Apparent Mineralocorticoid Excess: Report of Six New Cases and Extensive Personal Experience Department of nephrology R1 Choi In-Ah Gilles Morinequ, Veronique Sulmont, Remi Salomon, Beatrice Fiquet-Kempf, Xavier Jeunemaitre, Jerome Nicod, and Paolo Ferrari

1 st case report of mineralocorticoid excess - More than 30 year ago, Werder et al. - A case of 3-yr-old girl - Short stature, polydipsia, polyuria without obvious external abnormalities (including genitalia) - Mineralocortcoid hypertension with hypokalemia and metabolic alkalosis - Suppressed plasma renin and aldosterone - Gas-chromatographic analysis of urinary steroid profile :Exclude hypertensive forms of congenital adrenal hyperplasia but showed an abnormal steroid profile  failed to realize the true nature at that time ObjectivesMaterials and MethodsResultsBackgroundDiscussion

New et al. and Ulick et al Apparent mineralocorticoid excess (AME) : Inherited deficiency in the enzyme 11 β -hydroxysteroid dehydrogenase type 2 ( 11βHSD2 )  Cause a disorder in the peripheral metaboilism of cortisol  Hypokalemic hypertension and suppressed circulating renin and aldosterone : Low birth weight and failure to thrive, AR inherited ObjectivesMaterials and MethodsResultsBackground Discussion

Pathogenesis of AME 11βHSD2 enzyme: cortisol  inactive cortisone Mutations in the HSD11B2 gene  Subsequent 11βHSD2 deficiency  Excess cortisol saturate MR  Hypokalemia, sodium retention, volume expansion with suppression of plasma renin and aldosterone secretion ObjectivesMaterials and MethodsResultsBackground Discussion

Pathogenesis of AME Hallmark of the disease - State of mineralocorticoid excess - In the absence of aldosterone - In the presence abnormal urinary steroid profile with increased ratio of cortisol to cortisone Gas chromatographic analysis of urinary steroid profile: - ↑urine free cortisol to cortisone - ↑THF+5αTHF/THE ObjectivesMaterials and MethodsResultsBackground Discussion

Highlight the importance of 11betaHSD2 in understanding of a new biologic principle in hormone action : target tissue specificity of mineralocorticoid action is enzyme mediated, not receptor.ObjectivesMaterials and MethodsResults Discussion Background

Case Histories and Clinical and Biochemical Analysis 1) Birth - Boy, normal birth weight/height, unrelated parents, France 2) Onset symptom - At age 13 - Severe hypertension, morning headaches, limb paresthesia, cramps, palpitations, tetanic convulsions 3) Family Hx - His father and paternal grandfather : hypertension 4) Other finding - Relatively short stature - Kidney: normal, Echo: LVH Objectives Materials and Methods Results Discussion Background Case 1 (M.H)

Case Histories and Clinical and Biochemical Analysis 5) Biochemical investigations - Low to normal serum potassium without metabolic alkalosis - Low plasma renin, low aldosterone - Normal: deoxycorticosterone (DOC), corticosterone (B), 18- hydroxydeoxycorticosterone (18OHDOC), 18- hydroxycorticosterone (18OHB), 18-hydroxycortisol  Rule out other forms of mineralocorticoid excess - Abnormally high urinary ratio of (THF + 5alphaTHF)/TGE 6) Treatment - Dexamethasone (1.5mg/d) and spironolactone (50mg/d)  Well controlled BP, 2yr F/U Objectives Materials and Methods Results Discussion Background Case 1 (con’t)

Case Histories and Clinical and Biochemical Analysis 1) Birth - Boy, normal birth weight, consanguineous mother and father, Moroccan 2) Onset symptom - At age 2 - Hypertension 3) Family Hx - His father and mother: normal BP 4) Other finding - Growth: normal - Kidney: extensive nephrocalcinosis, Echo: mild LVH Objectives Materials and Methods Results Discussion Background Case 2 (H.E.M.)

Case Histories and Clinical and Biochemical Analysis 5) Biochemical investigations - Hypokalemia and metabolic alkalosis - High BP excluded Bartter syndrome - Low plasma renin, low aldosterone - Normal: DOC, B, 18OHDOC, 18OHB, 18-hydroxycortisol  Rule out other forms of mineralocorticoid excess - Abnormally high urinary ratio of (THF + 5 α THF)/TGE 6) Treatment - Salt restriction and hydrochlorothiazide - 3yr F/U, normal BP control Objectives Materials and Methods Results Discussion Background Case 2 (con’t)

Case Histories and Clinical and Biochemical Analysis 1) Birth - Boy, normal birth weight, Algerian 2) Onset symptom - At age 11 - Severe dehydration, polyuria, polydipsia, hypercalciuria 3) Family Hx: ? 4) Other finding - Growth: normal - Kidney: nephrocalcinosis, Echo: moderate LVH Objectives Materials and Methods Results Discussion Background Case 3 (A.L.)

Case Histories and Clinical and Biochemical Analysis 5) Biochemical investigations - Hypokalemia - Low plasma renin, low aldosterone - Normal: DOC, B, 18OHDOC, 18OHB, 18-hydroxycortisol  Rule out other forms of mineralocorticoid excess - Abnormally high urinary ratio of (THF + 5αTHF)/THE 6) Treatment - Salt restriction - At 5 yr of age, normal BP Objectives Materials and Methods Results Discussion Background Case 3 (con’t)

Case Histories and Clinical and Biochemical Analysis 1) Birth - Girl, 36wk gestation, low birth weight (1400g), French Guyana 2) Onset symptom - At 4months - Arterial hypertension 3) Family Hx: ? 4) Other finding - Kidney: normal, Echo: LVH Objectives Materials and Methods Results Discussion Background Case 4 (K.I.)

Case Histories and Clinical and Biochemical Analysis 5) Biochemical investigations - No hypokalemia, metabolic alkalosis - Low plasma renin, low aldosterone - Abnormally high urinary ratio of (THF + 5αTHF)/THE with very low cortisone compared with cortisol 6) Treatment - Fail to control BP by nifedipine  hydrochlorothiazide - At 5 yr of age, normal BP with dietary salt restriction and hydrochlorothiazide Objectives Materials and Methods Results Discussion Background Case 4

Case Histories and Clinical and Biochemical Analysis 1) Birth - 2 brothers, boys, normal birth weight, consanguineous parents, Portuguese 2) Onset symptom (1) One boy (A.G) - At age 2 - High BP, hypokalemia (2) The other boy (J.C.G) - At age 3 - Hypokalemia and delayed growth, but normal BP - Hypertension at 6 yr old age Objectives Materials and Methods Results Discussion Background Case 5

Case Histories and Clinical and Biochemical Analysis 3) Radiologic exam and biochemical investigations - Low plasma renin, low aldosterone - Elevated ratios of cortisol to cortisone in the urine - No metabolic alkalosis (1) A.G - Discrete renal cortical atrophy and LVH (2) J.C.G - Mild proteinuria and hypercalciuria 4) Treatment A.G.: dexamethasone or spironolactone J.C.G.: necessary to associated calcium channel blockers, ACE inhibitors, dihydralazine, salt restriction Objectives Materials and Methods Results Discussion Background Case 5 (con’t)

Case Histories and Clinical and Biochemical Analysis 1) Birth - 37 yr old woman with a history of low birth weight (2150g, 43cm), France 2) Onset symptom - At age 4 - Hypertension, hypokalemia, metabolic alkalosis - Low plsma renin and aldosterone levels 3) Family Hx - Her mother: 3 misscariages, hypertension during 1 st of 2 pregnancy - Her grandmother: hypertension - Father: hypertension, die due to CVA Objectives Materials and Methods Results Discussion Background Case 6 (I.V.)

Case Histories and Clinical and Biochemical Analysis 4) Other finding - Kidney: nephrocalcinosis 5) Biochemical investigations - High cortisol/cortisone ration in serum - Abnormally high urinary ratio of (THF + 5 α THF)/TGE 6) Treatment - Hypertension responded first to spironolactone - Subsequent dexamethasone and beta blocker - After renal failure, hemodialysis was started  without antihypertensive drugs, only fluid control Objectives Materials and Methods Results Discussion Background Case 6

Case Histories and Clinical and Biochemical Analysis Objectives Materials and Methods Results Discussion Background

Urinary Steroid profile - By gas chromatography-mass spectrometry - All steroid metabolites to be measured Objectives Materials and Methods Results Discussion Background Analysis of Genomic DNA - Genomic DNA was extracted from peripheral blood leukocytes - primer  PCR  gel running  variants products were verified by sequencing

Objectives Materials and Methods Results Discussion Background Analysis of Genomic DNA - primer  1 st PCR  gel running  purify PCR products  2 nd PCR  gel running  variant products were verified by sequencing - Not identifiable by direct sequencing - Amplified PCR fregments  subcloned into a TOPO TA cloning vector  transformed in DH5α competent cells - pUC19 plasmid: control

Objectives Materials and Methods Results Discussion Background Assay for 11 β HSD2 Activity - cDNA construct was subcloned in the expression plasmid pcDNA3  HEK-293 cells, devoid of endogenous 11βHSD2 activity were cultured with tranfected plasmid  Harvested after 72hrs  Oxidative activities of 11βHSD2 constructs were measured using cell lysates - Steroids were analyzed by thin-layer chromatography - Experiments were performed in triplicate

Objectives Materials and Methods Results Discussion Background

ObjectivesSubjects and methodsResults Discussion Background Clinical and Biochemical Data * Affected children displayed varying degrees of signs and features of AME (table 1) - Hypokalemia and metabolic alkalosis - Presence of LVH and nephrocalcinosis - One patient received a diagnosis of AME at 37 yr of age, later developed hypertensive ESRD

ObjectivesSubjects and methodsResults Discussion Background Genotypic studies HSD11B2 Identified in Each AME Kindred

ObjectivesSubjects and methodsResults Discussion Background In Vitro Enzymatic Activity - Completely abolished enzymatic activity : Arg213Cys, Ala328Val, Glu342Fs mutation - Marked reduced : Asp144Val, Phe367del (20% of the WT) Phe135Ser (30% of the WT)

ObjectivesSubjects and methodsResults Discussion Background Genotypic-Phenotype Correlation

ObjectivesSubjects and methodsResults Discussion Background

Objectives Subjects and methodsDiscussionBackgroundResults HSD11B2 Gene Mutations - Autosomal recessive trait : homozygous carriers with consanguineous parents - Mutations in exon 3,4,5 of the HSD11B2 gene with the exception of the Arg74Gly and Pro75,∆1nt in exon 1 and Leu114, ∆6nt mutant in exon 2 - Arginine is the residue that most frequently mutated

Objectives Subjects and methodsDiscussionBackgroundResults Genotypic-Phenotype Correlation - Variable phenotypic expression of HSD11B2 gene mutations : vary from classic AME syndrome to low renin hypertension without hypokalemia - Relation bewteen hypertension or LVH : less evident : difference in the age of reported case, drug treatment used before diagnosis, duration of hypertension Hypokalemia, metabolic alkalosis, hypercalciuria: complete lack of 11βHSD2 activity Absence of nephrocalcinosis and metabolic alkalosis: some residual in vivo activity

Objectives Subjects and methodsDiscussionBackgroundResults 11 β HSD2 and Essential Hypertention - Mutation or inhibition of 11βHSD2→mineralocorticoid excess - What subtle abnormalities in MR/11βHSD2 contribute? : Heterozygote state might predispose to “essential” low renin HTN : Subset of patients among the essential hypertntion may have subtle form of AME - Impaired 11βHSD2 activity is associated with an increased susceptibility of BP to salt load →Low renin hypertension is a salt sensitive form of high BP - Case of ESRD :Low renin HTN → leading cause of ESRD in black patient → impaired 11βHSD2 activity might be more prevalent

In Conclusion…. Local metabolism of the glucocorticoid hormone into inactive derivatives by the enzyme 11βHSD2 is one of the mechanisms that intervene to allow specific aldosterone regulatory effect