excellent photographs; if you can obtain a

copy of this, do so (it is not available for sale).

Urinalysis is an inexpensive laboratory procedure that is central to the investigation of urinary tract

disease.

It requires a minimum of equipment (centrifuge, decent microscope, refractometer, dipsticks).

Although not difficult, the microscopic examination of urine does take some practice.

Urine quality and/or quantity are affected by a range of primary problems outside the urinary system; these include endocrine disorders, cardiovascular, muscular and hepatic diseases,

abnormalities of the hemopoietic system and genital inflammation or infection.



Specimen Collection & Storage

1. Timing & Storage

If the urine cannot be analysed within 2 hours the sample should be held at 4oC for < 8-12 hours (longer if necessary) and brought to room temperature before analysis.

RBCs remain stable in refrigerated acid urine for several days.

Leukocytes remain stable in refrigerated urine for at least 3 days.

pH increases after collection due to escape of CO2 as well as proliferation of bacteria which may be urease producers; these effects are reduced greatly by refrigeration.

Frozen urine can be used for biochemical analysis, but NOT for sediment examination.

Although there is a risk that refrigeration will kill fragile organisms, refrigeration for



Polyuria is confirmed absolutely at water intake of 100 mL/kg/day (according to published information); however in between 50 and 100 is a sliding scale and many of the

dogs investigated for polyuria have water intakes in the 50-100 mL/kg/day range rather

than >100 mL/kg/day.

PU/PD should be included on the problem list if the owner reports increased water intake or urine output relative to what she/he considers normal for that animal.

If the urine SG if found to be persistently low, PU/PD should be included on the problem list, even if the owner does not report a problem.

2. Colour

Normal urine is amber yellow due to the presence of urochromes and clear to slightly turbid, with more concentrated urine usually being darker and less concentrated lighter.

Equine urine darkens when it is left to stand.

Hematuria, hemoglobinuria and bilirubinuria are the most common causes of discoloured urine.

Pyuria is the most common cause of turbid urine, but any increase in cells, bacteria, crystals, semen or mucin will increase turbidity (equine urine contains mucus).

3. Transparency

Freshly voided, normal urine is transparent. Horses may excrete slightly turbid urine

Crystals, RBCs, WBCs, epithelial cells can cause turbidity

4. Odor

Normal urine has a characteristic odor. Male cats excrete pungent urine. Urine of horses has an aromatic odor

Ammoniacal odor is caused by release of ammonia from urea on the effect of bacteria with urease activity

Sweety/fruity odor may be associated with ketonuria

5. Specific Gravity (SG)

The SG is an expression of the concentration of solutes in urine which in turn is a reflection

of the concentrating ability of the renal tubules (also measured by urine osmolality). The

urine SG depends on the hydration state of the patient with minimally and highly

concentrated urine being appropriate responses to different hydration states; however, in

renal disease the urine concentration may be inappropriate to the hydration state of the

animal; e.g. dilute urine is appropriate in the over-hydrated patient.

The refractive index of urine is determined by the concentration of all solutes in the sample and is measured relative to water using a simple refractometer (the SG is a unit

less ratio).

For a refractometer to give reliable results it is essential to:

Keep it clean and look after it, never allow urine or other biological fluid to dry on the glass surface.

Avoid scratching the glass surface with the broken ends of microhematocrit tubes. Calibrate it regularly on distilled water if the refractometer has this capacity Keep in a constant temperature area. Be aware that the temperature of the sample may affect the results.

Some dipsticks have a pad for SG, but for animal samples these do not correlate well with the measurement of SG by a refractometer.



Some of the veterinary refractometers on the market have separate scales for cats and dogs; this was done in an attempt to make cat and dog SGs the same for the concentrated

range. All SG measurements that are given in these notes relate to the SG read on the

"human" standard scale. It does not matter which refractometer you use, as long as you

know which one you are using. The human scale is very similar to the dog scale.

Urine SG readings are also affected by marked proteinuria and/or glucosuria, resulting in an overestimation of urinary concentrating ability.

Interpretation of Urine SG

Interpretation of any urine SG value is dependent on knowledge of the animals hydration status, blood urea and creatinine, knowledge of medications and/or fluid therapy, age and

diet. Random urinalysis in healthy animals will give a wide range of SG of results. The

values below are NOT a reference range and there is no normal value for SG.

Dog: 1.015 1.045 Cat: 1.015 1.065 Horse: 1.020 1.050 Cattle: 1.025 1.045

There are no hard and fast rules for interpretation of urine SG. The view presented below is

the more traditional but is not the only approach. The important message is that the whole

animal and all information must be taken into account.

Concentrated urine Dog: SG 1.030

Cat: SG 1.035

suggests adequate or normal renal tubular concentrating ability

a patient may still have another disease which causes PU-PD

does not rule out glomerular dysfunction

Dilute urine Dog: SG



Urine Biochemistry

Urine biochemistry tests fall into two categories; those that can be done on dipsticks and those that require wet/dry chemistry analysers.

Dipstick Tests Urine pH

Glucose

Ketones

Bilirubin

Occult blood, hemoglobin & myoglobin

Proteinuria

X Urobilinogen

X Nitrituria

X Leukocytes

X Urine SG

Can be used in animals X Do not use in animals

Analyser-based Tests Urine protein:creatinine ratio (UPC)

Fractional excretion/clearance ratios (FER)

GGT:creatinine clearance

Microalbumin, ERD

Various companies market "dipstick" test strips (reagent strips) for routine urinalysis; these are more

complex systems than they look and must be stored and used according to the manufacturer's instructions.

Many in use (e.g. Multistix) are designed for the human market, but the Bayer Petstix are designed for the veterinary market.

Although often read visually, these can also be read automatically by machines such as the Clinitek. It is useful to know if the urine sent to a laboratory is read visually or automatically as this will affect the

results slightly and therefore how you interpret them. These machines are literal and in very cloudy and/or discoloured urine are unable to separate the colour changes due to real chemical reaction and the

background (visual reading is not necessarily more accurate in this situation).

1. Urine pH

The urine pH is determined by renal regulation of blood HCO3- and H

+ levels (reabsorption

of HCO3- and excretion of H

+ ), but urine pH does not always accurately reflect body pH

and urine pH has a very limited role as guide to body acid-base balance except ruminants.

pH 5 to pH 8.5 is measured on a dipstick pad if the strip is only dipped into the urine for 1 second and the excess urine tapped off immediately (to avoid buffers from adjacent pads running onto the pH pad). However, recent work indicates that there is a rather

poor correlation between dipsticks and results with an accurate pH meter and it has been

suggested that if an accurate urine pH is critical for clinical decision making, then the

dipstick pH should not be relied on (although it is claimed that Petstix reads within 0.5 pH units of a pH meter).

Instrumentally read strips will tend to underestimate below 6.5 and overestimate over 7.5 relative to visually reading; i.e. a wider range of results will be seen.

Most dogs and cats have urine pH 5.5 - 7.0 as they excrete predominantly acid salts of phosphates, but it is important to understand that any urine pH can be normal.

Adult herbivores produce alkaline urine (pH 7.0 9.0) due to obligatory excretion of alkaline salts of carbonates.



Normal suckling foals have acidic urine pH



3. Ketones (acetoacetate, acetone and -OH-butyrate)

Dipsticks detect acetoacetate, are less sensitive to acetone and do not detect -OH-butyrate, therefore samples from all species need to be fresh, as acetoacetate is converted to the other ketone bodies with time.

Urine is negative for ketone bodies in normal animals.

Only diabetic ketoacidosis will be associated with significant ketonemia in cats and dogs.

Ketones are commonly found in ruminant urine in starvation or following any cases of negative energy balance, but rarely found in the dog or cat during starvation.

The severity of the ketoacidosis is not correlated with the degree of ketonuria.

Practical Hints

Ketones are volatile and samples should be tested immediately or refrigerated.

Dipsticks are readily adversely affected by effects of moisture, heat and light.

False increases can occur with a range of drugs, e.g. aspirin.

False trace/1+ positive results are relatively common with automatic readers when the urine is very cloudy or strongly discoloured.

4. Bilirubin

Bilirubin formed from heme and then conjugated in the liver is readily filtered by the glomerulus and appears in the urine (dipsticks only detect conjugated bilirubin).

Dogs, especially males, may normally have small amounts of bilirubin if the urine SG is > 1.020.

Bilirubinuria is uncommon in healthy cats and should not be ignored.

The most common causes of bilirubinuria in dogs and cats are hepatic disease, post-hepatic bile duct obstruction and hemolytic disease, but note that bilirubinuria is

often detected before clinical jaundice or an elevation in serum bilirubin.

Practical Hints

Bilirubin is inherently unstable and false negatives may occur in urine that has been exposed to the light.

Dipstick pads are readily adversely affected by effects of moisture, heat and light.

False trace/1+ positive results are relatively common with automatic readers when the urine is very cloudy or strongly discoloured (this is

a significant problem in cats with hematuria).

5. "Blood" - Hemoglobinuria/Myoglobinuria

The test pad for "blood" on dipsticks is actually a test for heme pigments and therefore it detects hemoglobin (Hb), myoglobin and to a lesser extent, the Hb in intact

erythrocytes.

Very sensitive, for example Multistix detect 5-20 RBC's/L of urine or 150-620 g/L of hemoglobin or myoglobin. In contrast, gross visual detection of RBCs requires

approximately 2500 RBCs/L.

Sensitivity is less for intact RBCs than for hemolysed blood.



Hematuria

Non-specific indicator of a problem in the urinary or genital tracts; a diagnosis of hematuria is confirmed by the presence of RBCs or RBC ghosts in the urine sediment

(see wet microscopy section for comments on numbers expected in normal urine).

May be gross or microscopic; pathologic or nonpathologic. Hemoglobinuria

Hemoglobinuria is a consequence of hemolysis; but hemoglobinuria is not always accompanied by hemoglobinemia as the hemolytic episode may have resolved,

leaving Hb only in the urine.

RBCs may lyse in very dilute (SG < 1.008) or alkaline urine (urinary hemoglobinuria); they may be apparent as "ghost" cells or be invisible.

Myoglobinuria

Myoglobinuria is the result of significant myonecrosis and if suspected, it is most practical to use plasma creatine kinase (CK) levels to look for evidence of myonecrosis.

Practical Hints

False negative results can occur in hematuric samples in which the RBCs have settled to the bottom on the tube.

The presence of large numbers of bacteria may decrease the reaction.

RBCs may lyse in very dilute (SG < 1.008) or alkaline urine (they may be apparent as "ghost" cells or be invisible).

6. Protein

A small amount of protein may be present in the urine of all species (trace or 1+ reactions).

Urine containing a large amount of protein will "froth" if shaken.

Positive results for protein imply glomerular leakage or inflammation in the genitourinary tract and the protein reaction must be interpreted in conjunction with the

urinary sediment.

Proteinuria must also be interpreted in light of the SG; if the urine is very dilute, a large amount of protein has to be excreted before it may be detectable and animals with

isosthenuria or hyposthenuria and trace protein reactions may still be losing significant

amounts of protein. This is largely overcome by the use of the urine protein:creatinine

ratio (see below).

Physiologic/functional proteinuria is usually transient and associated with strenuous exercise such as racing or convulsions.

Glomerular proteinuria is associated with leakage of plasma protein through damaged glomeruli at a rate which exceeds the tubular capacity to reabsorb protein. Minimal

damage is associated with the loss of low molecular weight proteins (e.g. albumin,

causing firstly microalbuminuria) but as damage becomes more severe, the proteinuria

becomes less selective and larger as well as smaller proteins are lost. Prolonged mild or

severe glomerular proteinuria will cause hypoproteinemia (selective hypoalbuminemia

or hypoproteinemia).

Tubular proteinuria may also result in proteinuria as the result of impaired reabsorption and catabolism of the low molecular weight proteins that are normally filtered by the

glomeruli; however tubular proteinuria is milder. It is most often associated with acute

renal diseases, but can also be congenital. These do not cause hypoalbuminemia.

Nonrenal proteinuria is due to contamination of urine with inflammatory exudate or blood may be associated with mild to moderate proteinuria; i.e. proteinuria must be

interpreted in conjunction with examination of the urine sediment.



Measurement of Protein in Urine:

Dipstick/Test Strips

The test pad on the dipstick primarily detects albumin and is less sensitive to globulins and Bence-Jones proteins (myeloma).

The dipstick method detects >30 mg/dL of protein (0.3 g/L), but is more sensitive to albumin and will detect 14-21 mg/dL of albumin.

The dipstick uses a method in which the protein concentration causes a pH change which is read as a colour change.

Chemical Measurement

Urinary protein (both total and albumin) can be precisely quantitated by specific chemical or immunoassay techniques.

This is used when the protein content must be quantitated such as for a urine protein:creatinine ratio (see below under tests of urinary protein loss).

Sulphosalicylic Acid Precipitation (SSA)

This is a turbimetric test which is not used all that often. Apply when false positive dipstick reaction is suspected.

Practical Hints

The dipstick method is sensitive to interference by very alkaline urine and false positives may occur with very alkaline urine or some

drugs/chemicals.

False negatives with the dipstick method may occur with very acidic urine, but this is less common than interference due to alkaline urine.

7. Urobilinogen do not use

8. Nitrituria ..do not use

9. Leukocytes do not use

Canine Urine: Although this test in canine urine is specific (i.e. minimal false positives) they are very

poorly sensitive (detect only 25-75 leukocytes/L) and false negatives are common. The test is more sensitive for the detection of bactiuria and is specific for bactiuria, but

overall this test is not recommended for general use with canine urine.

Feline Urine: This test is very nonspecific in feline urine and most samples without pyuria will give

false positive results. Do not use this test in cats.



Microscopic Examination of Urine

1. Methodology

Estimation of cells/hpf:

Uses a standard volume (usually 5 or 10 mL which is centrifuged, then all except 0.5 mL of supernatant is removed; the resuspended sediment is examined (usually

unstained) under subdued light at x10 and x40 with a lowered condenser.

Air-dried Wright's stained smears or cytospin preparations can be made if neoplasia is suspected, but are of no advantage for the diagnosis of routine urinary tract

inflammation.

It is recommended to semi-quantify results; e.g. number of cells/high power field (hpf, = x 40). The major limiting factor in this methods is that the volume of urine and

sediment used is not standardized and it is not possible to compare results when for

example the sediment from 10 mL of urine is used compared to the results obtained

from a direct preparation used if only a small volume of urine is available.

Although not difficult, examination of urine sediments takes considerable practice; it is easy to recognise a bacterial cystitis, but less dramatic changes are more challenging.

There are definite advantages to examining urine while it is fresh as generally cells and structures do not last well in urine (unless there is a lot of protein present).

Quantitative cell counts (cells/L) These are done using a system such as the Kova Glasstic Slide system which is a

disposable hemocytometer system.

This method gives much more reproducible results and should allow results to be compared between different samples.

2. Erythrocytes (RBCs)

< 5 RBC/hpf are occasionally seen in normal animals (this is the published normal but

usually healthy animals have 3/hpf.). It should be assumed that is from 10 mL of urine that has been centrifuged and the sediment resuspended as described above.

Quantitative counts are



Pyuria occurs because of contamination with preputial or vaginal secretions, urinary tract infection, sterile cystitis, neoplasia, calculi, fever or exercise (transient for the latter

two).

4. Microorganisms

Includes bacteria, yeasts, algae, parasites and fungi; cystocentesis is the preferred method of collection to avoid contamination.

Whilst bacilli are quite easy to see in an unstained preparation, cocci can be much more difficult to see or distinguish from the amorphous debris that is common in urine.

If found in a cystocentesis sample, or accompanied by WBCs, organisms can be assumed to be significant.

Significant bactiuria can occur without evidence of inflammation; this is particularly likely in animals with diabetes mellitus or hyperadrenocorticism.

The presence of significant bactiuria must always be confirmed by quantitative culture.

In looking for fungal hyphae (e.g. suspected Aspergillus infection) it may be necessary to let the urine stand overnight at room temperature before reexamination to allow low

numbers of organisms to multiply, increasing the chances of detection.

5. Epithelial Cells

Small numbers of transitional and squamous epithelial cells are normal and samples collected by catheterisation will tend to contain sheets of normal-looking epithelial cells.

Large numbers of transitional epithelial cells, especially in cohesive clusters with a high

nuclear:cytoplasmic ratio, suggest dysplasia or neoplasia which must be confirmed on

cytology.

6. Urinary Casts

Under the right conditions precipitated protein cellular elements may combine to form casts of the inside of the renal tubules.

Casts are cylindrical-shaped slightly refractile structures which have round, square, irregular or tapered ends.

Hyaline protein casts are relatively common in acid urine. They are clear and colourless and low numbers are not usually significant.

Fatty casts and granular casts are relatively common and not usually significant.

Casts may also contain RBCs, WBCs and/or epithelial cells but cellular casts are less common.

Casts occur in tubular lesions (tubulonephrosis)

Casts are best identified in fresh urine.

7. Lipid Droplets

Commonly seen in the urine of normal carnivores.

8. Crystals

Several types of crystals are seen in the urine of domestic animals; these are formed from either normal components of urine OR exogenous compounds, abnormal metabolism or

abnormal excretion.

The degree of crystalluria depends on pH, concentration of ions, and temperature of the urine.



It is important to note that crystals may be seen quite commonly in the urine of healthy animals with no evidence of urolithiasis.

dogs and cats: particularly struvite (triple phosphate) and Ca oxalate Dalmatian dog: (bi)urate i.e. uric acid; but not ammonium-biurate horses: Ca carbonate

Increased numbers of crystals in the urine sediment may suggest conditions that are affecting the pH or ion concentration.

Crystalluria is considered a risk factor for urolithiasis, but does not confirm the diagnosis of urolithiasis.

Enhanced crystal formation often occurs in stored urine because of temperature effects with refrigeration, evaporation and changes in pH, however there are some crystals that

may dissolve with storage.

Over recent years, calcium oxalate urolithiasis in dogs and cats has become much more common; it is thought that this increased incidence is associated with the feeding of

urinary acidifying diets designed to decrease struvite crystal formation. Recent evidence

suggests that trend is being corrected by the changing formulations of adult maintenance

diets and the prevalence of struvite crystals is increasing again slightly.

Practical Hints for Urine Wet Micro

No matter how much practice and skill you have, not all cells and material in urine sediments can be precisely identified!

Many medications are excreted by the kidneys and may be seen in the urine as crystals.

It is very easy to confuse Brownian motion of fine particulate material with bacteria, especially cocci.

Bacteria proliferate rapidly in urine, especially at room temperature.

Bladder Tumour Antigen (BTA) Urine Dipstick Test (V-BTA, Canine Urine)

This is not a biochemical or routine test, but deserves a mention as the interpretation of the result

of this test is significantly influenced by the results of the tests described above. Transitional cell

carcinoma is the most frequently occurring malignancy of the lower urinary tract in the dog. Most

cases present with typical signs of lower urinary tract disease (hematuria, dysuria, and pollakiuria).

The Bard BTA (bladder tumour antigen) test detects a glycoprotein complex that is present in the urine of patients with transitional cell carcinoma (TCC). Veterinary version is V-BTA

test.

The first generation human test is a qualitative rapid latex agglutination test that is run on 0.5 mL of urine 30-40 WBC/hpf) and hematuria (>30-40 RBC/hpf).

The negative predictive value is 98.6%, making this a very good test for ruling out TCC, but the positive predictive value is much lower (64%) and positive results need to be interpreted

relative to the other urinalysis findings.

It is sensible to do urine wet microscopy first and then decide whether or not to proceed with the BTA.

Later generation human tests do not appear to work with canine TCC which is why there is a specific canine test, V-BTA (there is no feline BTA test). For details see Henry et al.: Am.

J. Vet. Res. 2003. 64. 1017-1020.



EVALUATION OF RENAL FUNCTION

A. Azotemia

B. Urine Concentration Tests

C. Tests of Urinary Protein Loss

D. Urine Fractional Clearance/Excretion Ratio (FCR/FER)

E. Urinary Enzymes

A. Azotemia

Azotemia is defined as elevation of the plasma/serum non-protein nitrogenous (NPN) compounds (urea

and creatinine).

1. Urea Metabolism, Excretion & Measurement

Endogenous urea synthesis provides a means of excreting the ammonia produced during catabolism of amino acids (from proteins) in mammalian species.

Urea appears in the glomerular filtrate in the same concentration as in the plasma; as it passes along the renal tubules it diffuses passively according to concentration gradients

and membrane permeability (influenced by AVP/ADH in the CD).

Because of the physicochemical properties of urea, it is not reabsorbed as efficiently as water and the rate of reabsorption is related to the rate of flow through the tubules; high

rates of urine flow are related to decreased reabsorption and low rates of urine flow

result in high rates of urea reabsorption.

Anything that decreases the GFR will increase the blood urea.

Processes which induce protein catabolism can increase urea production: hemorrhage into the small intestine, fever, burns, corticosteroids, starvation, infection.

Increased nitrogen intake in ruminants significantly increases urea synthesis in the liver leading to development of mild, prerenal azotemia.

Decreased urea synthesis may occur with decreased protein intake or severe hepatic insufficiency.

Chronic polyuria may also result in a decreased blood urea (washout effect).

Urea can be measured in serum, some plasmas and urine (the latter requires specific analyser settings).

Some references refer to BUN (blood urea nitrogen); if using SI units this does not affect interpretation, but if converting from mg/dL to SI units it is important to use the correct

conversion factor.

The assay is quite robust and between run CV is expected to be < 4.0% with lower values having a CV closer to the upper end of this range.

Azostix reagent test strips can be used to get an estimate of the blood urea. These are useful for ruling out azotemia as they are effective at detecting "normal" blood urea

concentrations, but they tend to underestimate values. In one trial, the Azostix only

identified 65% in the group that had elevated blood urea levels; i.e. very low sensitivity,

but high specificity.

2. Creatinine - Metabolism, Excretion & Measurement

Small quantities of creatinine are ingested, but the majority of creatinine is derived from the spontaneous non-enzymatic breakdown of muscle phosphocreatine; the quantity of

creatinine formed each day depends on factors such as muscle mass, age and gender.

In all mammalian species creatinine is freely filtered through the glomerulus and appears in the glomerular filtrate in essentially the same concentration as in the plasma.



It is generally assumed that there is no significant renal tubular processing of creatinine; this assumption is used frequently in tests of renal (glomerular) function.

Creatinine can be measured in serum, some plasmas and urine (the latter requires specific analyser settings).

The most commonly used method (alkaline picrate) does sometimes give false increases due to cross-reaction with noncreatinine chromagens that may be present in some blood

samples.

The assay is quite robust and between run CV is expected to be < 4.0% with lower values having a CV closer to the upper end of this range.

Enzymatic measurement of creatinine is more accurate than the non-enzymatic picrate method

3. Pancreatic enzymes

Amylase and lipase is excreted in the urine, their activity may increase in the serum in azotemic animals

4. Interpretation Of Azotemia

Reference ranges for urea and creatinine are quite wide because of the numerous variables that affect them.

When renal disease causes a reduction in the number of functioning nephrons, the remaining nephrons hypertrophy and take on a bigger load to maintain the overall GFR

and keep urea and creatinine within reference limits.

In most domestic species, urea and creatinine are crude estimates of the GFR. Creatinine is considered to be a slightly more accurate indicator of GFR than urea because of its

lack of renal tubular processing but it is possible that increases in urea correlate better

with clinical signs of uremia than increases in creatinine.

Azotemia is always a reflection of decreased GFR; the question then is what has caused the decrease in GFR?

Azotemia should always be interpreted in light of concurrent urinalysis

Ure

a m

mo

l/L

Cre

ati

nin

e

mo

l/L

-600

-500

-400

-300

-200

-100

60-

50

40-

30-

20-

10-

4.0 3.0 2.0 1.0

Glomerular filtration rate mL/min/kg

Upper limit of reference range



Prerenal Azotemia

Prerenal azotemia is more common than renal azotemia and (almost always) results from a decrease in GFR resulting from events that occurred prior to glomerular

filtration.

e.g. Decreased renal perfusion is a common cause of prerenal azotemia: shock,

dehydration, cardiovascular insufficiency (may be accompanied by increased PCV

and TS protein or total protein).

e.g. Increased protein catabolism secondary to small intestinal hemorrhage,

infection, fever, corticosteroids (mild azotemia).

e.g. Increased nitrogen intake (protein or NPN) in ruminants

The blood urea may increase more rapidly than creatinine (increased urea:creatinine). Adequately concentrated concurrent urine indicates that renal tubular function is

adequate; this usually differentiates prerenal from renal azotemia (but not always).

dog: urine SG >1.030

cat: urine SG >1.035

cattle: urine SG > 1.025

horses: urine SG> 1.025

When prerenal azotemia occurs as the result of bacterial deamination of exogenous (dietary) or endogenous (hemorrhage) proteins in the gut, the urea will increase

without a concurrent increase in creatinine; this is one of the few times that azotemia is

not the result of a decrease in GFR.

Renal Azotemia

Both urea and creatinine are relatively insensitive in detecting renal dysfunction; 75% of nephrons must be nonfunctional before GFR is sufficiently decreased for values to

increased to above the reference range.

Factors which cause prerenal or postrenal azotemia may eventually cause renal azotemia if the kidneys become secondarily involved.

Renal concentrating ability has already been affected by the time azotemia develops and urine is fixed or minimally concentrated (i.e. loss of concentrating ability occurs

before azotemia in renal disease):

dog: urine SG 1.008 - 1.029

cat: urine SG 1.008 - 1.034

Cats in early renal failure may have SG >1.035 and dogs in early renal failure may have SG < 1.008.

The patient may be polyuric, oliguric or anuric. It is best to do serial evaluations to determine prognosis, single estimations do not give

a good indication of prognosis (see later also).

Postrenal Azotemia

Postrenal azotemia is most often associated with a decrease in GFR caused by obstruction (most common) or leakage distal to the kidneys; it is differentiated from

renal azotemia by clinical signs of oliguria/anuria as well as physical and radiologic

examination.

Urine SG may be any value, including in the isosthenuric range.



When postrenal azotemia is due to rupture of the lower urinary tract without significant obstruction, the GFR is not impaired. To confirm that peritoneal fluid contains urine,

measure the creatinine in the fluid; a 2x increased creatinine in the peritoneal fluid

compared to the serum creatinine is a reliable indicator of uroperitoneum.

Measurement of the urea in peritoneal fluid cannot be used because urea very rapidly

equilibrates with the blood (creatinine is poorly diffusible); it is the urea diffusing back

into the blood which may increase the blood urea.

Urea and creatinine should return to normal within days of relief of an obstruction or repair of leakage (unless secondary renal damage occurred).

Hyperkalemia (often very severe) is a common finding in postrenal azotemia due to decreased ability to excrete K

+.

5. Other Biochemical Findings associated with Azotemia

Serum Phosphorus

Hyperphosphatemia is a fairly consistent finding in dogs, cats and horses (decreased GFR).

Hyperphosphatemia may or may not occur in cattle (there are many alternative routes of excretion)

Some azotemic horses have low or low-normal phosphorus.

Serum Calcium

Total calcium may be low, high or within range in all species. Dogs, cats and cattle tend to have low-normal calcium. Horses are more likely to be hypercalcemic (they normally excrete large amounts of

dietary calcium via the kidneys).

Serum Potassium

In dogs, cats and horses, hyperkalemia occurs with acute decreases in renal function, especially oliguric or anuric failure (eg post-renal azotemia).

Cattle tend to have hypokalemia due to alkalosis, decreased intake or increased excretion in saliva.

B. Urine Concentration Tests

Urine concentration tests are indicated in nonazotemic animals with polyuria; they are contraindicated in

animals with unequivocal azotemia or any other evidence of disease. However, these tests do not always

give clear-cut results and many consider them best avoided; it is not without reason that papers with the

title Urinary Concentration Tests How to Avoid Them exist.

1. Water Deprivation Tests

The aim is to stimulate endogenous AVP/ADH (normally occurs with a body water deficit

of 2-3%); i.e. the urine SG should increase. Water deprivation is contraindicated in

azotemic and/or dehydrated animals and must be discontinued if body weight decreases by

>5%. Animals should always be given a thorough clinical examination and have a CBC,

biochemical profile and urinalysis done before a water deprivation test. Like most tests,

there are different protocols for both abrupt and gradual water deprivation tests, depending

on the source; all require very careful monitoring of the animal. See e.g. Willard MD et al.

in Small Animal Clinical Diagnosis by Laboratory Methods for suggested protocols and

interpretation.



2. Exogenous AVP/ADH Test

This supplies an exogenous source of vasopressin; regardless of hydration status,

concentrated urine should be produced in response to vasopressin if the kidneys are normal

and capable of concentrating urine.

3. Summary of Urine Concentration Tests

Normal response to water deprivation: indicates the patient is either normal or has psychogenic polydipsia.

Inadequate response to water deprivation, adequate response to vaspressin: indicates central diabetes insipidus (the patient is not able to release adequate amounts of

AVP/ADH).

Inadequate response to water deprivation, inadequate response to AVP/ADH: indicates nephrogenic diabetes insipidus (inability to respond to AVP/ADH).

Medullary washout may occur in patients with prolonged PU/PD, impairing their ability to respond to urine concentration tests and giving results that look like renal diabetes

insipidus. To try to eliminate washout as a factor prior to doing these tests, water

deprivation can be gradually increased and/or the patient can be treated with ADH for a

few days prior to testing in an attempt to reestablish medullary hypertonicity.

C. Tests Of Urinary Protein Loss

Qualitative tests such as that used on dipsticks and the sulphosalicylic acid precipitation test are very

sensitive screening tests, but are affected by urine volume and concentration. Even with a timed, e.g. 24

hour, urine collection, it can be difficult to accurately assess urinary loss of protein (and not practical to

do).

Proteins in the urine may originate from plasma, or from renal tubular cells.

Proteins from the plasma may occur due to glomerular and/or tubular damage.

Mild glomerular lesion causes loss of small MW proteins such as albumin (selective proteinuria)

With more severe, usually irreversible, glomerular damage, more of the high molecular weight proteins are filtered and this is termed nonselective proteinuria.

With tubular damage, there is decreased tubular reabsorption of filtered proteins; if the glomerulus is undamaged these are usually low molecular weight proteins.

With time, patients with progressive glomerular disease will also develop tubulointerstitial disease and this corresponds to the appearance of low molecular weight proteins in the urine in

addition to the larger proteins.

Much of the work that has been published on proteinuria in veterinary medicine has related to canine CKD.

In cats, tubulointerstitial disease is more common than glomerular disease and proteinuria is less common in cats than in dogs.

A recent study found that proteinuria (as measured by the UPC) was a good predictor of reduced survival time in cats in spite of the relatively low concentrations of proteinuria typical

of chronic renal disease in cats.

There has been a vast amount published in the area of proteinuria in dogs and cats in recent years.



1. Urine protein:creatinine Ratio (UPC)

< 0.5 normal

0.5 -1.0 questionable

> 1.0 abnormal (increased urinary protein loss)

The urine protein:creatinine ratio (UPC) test overcomes many problems as it is not affected by urine concentration and volume; it is a sensitive, rapid and convenient means

of detecting and quantifying proteinuria (protein-losing nephropathy).

The UPC on a single urine sample correlates well with the protein content of 24-hour collections.

The test is based on the assumption that glomerular filtration and concentration mechanisms affect protein and creatinine similarly.

mol/L creatinineurinary

8840 x g/Lprotein urinary UPC

Proteinuria resulting from glomerular disease is the most commonly recognised and the most significant proteinuria (use urinalysis to rule out proteinuria caused by lower UT

infection).

If hyperproteinemia is not present and there are no abnormal cells on the urine sediment, an increased UPC is strong evidence of glomerular dysfunction.

When monitoring UPC ratios it is important to remember that the UPC varies considerably from one day to the next due to nonrenal factors such as diet, exercise,

stress/excitement and blood pressure, even when these factors are relatively

well-controlled.

Although the range above indicates



Routine analyser methods and semiquantitative test strips for measuring albumin in human urine are NOT suitable for veterinary use.

Hematuria alone does not usually affect the results of this test unless it is grossly visible hematuria.

Many dogs with pyuria do not have albuminuria or proteinuria, but albuminuria is more likely in dogs with pyuria and concurrent hematuria or bactiuria.

D. Urine Fractional Clearance/Excretion Ratio (FCR/FER)

Clearance is the more correct term, but the term excretion is used widely.

The FCR of various solutes is used to determine the fraction of material filtered that appears in urine (sodium, calcium, potassium, chloride, phosphorus); it reflects the efforts of the kidney

to maintain homeostasis or defects in its ability to do so.

Those electrolytes/substances that are conserved (e.g. Na, Cl) have very low FCR values.

Values are affected by diet as well as time since last meal; this is more important in carnivores where food intake is less constant than in herbivores.

Uses include:

the diagnosis of primary hyperparathyroidism (increased FCR-P, but direct measurement of PTH is becoming more common),

monitoring of renal secondary hyperparathyroidism (increased FCR-P, decreased FCR-Ca) in CRF cases,

diagnosis of tubular dysfunction (eg increased FCR-Na)

FCR based on spot measurements should be done after a 12 to 15 hours fast because dietary intake and intestinal absorption can affect the results. Ideally, feed a standardised diet for

several days before the sample is collected.

Measurement of any FER requires collection of a urine sample followed immediately by collection of a blood sample (the values below are a guide only).

100% x e][creatinin urine

e][creatinin plasma x

][substance plasma

][substance urine FCR

Dog Cat Horse Cattle

FCR-Na % 0 0.7 0 0.8 0 0.5 0 3.6

FCR-K % 6.0 - 20 3.0 - 37 23 48 40 60

FCR-Ca % 0 - 0.4 0 - 0.1



F. EARLY DIAGNOSIS OF CHRONIC KIDNEY DISEASE (CKD)

ADDITIONAL READING, NOT PART OF LECTURES

This has been the area of greatest change in the management of CKD in dogs and cats in recent years.

Why does it matter?

There is increasing evidence that proteinuria itself can cause glomerular and tubulointerstitial damage and result in progressive nephron loss.

Proteinuria/microalbuminuria can occur secondary to glomerular damage or secondary to intraglomerular hypertension/hyperfiltration due to compensatory hypertrophy that occurs in any

CKD.

Increased phosphorus levels and decreased potassium levels may also accelerate progression of renal damage.

Any intervention will have a much greater effect on progression of disease, morbidity and survival if it is instituted early rather than late; the earlier the intervention, the greater the benefit.

There are effective therapies available.

Early CKD may be detected prior to the onset of suboptimal urine concentration and azotemia.

By definition, early detection also includes mildly azotemic animals that are still clinically well without clinical signs of CKD.

Dilemmas

Selection of cut-off values for interpretation of screening tests always creates a dilemma with sensitivity vs. specificity; screening tests are generally selected for high sensitivity with

follow-up tests to weed-out false positives. For example, if using a UPC cut-off of 0.5 there will be some false positive results for detection of CKD (hence the need for repeated testing

defined below to confirm that the proteinuria is persistent).

There is now recognition that mild, non-progressive renal disease may exist; this may or may not progress and there is no means of determining this except by regular monitoring. Many dogs and

cats that have lesions in their kidneys never develop clinically renal dysfunction.

Aids in detecting Early CKD

microalbuminuria proteinuria decreased SG azotemia clinical signs

1. Urinalysis

Essential to provide a context in which other results are interpreted (eg proteinuria, azotemia).

Rules in/out non-urinary disease and lower UT disease.

2. Detection of Microalbuminuria and Proteinuria

All positive dipstick reactions should be investigated further, regardless of SG.

Microalbuminuria is an albumin concentration in the range of >1 mg/dL but 30 mg/dL will be detected by a UPC (30 mg/dL = 0.3 g/L)

In humans, persistent microalbuminuria indicates the presence of intraglomerular hypertension and/or generalised vascular damage and endothelial damage.

Recent work indicates that microalbuminuria is a good indicator of early renal disease in dogs, especially glomerular disease.

Persistent microalbuminuria /proteinuria usually indicates the presence of CKD which

may or may not be progressive (persistent = present on 3 urinalyses 2 weeks apart).



Microalbuminuria that is persistent and increasing with time is of greater concern than stable microalbuminuria.

Persistent proteinuria detects animals at significant risk of an adverse health outcome.

Microalbuminuria may also occur with range of systemic diseases in dogs and cats (IBD, infectious, neoplastic, cardiovascular, metabolic, inflammatory).

The appropriate respons

Documents

VET343 Urinary Lecture Notes 2014