GFR is the best indicator of renal function. Numerous tests have been proposed for measurement of GFR, all requiring the calculation of urine or plasma clearance of a suitable indicator. Clearance is a proportionality constant describing the relationship between the rate of transfer of a substance, in amount per unit of time, and its concentration in an appropriate reference fluid which could be urine or plasma (Figure 4.1).
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Figure 4.1 – Principle of plasma clearance
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Urine clearance tests
Urinary clearance of different markers has been recommended in the past. These tests most often use creatinine or inulin as indicators. Urine inulin clearance is generally regarded as the reference method for determining GFR. These techniques have been valuable in research, but are not feasible in veterinary practice. They are labour-intensive, time consuming, they require the accurate collection of timed-urine samples and have the added risk of urinary infection from repeated urethral catheterisation.
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24-hour urine clearance tests require cage rinsing to maximise the recovery of the test substance in a similar way to drug metabolism studies. Failure to rinse the cage can result in a 45% underestimation of urine creatinine clearance, which would lead to a misinterpretation of the renal function of the animal (Watson & al, 2002). The principle of the calculation is explained in Figure 4.2.
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Figure 4.2 – Principle of urinary clearance
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Single-bolus injection tests
In single injection tests, a marker is injected intravenously and the GFR is determined from plasma clearance (assuming that extra-renal clearance is negligible). A variety of markers have been used, such as inulin, iodine-containing contrast agents (iohexol and iothalamate), various radionucleides and creatinine. However, many of these tests have limitations, such as the requirement for large sample volumes (up to 3-4 mL of plasma for iohexol assay by x-ray fluorescence), the assays can be difficult or expensive or not readily available, and the use of radioactive markers is undesirable.
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Plasma Exogenous Creatinine Clearance Test (PECCT)
This test has been developed recently in dogs (Watson & al, 2002), but preliminary data indicates that it may also be useful in cats (Le Garreres & al, 2001).
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The determination of plasma clearance requires only blood sampling. Creatinine is an endogenous compound that is continuously produced by the metabolism of creatine in muscle. Creatinine is excreted solely by the kidneys, is filtered completely by glomeruli, and undergoes negligible tubular secretion. Therefore, plasma creatinine clearance and renal creatinine clearance both reflect GFR (Figure 4.3).
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Assessment of plasma clearance of creatinine is much better for assessing renal function than measuring the plasma creatinine concentration alone. The latter is really only an indirect and relatively rough indicator of GFR because it depends on the production of creatinine from muscle, its volume of distribution and its elimination from the body. When the production and volume of distribution of creatinine are constant, any increase in plasma creatinine concentration is an indicator of an inverse change GFR. In those circumstances, monitoring plasma creatinine concentration may be helpful to detect any improvement or worsening of renal function during long-term patient assessment. The main advantage of PECCT is that it provides a direct estimate of GFR, independent of the volume of distribution and the endogenous production of creatinine.
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Figure 4.3 – Measurement of GFR
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If the marker is not metabolized and if it is totally cleared by the kidneys, it disappears as the same rate from plasma as it appears in urine.
• GFR = Plasma clearance
• GFR = Dose / Area Under the Curve
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The steps involved with the PECCT (see Appendix for detailed description) are:
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- determination of basal plasma creatinine concentration
- intravenous bolus administration of a known amount of creatinine
- repeated blood sampling following administration of creatinine
- determination of plasma creatinine concentrations
- calculation of plasma clearance of exogenous creatinine.
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• Validation of the test:
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The GFR values determined with PECCT are similar to those obtained with other validated tests for GFR assessment such as urinary inulin clearance, endogenous creatinine clearance and plasma iothalamate clearance. It has been verified in healthy and renal-impaired animals (Figure 4.4)
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• Indications for the test
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PECCT should be performed in the following situations.
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- When CRF cannot be clearly confirmed by clinical signs and biochemical features: for example in the case of a 10-year old German Shepherd dog with inappetence, a plasma creatinine value of 190 µmol/L (2.15 mg/dL) and USG 1.025.
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- When drug dosage adjustment is recommended in renal-impaired patients: for example the drugs that are essentially cleared by the kidneys and have a low therapeutic index may accumulate in CRF conditions. This higher level of drug within the body may lead to adverse effects.
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- When GFR is a primary factor in a clinical trial for assessment of the efficacy of a new drug or diet indicated for treating CRF. In that case, repeated measurements of GFR could be performed over time in the same patients.
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Figure 4.4 - Plasma exogenous creatinine clearance is comparable with urine inulin clearance
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This graph shows mean clearance values obtained in 6 healthy dogs for plasma (P) and urine (U) urine clearances of inulin, exogenous creatinine (exo) administered at 3 different doses (40, 80 and 160 mg/kg) and plasma iothalamate (P-iothal) clearance.
A clear difference in plasma and urine inulin clearance was seen (the reference method). This is because inulin is also cleared by extra-renal routes.
Plasma exogenous creatinine clearances provides an adequate estimate of GFR, whatever the dose administrated (Watson & al, 2002).
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- PECCT can be performed in routine veterinary practice.
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- Only 1mL blood samples are required, which allows repeated blood sampling to occur even in small patients such as puppies or miniature dogs.
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- It does not require specialised assays that are available in only few laboratories (unlike iohexol). Plasma creatinine concentrations can be assayed with routine biochemical analysers available in most veterinary practices.
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- Results are available immediatly after the test has been performed.
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- Determination of creatinine clearance does not require complex calculations to be performed. It can be calculated easily using specific software such as the "Creatinine clearance calculator" which has been created specifically for this purpose.
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- Creatinine is a safe test agent. Values up to 8,000 µmol/L (90 mg/dL) have been observed after intravenous bolus of large doses without adverse effects in dogs suffering from CRF.
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- PECCT is not expensive.
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- There is no commercially available solution made up ready to use. Nevertheless, anhydrous creatinine powder is available from providers of chemicals for biology. A special formulation for veterinary use in currently under development.
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- The last blood sample should be taken at least 6 hours after creatinine administration. The need to keep a dog hospitalised for one day is a relative limitation, but such hospitalisation periods are very common when further investigations are required. Moreover, the major limit for such a diagnostic test is more usually the time to obtain the result, which is very short here (within one day), compared with other proposed tests.
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- Proper reference intervals are not yet available for any method of GFR assessment. Currently, data obtained from only a limited number of animals (most often Beagle dogs) are cited in the literature. Use of the PECCT in larger canine and feline populations will allow development of better reference intervals.
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