nephrology grand rounds acid-base conference

Nephrology Grand Rounds Acid-Base Conference

Vasishta Tatapudi, MD 21st May, 2013

Contents

• Case vignette – Metabolic acidosis. • Role intercalated cells in acid secretion. • Adaptive changes in the distal nephron. • Role of Hensin/DMBT1. • Conditional knock-out of hensin. • Notch signaling – origin on Principal cells and Intercalated cells. • Terminal differentiation.

Case Vignette

• 44 year old female patient with complaints of generalized fatigue. No past medical or surgical history. Takes no medications. No smoking, alcohol or drug use. Family history of rheumatoid arthritis in mother.

• Vital signs and physical exam unremarkable.

Na 140

K 3.0

Cl 115

HCO3 15

BUN 22

Cr 1.4

Glu 92

Ca 9.0

PO4 3.0

AG 10

Uric acid

5

pH 7.32

pCO2 30

HCO3 15

Lact. 0.3

ABG

BMP

Color Yellow

SG 1.010

pH 6.0

Glucose Negative

Bilirubin Negative

Ketones Negative

Blood Negative

Protein Negative

Leuk. Est. Negative

Nitrite Negative

RBC 0-2

WBC 0-2

Casts Negative

Crystals Negative

Urinalysis U. Na 35

U. K 40

U. Cl 18

24h U.Ca 150mg 24h U uric acid 300mg

F e PO4 3

U/ Pr/Cr 0.15g/g

U. Electrolytes & Prt.

Hyperchloremic/non-anion gap metabolic acidosis with adequate respiratory compensation, high urine anion gap, urine pH and hypokalemia.

Comp. Clin. Neph. 2010, Biff F. Palmer, Robert J. Alpern

Acid secretion in the collecting duct

Dietary Acid and Alkali

Clin. Physiol. Of Acid Base and Electrolyte disorders, Rose. B.D, Post. T.W.

Homeostatic Response

• Chemical buffering by extracellular and intracellular buffers: HCO3

-. Intracellular proteins and phosphates. Bone carbonates*.

• Changes in alveolar ventilation to control pCO2.

• Alterations in renal H+ excretion to regulate plasma HCO3

-.

Clin. Physiol. Of Acid Base and Electrolyte disorders, Rose. B.D, Post. T.W. *Irrelevance of bone buffering to acid-base homeostasis in chronic metabolic acidosis.Oh MS. Nephron. 1991;59(1):7-10.

Renal H+ Secretion

• Acid production by oxidation of dietary foodstuff would be lethal were it not for the kidney, which restores the blood’s buffering power by producing new HCO3.

• The proximal tubule reabsorbs the filtered HCO3, and the distal nephron produces new HCO3.

• Both processes are needed to titrate the generated metabolic acid.

Qais Al-Awqati, Annu. Rev. Physiol. 2011. 73:401–12

Comp. Clin. Neph. 2010, Pages 149-154, Biff F. Palmer, Robert J. Alpern

Collecting Duct

• The CCD of the kidney has two cell types: Principal cells, which transport Na+, water, and K+. Intercalated cells, which mediate acid-base transport.

• Two distinct subtypes of intercalated cells have been identified in

the CCD: The α intercalated cell secretes H+ by an apical H+ ATPase and by a

basolateral Cl−:HCO3(AE1). The β type secretes HCO3 by an apical Cl:HCO3 exchanger

(Pendrin) and a basolateral H+ATPase.

Comp. Clin. Neph. 2010, Pages 149-154, Biff F. Palmer, Robert J. Alpern

Response to acidosis

• α-IC cells: Change in cell pH induced by CO2 or a weak acid results in exocytosis of vesicles whose membrane is enriched in H+ATPases, thereby stimulating the rate of transepithelial H+secretion.

• This is mediated by increased intracellular calcium.

• Feeding animals an acid diet results in an increase in the number of α-IC cells and a decrease in the number of β-IC cells, while the total number of IC cells remained constant.

• Are β IC cells the progenitors of the α IC cells ? – Plasticity of functional epithelia.

van Adelsberg J,.J Cell Biol 102: 1638–1645, 1986. Schwartz GJ, Barasch J, Al-Awqati Q. Nature 318: 368– 371, 1985.

Hensin/DMBT1

• When immortalized β-IC were induced to convert to the α-IC phenotype they deposit in their ECM a new extracellular matrix protein (ECM) that is capable by itself of inducing the change in phenotype.

• This ECM protein was purified, cloned and called ‘Hensin’.

• ‘Henshin’ – Japanese for transform.

• Called DMBT1 by the Mouse Genome Project-Deleted in malignant brain tumor (medulloblastoma).

Physiology 26:266-272, 2011. ;Qais Al-Awqati and Xaio

α vs β

Physiology 26:266-272, 2011. ;Qais Al-Awqati and Xaio

Role of Hensin

• Hensin/DMBT1 is expressed in most epithelia, often in alternately spliced forms.

• Hensin might be involved in the differentiation of other epithelia as well.

• Global deletion of hensin resulted in early embryonic lethality at the time of appearance of the first columnar epithelium, visceral endoderm.

Gao X, Eladari D, Al-Awqati Q. Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21872-7

Qais Al-Awqati, Annu. Rev. Physiol. 2011. 73:401–12

• Conditional deletion of hensin from intercalated cells resulted in an almost complete absence of typical α-intercalated cells.

• Deletion of β1 integrin in the intercalated cells also prevented the conversion of β- to α-intercalated cells.

• Presence of only HCO3-secreting β-cells, as expected produced complete distal renal tubular acidosis (dRTA).

Cre-Lox Recombination

• Site-specific DNA recombination. • Discovered in P1 bacteriophage. • Bacteriophage uses Cre-lox recombination to circularize and

facilitate replication of its genomic DNA. • Cre recombinase: an enzyme that catalyzes recombination between

two loxP sites. • LoxP sites: specific 34-base pair (bp) sequences.

Sauer, B.; Henderson, N. (1988). "Proc. Natl. Acad. Sci. USA 85: 5166–5170.

Application of Cre-Lox Recombination

• Developed as a technology for genome manipulation applied in mammalian cell cultures, mice, and other organisms.

• Widely used to carry out deletions, insertions, inversions and translocations at specific sites and produce general knockouts, conditional knockouts.

Cre-Lox System

Conditional deletion of hensin from intercalated cells

• To delete hensin from the intercalated cells, loxP hensin mouse line was crossed with one expressing Cre under the control of the VB1 promoter (B1 subunit of V ATPase), resulting in deletion of hensin in collecting duct cells.


VB1 Cre Line


HOXB7 Cre Line


β1 Integrin-VB1 Cre Line


Hensin expression - Western blot


• VB1Cre: 69% of the wild-type levels. • HoxB7: 34% of the wild-type levels. • β1 integrin Cre: no change.

Hensin Knockout Causes Distal Renal Tubular Acidosis


Blood gas analysis in retro-orbital mixed venous blood taken from awake and spontaneously breathing mice.

Metabolic acidosis


Metabolic Acidosis


Respiratory compensation


• Studied a family exhibiting dominant inheritance and defined a

mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs.

• Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function.

• AE1-M909T localized to both the apical and basolateral membranes in MDCK cells.

• AE1-M909T acquired class 1 PDZ ligand activity.

Andrew C. Fry,Howard Trachtman et al J Am Soc Nephrol. 2012 Jul;23(7):1238-49.

Andrew C. Fry,Howard Trachtman et al J Am Soc Nephrol. 2012 Jul;23(7):1238-49.

AE1 mutation vs Hensin knockout

• In patients with AE1 mutation, acidosis will result in conversion of most cortical β-intercalated cells into α-intercalated cells, albeit ones that are incapable of pumping protons.

• In hensin knockout mice almost all intercalated cells have apical pendrin.

• Continued HCO3 secretion explains the appearance of significant spontaneous metabolic acidosis.

• Whether human syndromes of hensin deficiency exist will require direct tests in families with distal RTA.


Quantification of intercalated cells

• Aqp 2 - used as a marker for principal cells. • B1 subunit of the V-ATPase for intercalated cells. • More than 5,000 cells counted in the cortical collecting duct and

medulla in each line of mice. • Wild-type mice: intercalated cells represented 39% of collecting

duct cells in the cortex and 38% in the medulla. • In mice deleted for hensin – almost identical findings. • Hence, deletion of hensin did not reduce the number of intercalated

cells in the kidney.


Quantitative analysis of the number of α and β cells


Results

• Essentially all of the intercalated cells in the cortex of the mutant mice are canonical β-type cells, with apical pendrin and basolateral V-ATPase.

• In the medulla, however, a previously undescribed cell type has been uncovered, which resembles the cortical β-intercalated cell in ultrastructure, but does not express pendrin.

• β1 integrin deletion from the intercalated cell caused a phenotype that was identical to the deletion of hensin itself, supporting its critical role in hensin function.


Medullary IC cells – wild type mice


Medullary IC cells – VB1Cre line


Medullary IC cells – HOXB7 line


Interpretation

• Hensin is required for the generation of the α-intercalated cell phenotype in cortical collecting ducts.

• Supports the hypothesis that during acidosis, β-intercalated cells can convert to α-intercalated cells in a hensin-dependent manner.

• There is a path of differentiation in which the β-cell is less differentiated than the α-type.

• Medullary α-intercalated cells appear to be derived from a different type of cell, which, unlike ß-intercalated cells, does not express pendrin. They are progenitors of the medullary α-intercalated cell.


Physiology 26:266-272, 2011. ; Qais Al-Awqati and Xaio

Epithelia

Embryonic stem cells

Proto-epithelial cells

Salt and pepper distribution

Yan Liu et al, Development 134, 1111-1122 (2007)

Terminal Differentiation

• The concept of terminal differentiation implies that once a cell reaches that phenotype its fate is fixed as it becomes “postmitotic.”

• During embryonic development a terminally differentiated cell could readily convert to a different phenotype.

• Epithelial-to-mesenchymal and mesenchymal-toepithelial transitions.

• α- and β-intercalated cells are in different states of differentiation with their acidosis-dependent conversion being a shift from one differentiated state to another.


Thank you.

nephrology grand rounds acid-base conference

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