Urolithiasis biochemistry

1. URINARY LITHIASIS-
PHYSICOCHEMISTRY
Dept of Urology
Govt Royapettah Hospital and Kilpauk Medical College
Chennai

2. Moderators:
Professors:
• Prof. Dr. G. Sivasankar, M.S., M.Ch.,
• Prof. Dr. A. Senthilvel, M.S., M.Ch.,
Asst Professors:
• Dr. J. Sivabalan, M.S., M.Ch.,
• Dr. R. Bhargavi, M.S., M.Ch.,
• Dr. S. Raju, M.S., M.Ch.,
• Dr. K. Muthurathinam, M.S., M.Ch.,
• Dr. D. Tamilselvan, M.S., M.Ch.,
• Dr. K. Senthilkumar, M.S., M.Ch.
Dept of Urology, GRH and KMC, Chennai. 2

3. CONCENTRATION PRODUCT
A solution containing ions or molecules of a
sparingly soluble salt is described by the
concentration product, which is a mathematic
expression of the product of the concentrations
of the pure chemical components (ions or
molecules) of the salt
3
Dept of Urology, GRH and KMC, Chennai.

4. SOLUBILITY PRODUCT
• A pure aqueous solution of a salt is considered
saturated when it reaches the point at which
no further added salt crystals will dissolve.
• The concentration product at the point of
saturation is called the thermodynamic
solubility product, Ksp, which is the point at
which the dissolved and crystalline
components are in equilibrium for a specific
set of conditions
4
Dept of Urology, GRH and KMC, Chennai.

5. METASTABLE RANGE
In urine, despite concentration products of stone forming
salt components such as calcium oxalate that
exceed the solubility product, crystallization does not
necessarily occur because of the presence of inhibitors
and other molecules that allow higher concentrations of
calcium oxalate to be held in solution before precipitation
or crystallization occurs. In this state of saturation,
urine is considered to be metastable with respect to
the salt
5
Dept of Urology, GRH and KMC, Chennai.

6. FORMATION PRODUCT
As concentrations of the salt increase further,
the point at which it can no longer be held in
solution is reached and crystals form.
The concentration product at this point is called
the formation product, Kf.
6
Dept of Urology, GRH and KMC, Chennai.

7. The solubility product and the formation
product differentiate the three major states of
saturation in urine:
1. undersaturated,
2.metastable,
3.unstable
7
Dept of Urology, GRH and KMC, Chennai.

8. Nucleationwill occur
Inhibitorsnot generally effective
Unstable FORMATIONPRODUCT
Meta stable CONCENTRATIONPRODUCT
Crystal growth will occur
Crystal aggregation will occur
Inhibitorswill impede or
prevent crystallization
De novo nucleation is very slow
Heterogeneousnucleation may occur
Matrix may be involved
Undersaturated SOLUBILITYPRODUCT
Crystals will not form
Existing stones may dissolve
T
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E
R
A
P
Y
8
Dept of Urology, GRH and KMC, Chennai.

9. In Metastable range
Crystal growth can occur on existing crystals
Denovo formation of crystals occur in the following
situations
1. In parts of the nephron local concentration
products may exceed the formation product for
long enough time periods to allow nucleation to
occur.
2. local areas of obstruction or stasis in the
upper urinary tract
Microscopic impurities or other constituents
facilitating heterogenous nucleation
9
Dept of Urology, GRH and KMC, Chennai.

10. In normal human urine, the concentration of
calcium oxalate is four times higher than its
solubility in water.
Urinary factors favoring stone formation
including low volume and citrate and
increased calcium, oxalate, phosphate, and uric
acid
all increase calcium oxalate supersaturation
10
Dept of Urology, GRH and KMC, Chennai.

11. in the presence of urinary inhibitors
and other substances, calcium oxalate
precipitation occurs only when
supersaturation exceeds solubility by 7 to 11
times.
11
Dept of Urology, GRH and KMC, Chennai.

12. • Homogeneous nucleation is the process by
which nuclei form in pure solution
• Nuclei are the earliest crystal structures that
will not dissolve
• Inhibitors, like citrate, destabilize nuclei,
• whereas promoters stabilize nuclei by
providing a surface with a binding site that
accommodates the crystal structure of the
nucleus
12
Dept of Urology, GRH and KMC, Chennai.

13. • In urine, crystal nuclei usually form through
heterogeneous nucleation by adsorption onto
existing surfaces of epithelial cells, cell debris
or other crystals.
• Within the time frame of transit of urine
through the nephron,estimated at 5 to 7
minutes, crystals cannot grow to reach a size
sufficient to occlude the tubular lumen
13
Dept of Urology, GRH and KMC, Chennai.

14. INHIBITORS
• Magnesium and citrate inhibit crystal aggregation
• Nephrocalcin, an acidic
glycoprotein made in the kidney, inhibits calcium
oxalate nucleation , growth, and aggregation.
• Tamm-Horsfall mucoprotein, the most abundant
protein in urine, inhibits aggregation
• Uropontin inhibits crystal growth
• Bikunin, the light chain of inter-α inhibitor, has
been shown to be an efficient inhibitor of crystal
nucleation and aggregation.
14
Dept of Urology, GRH and KMC, Chennai.

15. INHIBITORS
• inhibitors have been identified for calcium
oxalate and calcium phosphate,
• no specific inhibitors are known that affect
uric acid crystallization
15
Dept of Urology, GRH and KMC, Chennai.

16. INHIBITORS
• Inorganic pyrophosphate was found to be
responsible for 25% to 50% of the inhibitory
activity of whole urine against calcium
phosphate crystallization
• citrate, magnesium, and pyrophosphate
together were noted to account for
approximately 20% of the inhibitory activity of
whole urine , with citrate comprising the most
important factor of the three
16
Dept of Urology, GRH and KMC, Chennai.

17. CITRATE
• Citrate acts as an inhibitor of calcium oxalate and
calcium phosphate stone formation
1.First, it complexes with calcium, thereby reducing the
availability of ionic calcium to interact with oxalate or
phosphate
2. Second, it directly inhibits the spontaneous
precipitation of calcium oxalate and prevents the
agglomeration of calcium oxalate crystals . It has
potent activity in reducing calcium phosphate growth
than oxalate.
3. Lastly, citrate prevents heterogeneous nucleation of
calcium oxalate by monosodium urate
17
Dept of Urology, GRH and KMC, Chennai.

18. MAGNESIUM
• The inhibitory activity of magnesium is
derived from its complexation with oxalate,
which reduces ionic oxalate concentration and
calcium oxalate supersaturation
• magnesium reduces the rate of calcium
oxalate crystal growth in vitro
18
Dept of Urology, GRH and KMC, Chennai.

19. POLYANIONS
• Polyanions including glycosaminoglycans, acid
mucopolysaccharides, and RNA have been
shown to inhibit crystal nucleation and growth
• Among the glycosaminoglycans, heparin
sulfate interacts most strongly with calcium
oxalate monohydrate crystals
19
Dept of Urology, GRH and KMC, Chennai.

20. URINARY GLYCOPROTEINS
• Two urinary glycoproteins, nephrocalcin and
Tamm-Horsfall glycoprotein, are potent
inhibitors of calcium oxalate monohydrate
crystal aggregation
20
Dept of Urology, GRH and KMC, Chennai.

21. NEPHROCALCIN
• Nephrocalcin is an acidic glycoprotein
containing predominantly acidic amino acids
that is synthesized in the proximal renal
tubules and the thick ascending limb
• Has four isoforms with two isoforms having
inhibitory activity containing
γ-carboxyglutamic acid residues that were
lacking in the isoforms isolated from stone
formers.
21
Dept of Urology, GRH and KMC, Chennai.

22. Tamm-Horsfall protein
• Tamm-Horsfall protein is expressed by renal epithelial
cells in the thick ascending limb and the distal
convoluted tubule as a membrane-anchored protein
that is released into the urine after cleavage of the
anchoring site by phospholipases or proteases.
• Tamm-Horsfall is the most abundant protein found in
the urine and a potent inhibitor of calcium oxalate
monohydrate crystal aggregation, but not growth.
22
Dept of Urology, GRH and KMC, Chennai.

23. OSTEOPONTIN/UROPONTIN
• Osteopontin, or uropontin, is an acidic phosphorylated
glycoprotein
• expressed in bone matrix and renal epithelial cells of the
ascending limb of the loop of Henle and the distal tubule.
Osteopontin
• has been shown to inhibit nucleation, growth,
and aggregation of calcium oxalate crystals, as well as to
reduce binding of crystals to renal epithelial cells in vitro
• osteopontin may constitute an inducible inhibitor of
calcium oxalate crystallization that works in conjunction
with constitutively expressed Tamm-Horsfall protein to
prevent crystallization.
23
Dept of Urology, GRH and KMC, Chennai.

24. Inter-α-trypsin
• is a glycoprotein synthesized in the liver
• that is composed of three polypeptides (two
heavy chains and one light chain), of which
bikunin comprises the light chain
• Bikunin is a strong inhibitor of calcium oxalate
crystallization, aggregation, and growth in
vitro
24
Dept of Urology, GRH and KMC, Chennai.

25. Free crystal
particle growth versus fixed particle growth
• Fixed particle growth theory presupposes an
anchoring site to which crystals bind, thereby
prolonging the time the crystals are exposed
to supersaturated urine and facilitating crystal
growth and aggregation
• One favuored theory proposes that
oxalate-induced injury to renal tubular
epithelial cells promotes adherence of calcium
oxalate crystals
25
Dept of Urology, GRH and KMC, Chennai.

26. RANDALL’S PLAQUES
• the origin of the plaque is attributed to the
basement membrane of the thin limbs of the
loops of Henle and the plaque subsequently
extends through the medullary interstitium to a
subepithelial location (Fig. 45–3). Once the
plaque erodes through the urothelium, it is
thought to constitute a stable,
anchored surface on which calcium oxalate
crystals can nucleate and grow as attached
stones
26
Dept of Urology, GRH and KMC, Chennai.

27. RANDALL’S PLAQUES
• Using high-resolution Fourier transform infrared
microspectroscopy and electron diffraction, the
crystal component of plaque was determined to
be calcium apatite
• Deposits consisted of individual laminated
particles with mineral and organic layers All
crystals were coated with organic material, and
• osteopontin was indentified on the outer surface
of the crystal at the junction of the overlying
organic molecular layer
27
Dept of Urology, GRH and KMC, Chennai.

28. RANDALL’ PLAQUE-ENDOSCOPIC VIEW
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Dept of Urology, GRH and KMC, Chennai.

29. RANDALL’S PLAQUE-HISTOLOGIC VIEW
Ducts Ducts of Bellini
29
Dept of Urology, GRH and KMC, Chennai.

30. Association between
CNP and Randall plaque
• nanobacteria,or calcifying nanoparticles
(CNPs), which are self-propogating entities
that precipitate calcium apatite on their
exterior membrane
30
Dept of Urology, GRH and KMC, Chennai.

31. TUBULAR CALCIFICATIONS
• Patients with enteric hyperoxaluria due to
intestinal bypass for obesity demonstrate no
plaque but instead show apatite crystal
deposits plugging the inner medullary
collecting duct lumens, along with associated
epithelial cell damage with interstitial
inflammation and fibrosis
31
Dept of Urology, GRH and KMC, Chennai.

32. Tubular vs Interstitial calcifications
Brushite stone formers have been found to have
pathology intermediate between idiopathic calcium
oxalate stone formers and intestinal bypass patients,
demonstrating interstitial apatite plaque and apatite
plugging of the inner medullary and terminal collecting
ducts, along with associated collecting duct injury and
interstitial fibrosis
CYSTEINURIA ; although patients with cystinuria were found
to have plugging of the terminal collecting ducts of Bellini
with masses of cystine crystals, surprisingly apatite
deposits were also identified in the inner medullary
collecting ducts and in the thin ascending limbs of the
loops of Henle
32
Dept of Urology, GRH and KMC, Chennai.

33. Matrix
• The noncrystalline component of renal calculi is
termed matrix, which typically accounts for about
2.5% of the weight of the stone
• matrix comprises the majority of the stone (up to
65%), usually in association with chronic urinary
tract infection
• heterogeneous mixture consisting of 65% protein,
9% nonamino sugars, 5% glucosamine, 10%
bound water, and 12% organic ash
33
Dept of Urology, GRH and KMC, Chennai.

34. MATRIX
proteins incorporated into the matrix substance are
• Tamm-Horsfall protein,
• nephrocalcin,
• a γ-carboxyglutamic acid–rich protein,
• renal lithostathine,
• albumin,
• glycosaminoglycans,
• free carbohydrates, and
• a mucoprotein called matrix substance A
• significant number of inflammatory proteins(immunoglobulins,
defensin-3, clusterin, complement C3a, kininogen, and fibrinogen)
34
Dept of Urology, GRH and KMC, Chennai.

35. KEY POINTS
• Urine must be supersaturated for stones to form.
• Supersaturation alone is not sufficient for crystallization to
occur in urine, owing to the presence of urinary inhibitors.
• Nephrocalcin, uropontin, and Tamm-Horsfall protein are
important inhibitors of crystal nucleation, growth, or
aggregation.
• Urinary calcium and oxalate are equal contributors to
urinary saturation of calcium oxalate.
• Common calcium stones may originate from subepithelial
plaques composed of calcium apatite that serve as an anchor
on which calcium oxalate stones can grow.
• The noncrystalline component of stones is matrix, which is
composed of a combination of mucoproteins, proteins,
carbohydrates, and urinary inhibitors.
35
Dept of Urology, GRH and KMC, Chennai.