---start physio.lec.02.11.97----

Dr Bovee
Renal physiology continued.

renal blood flow: how is it controlled and measured?
glomerular filtration rate: process which leads to production of 
ultrafiltrate which pours into tubule.

bottom of p 3 handout:

TOTAL RENAL BLOODFLOW (RBF): total flow of BLOOD (not plasma) through 
kidney. to measure this, youhave to measure an arterial flow rate. you 
put something on renal artery and measure flow or use chemical method to 
measure flow.

electromagnetic flow meter: instrument placed around artery. movement of 
blood sets up EMF and meter can quantify blood flow precisely. so if you 
put one of htese on each renal artery you can measure total renal blood 
flow. this is invasive and is used in labs only.

doppler ultrasonic flow transducer: put ultrasound image - bombard artery 
with sound waves which are recorded and you can roughly determine flow of 
blood. this is a crude measurement, is off by 20-30% in general, but you 
can tell if flow seems more or less than normal.

most accurate and widely used method involves the CLEARANCE concept. 
clearance of Para-aminohippurate - solution is injected into blood, and 
clearance is measured (urine is tested) - this clearance represents RENAL 
PLASMA FLOW  (RPF)(not blood flow) but tyou can calculate blood flow 
accurately from this.

normal values for RPF

RBF = RPF/1-hct

12 +/- 2 mL/min/kg
260 +/- 20 mL/min/m^2 surface area (SA)

these RPF values are true for all species of mammals, and as shown are 
calculated either per body wt unit or per surface area unit.
um, how do you calculate surface area? there are tables...figured out via 
H2O displacement (human 1.73 m^2). generally, we express this value per 
1.0 m^2 as a standard. this makes little sense for small animals, so for 
them we express this per Kg. 

NON UNIFORM distribution of blood flow

recall how blood vessels are organized in kidney - there must be 
non-uniform blood flow since huge vessels are there in cortex, and huge 
plexi in cortex, and medulla is relatively avascular. 

in 1950 study was done showing differentiation of flow, leading to 
understanding of how flow is controlled. see fig 3.3 - inert gas washout 
method separates cortical flow from non-cortical flow. don't need to 
understand method in detail but know concept. you have time on x axis and 
radioactivity as seen as leaving kidney on y axis. so you put cath in 
renal artery and inject isotope. isotope goes with blood throughout 
kidney vasculature,and it is detected by sodium iodide crystal over the 
kidney. if there is high flow, isotope will disappear from that region 
quickly, if low flow, will leave slowly.
note that cortex and outer medulla had high flow, and medulla (inner 
medulla) had low flow, and then compartment 4 had very low flow. 
also can make inj, then clamp renal artery, freezing kidney blood in 
position, say at 5 min. then you slice and expose kidney to xray film to 
show where isotope is. don't do this to your patients, though. 
it turns out the fast component is all cortex - about 83% or 
radioactivity.
next fastest is outer medulla - 14%
inner medulla - 2 %
compt 4 - renal papillae - 1% - kind of nonfunctional component.

you can further determine nutrient flow - mL/100g/min

so this method as described above is a good way to determine regional 
blood flow in kidney, reminds us that flow is dramatically non-uniform 
and leads us to ask why flow is so nonuniform.

going a step further it was found that inside the cortex thee is also a 
non-uniform distribution of flow. parts of cortex have higher flow than 
others. MICROSPHERE method: measures intracortical compartments. involves 
injecting microspheres similar size as RBC into renal a., spheres enter 
glomerular capillaries and get stuck in there. so you do that, then you 
look at how many spheres ended up in different places: see fig 3-4 - flow 
greater in regions 2 and 3 than in 1 and 4 - this correlates well with 
the location of the glomeruli.
this method isn't accurate in outer medulla since there are no glomeruli 
there.

MEDULLARY BLOOD FLOW and so called WASHOUT OF RENAL MEDULLA
going back to  inert gas diagram...stop and think of flow represented by 
medulla - 16% of TBF. that flow is organized in that fashion for several 
reasons. the main reason is to keep solute trapped in interstitium of 
kidney, so it acts as osmotic force ot concentrate urine. so vessels in 
medulla must be held or fixed in a way to keep flow very low. "washout" 
is a pathologic state common in some dzs where the medullary flow 
increases, which allows blood to remove the solute (Na, Cl,urea) which 
decreases ability to concentrate urine. ths is very disadvantageous to 
the animal. it's important to make concentrated urine to control ECF 
volume.

renal bloodflow and OXYGEN CONSUMPTION fig 4-2
removal of oxygen from blood in kidney is different from all other organs 
in body. the massive blood flow to kidney that is ml/g is so high that 
kidney has HUGE quantity of O2 available, but it takes out a small 
portion of it. the O2 taken out is used for energy to reabsorb solutes - 
related to ATPase transport systems. and major solute kidney has to 
absorb from plasma is Na+. NaCl and NaHCO3. kidney is set up to carefully 
regulate and conserve sodium. so fig 4-2 correlates sodium absorption  (x 
axis) And O2 consumption (y axis). eg, renal tubular cells will take more 
O2 from blood if they need to absorb more sodium. if animal needs to lose 
more sodium to urine, will take less oxygen.this mechanism is unique to 
the kidney and is due to sodium regulation needs.

we'll talk about sodium transport later. then we'll see more about the 
above. note that all solutes are linked to O2 transport, not just sodium.


AUTOREGULATION of blood flow, fig 4-10

the kidney is going to decide itself how much blood it will recieve. 
kidney is presented with huge flow since we came from ocean, where sodium 
was everywhere. then we moved to land. so this is inefficient and kidney 
sees HUGE amt of blood/min. kidney knows it can get overloaded with this 
huge flow, so it needs local control. all organs do some autoregulation, 
but kidney has most complex system, involveing whole interrelated set of 
hormones. in general, the outline of this is seen in fig 4-10 - 
relationship of pressure (x) to flow (y). note this shows RPF and GFR, 
but look only at RPF right now. the renal arterial presesure is same as 
MAP mean arterial pressure. this curve represents flow seen in kidney at 
the given pressures of delivery. this is pressure IN RENAL ARTERY, the 
presenting pressure, which is usally 100mmHg. if MAP is below 100, flow 
falls dramatically. this region on graph is associated w/hypotensive or 
shocky state. a situation where something happened to vascular tone, 
relaxing it, or there has been volume loss, and the result is there isn't 
enough volume to maintain needed hydrostatic pressure. or else the heart 
has failed and isn't generating pressure.if any of the three factors 
decreases, kidney will be presented with lower pressure, and flow will be 
greatly reduced. this is called autoregulation but really, kidney has 
been abandoned. pressure gone down, flow down, kidney is under perfused, 
and is in big trouble. this is an ischemic kidney after a couple of hours 
and then you're REALLY in big trouble, with severe cell damage due to O2 
deficiency. this is not the oxygen that is carefully regulated with 
sodium, this is nutrient supply to renal tubular cells. those cells will 
die. so in this part of chart, there is NOT good autoregulation. we'll 
talk later about how kidney can adjust under low pressure and fight to 
maintain some plasma flow in this range as pressure goes to 70,60, 50.40. 
can't really do much if it goes below 40- cells will die.
real autoregulation occurs between about 80 and 220 mmHg - flow in this 
range does not change, remains constant. if kidney didn't have a system 
to autoregulate, kidney would see dramatic changes in flow every time you 
have a blood pressure change. you have bp changes all the time, so this 
would be bad. there's a second to second bp cycle, and a 10-15 min bp 
cycle, and a 24 hr bp cycle. you're constantly having bp changes. it's 
higher during day, is way higher when you eat or exercise or get 
emotional or sexually aroused. all of these cycles - plus dietary factors 
eg coffee - would result in increases in flow to the kidney and that 
would be bad for kidney. kidney not able to regulate all that solute. 
you'd be forced to urinate every 10 minutes if flow increased with 
pressure :)

so youhave a mechanism of control keeping flow stable between about 100 
and 220 and that's autoregulation. also, very high pressure - malignant 
hypertension above 220 mmHg causes breakdown of autoregulation, can no 
longer control, vascular tone is not able to resist pressure and vessels 
break down and get destroyed. blood gets extravascular. this can kill an 
animal inside an hour and is very rare.

mechanisms of control of autoregulation

MYOGENIC THEORY: there is wall tension which is influenced by pressure 
and internal diameter.
fig 4-10
T = transmural pressure P x internal vessel radius R
T = P R --->Laplace equation

myogenic control - smooth muscle control - this occurs in the afferent 
(preglomerular) arterioles, and the arterioles branching off the arcuate 
artery leading to afferent arteries.  these vessels follow this equation 
in a GENERAL sense. there are many smooth muscle components in the 
vessels, that are regulated by hormones which dilate or constrict. main 
one is ENDOTHELIN. endothelin is major regulator of tone in these 
vessels. the details of how endothelin and other regulators work is not 
well understood, and is hard to study.

TUBULOGLOMERULAR THEORY of control of renal blood flow: fig 10-1
we spoke of the piece of distal tubule that is between the afferent and 
efferent arterioles, containing the macula densa. the cells there are in 
close proximity with the afferent arterole and they seem to send a 
chemical signal to the arteriole telling it to constrict or dilate. the 
whole apparatus is called the juxtaglomerular apparatus. the endothelial 
cells which receive the signals are called juxtaglomerular cells.

the major mechanism at work here is renin-angiotensin system: both 
endocrine and autocrine. it works generally to control part of vascular 
tonea nd locally where it is thought ot influence 
vasoconstriction/dilation of afferent arteriole in conjunction w/release 
of renin, ATI and AT II. this has been looked at by many researchers 
since 1950. theory: amt of sodium left in tubule at macula densa region 
is seen as too much (or not enough,) causing the tubular cells to signal 
the juxtaglomerular cells where renin is produced, causing more 
production of renin, causing AT I and II prod, and vasoconstriction, 
which reduces flow to glom, reducing delivery of sodium downstream. this 
is a possible explanation of autoregulation but doesn't explain it fully. 
it plays a role but there are other factors.

--break---

influence of vasoactive agents on renal blood flow:
(total blood flow to kidney)

epi and norepi - catecholamines. epi is made in adrenal, norepi made in 
nerve endings all over. these two agents circulating and working locally 
are major vasoconstrictors which help maintain vascular tone minute to 
minute. wrt kidney, they are vasoconstrictors which maintian 
vasoconstrictive tone in main vessels leading to kidney and in aff. aa 
and glomerulus. may also work on efferent aa but those would be minor. 
these are tone controllers, boosters of pressure mainly on afferent side

isoproterenol: a peculiar molecule which affects blood flow differently 
depending on concentration.  (we used to call this isoprokillemall...) if 
you give a low dose, you increase renal blood flow. a high dose acts as a 
vasoconstrictor, decreasing flow. this is not an endogenous substance and 
is only important in studies...but it shows that agents can have opposite 
effects from what you'd expect.

ACH - vasodilator
bradykinin - and other kinins - vasodilator - made in vasculature all 
over body and also in kidney. these are major vasodilators in 
non-cortical region of kidney

dopamine: catecholamine: produced locally and centrally (in brain). is a 
major vasodilator in most vascular beds, moderate vasodilator in kidney.

angiotensin - AT I, AT II, AT III. AT II is the "classic" one and is 
potent vasoconstrictor. helps maintian role in vessels in kidney.most of 
it is produced in jg apparatus.

endothelin: the most powerful vasoconstrictor we have; made in afferent 
aa, and is exclusively autocrine: no endocrine functions.

prostoglandins: vasodilators in kidney. produced locally in kidney and 
will oppose the vasoconstrictor effects of catecholamines and AT II.

thromboxane: several of these but most important is thromboxane AII which 
is released by WBCs into circulation and also sometimes by damaged 
endothelium in kidney and it's powerful vasoconstrictor, constricts 
afferent aa, commmon in acute inflammm dz

ADH: is a vasoconstrictor in kidney in medulla. this hormone is made only 
in post. pituitary - classic endocrine substance.

NO, EDRF  - NO was "molecule of the year" in 1991, formerly endothelium 
relaxing factor. is an autocrine molecule, vasodilator, made all over the 
place, antagonizes endothelin. acts locally only

Natriuretic peptide: small peptides made in heart or brain. act as 
vasodilators in kidney. kidney may also produce some of thse, not much.

so there is a variety of substances. all of these work in harmony 
opposing each other at different times and stuff. it's kind of complex, 
because these also are involved in regulating transport, movement of 
water, solute, etc. highly integrated system with multiple competing and 
synergistic mechanisms resulting in highly controlled system that's hard 
to understand. try to not get confused. you will not understand the 
kidney after 10 lectures. it takes years.

RENAL NERVES

what about nerves? there are renal nerves innervating afferent 
arteriole...they are a mixture of andrenergic and cholinergic fibers 
coming from last thoracic and first few lumbar vertebrae. they follwo 
renal artery, and some end at aff. aa and some end in glomeruli and some 
end at proximal tubules.  it turns out these nerves aren't really 
involved in minute to minute regulation of bloodflow or transport. they 
are onnly essential in release of norepi in "fight or flight" situations, 
where they cause major vasoconstriction via norepi. we know this 'cause 
we can denervate the kidney (surgically or chemically) and kidney still 
works absolutely fine with all autoregulation intact. also, we can see 
the norepi released from nerves during "fright or flight" rxn. the first 
demo of "fright or flight" was in GSD at harvard in the 50-60s. one day 
in the secluded lab, dogs were being monitored, and a plumber walked in 
holding a big giant wrench, and dogs went nuts, trying to attack plumber. 
their renal blood flow dropped to 20% of normal as they chased out the 
plumber. it took about 1.5-2 hrs for renal blood flow to return to 
normal. they had massive increase in catecholamine release, bringing 
kidney flow down to about 20% of normal. this is "emergency" situation 
where renal nerves play a role. they also play a role in severe 
hypertension but we aren't going to cover that.

other factors affecting RBF

hemorrhage: hypotensive state is induced. keep in mind that once MAP is 
below 60, this RBF is dramatically reduced, falls to about 20-30% of 
normal, and kidney is in trouble. this state is induced by trauma, 
laceration of large vessel.

change in ECF volume: that volume is recognized by kidney all the time. 
kidney has been endowed with natriuretic peptides from brain and heart 
that recognize volume of ECF. they are increased and decreased in 
circulation depending on volume and they come to kidney and say we're 
over or under expanded and tell kidney to change amt of Na+ and H2O. say 
you expand animal's ECF volume. eg, give normal saline IV  - a big bolus 
of water. soon, this fluid is recognized by vascular stretch receptors, 
and heart and brain release peptide, which acts as peripheral 
vasodilator. comes to kidney and acts specifically at certain tubular 
sites to cause decreased Na+ and H2O reabsorbtion. ths happens w/in a few 
minutes. decrease in ECF volume also causes things to happen...whole 
bunch of signalling molecules come and cause vasoconnstriction in kidney 
and tell kidney to conserve H2O and Na+ and other solutes.

Diuretics: man made agents that we have devised in order to turn down Na+ 
reabsorption. these all reduce kidney function. people think they 
increase it but that's not true, though they increase urine output. 
they're mild vasoconstrictors that work by decreasing the efficacy of Na+ 
reabsorption. some don't change RBF, most decrease it, none increase it.

Exercise: species specific. people - RBF goes down 10-15% during 
exercise. but, sled dogs have no reduction in RBF while running until 
they become dehydrated.

Anesthesia: all anesthetics reduce RBF by 10-30% IF anesth is prolonged 
and animal gets cold, you can develop ischemic kidney.

GLOMERULAR FILTRATION AND CLEARANCE CONCEPT

glomerular filtration: a highly selective ultrafiltration process, unique 
to renal gloms, capillaries here are unique, and allow a great deal of 
plasma and small solutes to leave circulation and become ultrafiltrate. 
does not allow lg molecules to pass out of capillary. those go out 
efferent aa and back to core circulation.

number, size, uniformity of glomeruli. all glom are relatively similar - 
eg, glom of a mouse is the same size as glom of an elephant, except 
elephant has a lot more. small animals have fewer than large. we talked 
about two kinds of glomeruli....all glomeruli are open all the time. they 
don't close down and reopen. but they may function at somewhat different 
filtration pressures. eg flow going through them changes minute to minute 
or hour to hour. when you sleep and BP falls, and volume is stable, your 
GFR will fall during night and you'll make very little urine so you don't 
have to get up at night. so GFR reduction at night is big factor. doesn't 
turn off, just slows down.

how do we study glomeruli? in situ or in vitro. we can put pipettes into 
bowman's capsule, into the tubule, into the glom. capillary, into the 
arterioles, and we can measure concentrations of solute - MICROPUNCTURE 
METHOD - developed at Penn Med in 1927. very small glass pipettes were 
used, glomeruli were dissected out in situ in fish, and filtrate was 
sampled to determine content.

there is HUGE surface area of glomeruli.
glomerular surface area is huge due to great need for filtration of huge 
volume of plasma. it turns out the ECFV passes through gloms every few 
hours. ENTIRE extracellular fluid volume goes through kidney several 
times a day. surface area of glom dramatically exceeds body surface area. 
a 15 kg dog has 300 sq ft of glomerular capillaries. that's HUGE.

hydraulic permeability of glom caps compared to other caps. see table 
6-1. 
k=permeability in right column. k is quantified in terms of flow in 
microliters per minute * mmHg * sq.cm. you can see the volume that is 
produced - er, the permeability that is produced, is HUGE compared to 
other capillaries. 2.5 compared to .008-.5 in other capillaries. so a 
huge volume of stuff can pass through glomerular capillaries, but not 
through any other capillaries.

SELECTIVITY of glomerular membrane - table 4-21

Pore size: there are pores of about 75 angstroms. anything bigger than 
that can't pass through capillary wall.anything smaller can get into 
ultrafiltrate. we think of the filtrate as being protein free, because 
proteins are large and stay in capillary and enter efferent arteriole. 
the determinants of who passes and who doesn't are molecular weight and 
radius of molecule. see table 4-21. see that inulin clearance is 1.0, 
which is the highest. this could just as well be glucose which is also 
freely filtered. clearance (u/p) * (u/p) substance. so a freely filtered 
substance = 1.0. the other things can pass not as freely. water passes 
freely, glucose, sucrose freely pass. but...myoglobin, albumen, 
Hb...these do not pass freely. .75 of  myoglobin will pass, .25 kept in. 
albumin - .003 passes - essentially, no albumin passes. 

electrolytes pass like water, urea, and glucose unless they are bound to 
albumin or other large molecule. gammaglobulin is bigger than albumin, 
and won't pass.

FUNCTIONAL STRUCTURE OF GLOMERULAR MEMBRANE: 10-1

one capillary loop is shown with two associated cells - an epithelial 
cell on the outside, and endothelial cells on inside (labelled ep and 
end). if we make a cross section we see what we see in diagram. blood is 
inside capillary and has association w/endothelial cells - see endo cell 
labelled 5...fluid moves through and goes through slit processes of endo 
cells. then it contacts the trilayer basement membrane. the core of bm 
number 3 is major barrier made of glycoproteins, very dense in structure. 
then another thin layer of glom membrane 2 and then epi cell 1 with foot 
processes with slits in between, and fluid passes through slits into 
lumen of filtration space.


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