Experimentally induced renal insufficiency in laboratory animal Measurement of GFR based on kinetics of renal inulin excretion Ústav patologické fyziologie LF MU Kidney functions • regulation of • extracellular volume and blood pressure • ion balance • acid-base balance • excretion of waste • production of hormones Nephron – basic processes • nephron is the functional unit of the kidney • filtration • movement of fluid from bloodinto the lumen of nephron • only in the renal corpuscule • filtrate is produced • reabsorption • moving of substances from the filtrate into the blood • typicallysymport • Na/Glu, Na/AK… • transport maximum • renal threshold for glucose • secretion • adding selected molecules from the blood to the filtrate • active proces, more selective then filtration Filtration barrier • filtration takes place in the renal corpuscule • filtration barrier • glomerular capillaryendothelium • fenestrated • negatively charged proteins on the surface • basement membrane • layer of extracellular matrix • separates endothelium from epitelial cells of Bowman‘s capsule • glycoproteins, collagen, … • epitheliumof Bowman‘s capsule • podocytes • foot processes – long cytoplasmic extensions – leave narrow filtration slit closed by semiporous membrane • severalunique proteins • mesangial cells • between and around glomerularcapillaries • contraction • actin-like filaments • productionof cytokines Renal handling of different substances Glomerular filtration • determined by 3 pressures • hydrostaticpressure • blood flowing through glomerular capillaries • 55 mm Hg, higher than opposite pressures • colloidosmotic pressure • inside glomerular capillaries • 30 mm Hg, higher than that of fluid in Bowman‘s capsule • favors fluid movement back into capillaries • fluid pressure • pressure of fluids in Bowman‘s capsule • 15 mm Hg, opposes filtration • net driving force • 10 mm Hg • GFR is influenced by 2 factors • net filtrationpressure • filtrationcoefficient • surface area of glomerular capillaries • permeability GFR is subject to autoregulation • autoregulation • local controlproces • kidney maintainsa relativelyconstantGFR in the face of bloodpressure fluctuations • 80 – 180 mm Hg • mechanisms • myogenic response • intrinsic ability of vascular smooth muscle to respond to pressure changes • tubuloglomerularfeedback • fluid flow through tubule influences GFR Laboratory assessment of kidney disease • glomerular filtration rate (GFR) • the best overal index of kidney function • approx. 125 ml/min/1.73 m2 in young adult healthy individuals • indexing to body surface area reduces variation among healthy individuals • proteinuria • diagnostic and prognostic information • CKD, AKI, preeclampsia • often earliest marker of glomerular diseases • urinalysis • color • odor • relative density • pH • glucose • ketones Detection and diagnosis of kidney disease • lack of signs in early stages of chronic kidney disease (CKD) • tests for screening and diagnosis are critical in nephrology • measurement of kidney function • early detectionof abnormalities • specific diagnosis • determine prognosis • measurement of disease progression • effectiveness of therapy • CKD – independent risk factor for cardiovascular disease • identificationof patientsfor cardiovasculardisease risk factor management • acute and chronic diseases are common • acute kidney injury occurs in 10 – 20 % of hospitalised patients • chronic kidney disease is present in 10 – 15 % of adults in the general population KDIGO classification of CKD Kidney clearance • the volume of plasma from which a substanceis completely removed by the kidneys per time unit • unit: volume/time • indirect measure of glomerular filtration rate (GFR) and renal blood flow • GFR • clearance of filtered substance • perfusion • clearance of a substance that is filtered and also secreted (PAHA) • 2 approaches to determine GFR • endogenous marker with stable serum concentration • exogenous marker • plasma concentration decreases • not routinely available because of the complexity of the protocol Marker of GFR • requirements • stable plasma concentration • not protein bound • freely filtered by glomerulus • not secreted, reabsorbed, or metabolized by the tubules • not altering GFR per se • easy measurement • no interference Determinants of the serum level of endogenous filtration markers Relationship between clearance and GFR • in the steady state, for a substancehandled only be the kidneys that is neither reabsorbed nor secreted • amount filtered = amount excreted • GFR x Px = Ux x V • GFR = U x V/Px • cX = volume of plasma filtered (~ GFR) • UX = urine concentrationof X • V = urine output (ml/min) • PX = plasma concentrationof X • clearance of the substancecorresponds to GFR • reabsorption of a substanceX by tubules • clearance GFR Creatinine • proposed as a filtration marker in 1926 • produced by non-enzymatic degradation of phosphocreatine in the muscle • relatively constant production • depends on age, gender and muscle mass • possible GIT elimination at higher serum levels • kidney handling • free filtration • no reabsorption • secretion up to 10% • advantages • easy measurement • low cost • widespread availabilityof assays Serum creatinine vs. GFR Estimation of GFR from serum creatinine concentration • CKD-EPI • formerly • Cockroft-Gault • MDRD Cystatin C • cysteine protease inhibitor • produced by all nucleated cells • freely filtered at glomerulus • not secreted by tubules • reabsorbed and metabolized by tubules • urine levels are low • GFR is estimated from it‘s serum concentration • serum concentration • dependson age and sex • further affected by • inflammation, treatment with corticoids, smoking • hyperthyreosis, CRP • factors that influence cystatin C levelsare less defined • possible combination of cystatin C and creatinine for the estimation of GFR Measurement of glomerular filtration without collection of urine • intravenous application of a substance excreted only by glomerular filtration • plasma concentration measurementin corresponding intervals • plasma concentration decreases according to the exponential relation • concentrationswill be plotted with corresponding intervals on semi-logarithmic paper • we acquire line which we can use to identify biological half-life (t1/2) • express the time it takes for the blood plasma concentration of a substance to halve its initial concentration (Po) Inulin • polysaccharide isolated from roots of a variety of plants • meets all of the criteria for the ideal substanceto measure GFR • not practical for routine clinical applications • does not occur naturally in the body • inulin clearance can be measured based on it‘s kinetics in extracellular fluid GFR calculation • D = amount of inulin applied • 25 mg • P0 will be deduced from the curve • concentration in time when substancewas injected = projection of line with axis Y • extrapolated value in relation to time when the dose was administered • t1/2 will be deduced from the curve Experimental procedure • general anesthesia • middle laparotomy • experimental procedure • ligation of a. renalis sinistra (n = 6) • control group – without ligature (n = 6) • accessing v. jugularis and application of inulin • 25 mg/1,5 ml of physiologic solution) • blood sampling • thoracotomy after 5, 10, 15, 20, 25 and 30 min • heart puncture - 1,5 ml of blood • sample analysis • spectrophotometric determination of inulin concentration • calculation of P0 and t1/2