familial hypomagnesaemia with hypercalciuria and nephrocalcinosis ( fhhnc , omim # 248250 ) is a rare autosomal - recessive renal tubular disorder caused by mutations in the cldn16 or cldn19 gene , which , respectively , encode the tight junction - associated proteins , claudin-16 and -19 [ 13 ] .
these transmembrane proteins regulate the paracellular diffusion of selective cations along the thick ascending limb ( tal ) of henle 's loop ( figure  1 ) [ 4 , 5 ] .
fhhnc - associated tubulopathy is thus characterized by massive urinary losses of mg and ca , with subsequent hypomagnesaemia , bilateral nephrocalcinosis and rapid evolution to end - stage renal disease ( esrd ) .
a genotype / phenotype correlation regarding the severity of the disease has been proposed upon the impact of cldn16 mutations on protein function .
we report on an 18-year - old patient presenting with glomerular proteinuria associated with the typical fhhnc triad .
further investigations demonstrated severe tubular atrophy and interstitial fibrosis , as well as secondary glomerulosclerosis .
( a ) localization of members of the claudin family in a mammalian kidney . claudin-16 and -19 are specifically situated between epithelial cells lining the ascending limb of henle 's loop . adapted from angelow et al . .
( b ) schematic view of an epithelial cell lining the tal of henle 's loop . claudin-16 and -19 are positioned at the tight junctions ( tj ) between adjacent cells , where they selectively regulate the paracellular diffusion of ca and mg .
ion transporters implicated in generating and/or modulating the positive trans epithelial electrical gradient , including na / k - atpase , clc - kb / barttin , ca - sensing receptor , romk ( renal outer medullary k channel ) and nkcc2 ( type 2 na / k / cl cotransporter ) are depicted .
different k channels from four gene families have been identified within the tal basolateral membrane of various mammals . adapted from naderi and reilly ,
( a ) localization of members of the claudin family in a mammalian kidney . claudin-16 and -19 are specifically situated between epithelial cells lining the ascending limb of henle 's loop . adapted from angelow et al . .
( b ) schematic view of an epithelial cell lining the tal of henle 's loop . claudin-16 and -19 are positioned at the tight junctions ( tj ) between adjacent cells , where they selectively regulate the paracellular diffusion of ca and mg .
ion transporters implicated in generating and/or modulating the positive trans epithelial electrical gradient , including na / k - atpase , clc - kb / barttin , ca - sensing receptor , romk ( renal outer medullary k channel ) and nkcc2 ( type 2 na / k / cl cotransporter ) are depicted .
different k channels from four gene families have been identified within the tal basolateral membrane of various mammals . adapted from naderi and reilly , hamilton and devor .
the patient was referred for renal investigations after a fortuitous finding of increased serum creatinine levels ( 1.7 mg / dl ) , i.e. a glomerular filtration rate ( gfr ) of 52 ml / min per 1.73 m according to the modification of diet in renal disease ( mdrd ) equation .
his medical history included severe dehydration at birth , as well as persistent polyuria / polydipsia syndrome with nycturia since infanthood . no urinary tract infections or muscular cramps
one year prior to consultation , he developed acute kidney injury in a context of rhabdomyolysis and dehydration in a motorcycle crash . at that time
note the co - occurrence of hypomagnesaemia , hypermagnesuria and hypercalciuria , with heavy selective proteinuria .
pak 's oral ca load test led to a significant decline in parathormone levels , thereby ruling out primary hyperparathyroidism and supporting a renal origin for hypercalciuria .
abdominal ultrasound disclosed symmetric 10-cm kidneys , with nephrocalcinosis and multiple millimetric lithiasis as confirmed by computed tomography .
a kidney biopsy showed both atrophy and hypertrophy of renal tubules and interstitial fibrosis in association with focal and segmental sclerosis of glomeruli ( figure  2a and b ) .
the expression of claudin-16 in tal was lost , whereas the distribution of uromodulin did not appear to be significantly affected ( figure  2d  e ) . 

table  1.analysis of serum and 24-h urine samples at admissionserumsi unitsconventional unitsnormal valuessi unitsconventional unitscreatinine141.41.650110 mol / l0.61.2 mg / dlgfr54>60 ml / min per 1.73 muric acid517.58.7120420 mol / l27 mg / dlmagnesium0.61.40.751 mmol / l1.82.4 mg / dlcalcium2.39.22.12.6 mmol / l8.410.6 mg / dlsodium142142135145 mmol / l135145 meq / lpotassium3.23.23.14.9 mmol / l3.14.9 meq / lbicarbonate33332333 mmol / l2333 meq / lglucose4.4803.35.5 mmol / l60100 mg / dlintact pth2982981258 ng / l1258 pg / ml25-oh vitamin d89.836>80 nmol
/ l>32 ng / mlurinemagnesium8.51734 mmol / day68.5 meq / daycalcium / creatinine0.290.040.15 g / mgproteins18541.854<150 mg / day<0.150 g / daygfr , glomerular filtration rate according to mdrd equation ; pth , parathyroid hormone . 
 fig . 
2.kidney histology of a patient with c.340c > t ( nonsense ) and c.427 + 5g > a ( splice - site ) mutations of cldn16 gene .
eosin colouration ( a and b ) shows diffuse tubular atrophy and interstitial fibrosis , as well as perihilar segmental sclerosis of some glomeruli ( b , arrowhead ) .
immunostaining anti - claudin-16 ( d ) and anti - uromodulin ( e ) on serial sections ( cfr asterisk , * ) does not detect claudin-16 ( d ) in uromodulin - positive tubules lining the tal of henle 's loop ( e ) .
analysis of serum and 24-h urine samples at admission gfr , glomerular filtration rate according to mdrd equation ; pth , parathyroid hormone .
kidney histology of a patient with c.340c > t ( nonsense ) and c.427 + 5g > a ( splice - site ) mutations of cldn16 gene .
eosin colouration ( a and b ) shows diffuse tubular atrophy and interstitial fibrosis , as well as perihilar segmental sclerosis of some glomeruli ( b , arrowhead ) .
immunostaining anti - claudin-16 ( d ) and anti - uromodulin ( e ) on serial sections ( cfr asterisk , * ) does not detect claudin-16 ( d ) in uromodulin - positive tubules lining the tal of henle 's loop ( e ) .
medical treatment included thiazides and angiotensin - converting enzyme ( ace ) inhibitors , as well as oral supplementation of mg and active vitamin d. still , despite the complete resolution of proteinuria under treatment , the patient reached esrd at the age of 23 .
the slope of gfr decline was calculated to be 9 ml / min per 1.73 m / year .
the 1-year follow - up showed an uneventful evolution , with a stable gfr 50 ml / min per 1.73 m. the pre - transplant work - up prompted genetic testing , which allowed the identification of two novel mutations in the cldn16 gene : c.340c > t and c.427 + 5g > a .
his father , who presented with recurrent nephrolithiasis , could not be tested because of sudden death at the age of 52 .
hypercalciuria and nephrocalcinosis most probably have a negative impact . still , all inherited tubulopathies characterized by nephrocalcinosis do not uniformly lead to esrd , which suggests that the functional loss of claudin-16 may per se impair kidney architecture and function .
previous reports listed 40 missense / nonsense mutations , four splice - site mutations and 5 indels of the cldn16 gene .
genotype / phenotype correlation studies postulated an earlier onset of the disease and a more rapid decline of gfr in patients harbouring cldn16 mutations inducing a complete loss of function of the protein in comparison with patients with mutations associated with a partial dysfunction .
our patient presented at the age of 18 with stage 3a ckd and significant selective proteinuria .
symptoms obviously developed in early infanthood , and the slope of gfr decline was > 5 ml / min per 1.73 m / year , which suggests a complete loss of function of claudin-16 .
kidney histology further supports an advanced and chronic tubulopathy , with tubular atrophy and ca deposits .
in addition , sclerotic lesions were found in some glomeruli , with a particularly perihilar distribution ( figure  2 ) .
such a location is suggestive of secondary glomerulosclerosis , and accounts for the glomerular proteinuria highly responsive to ace inhibitor therapy .
the complete loss of claudin-16 , as demonstrated by immunostaining , may thus cause progressive nephron loss with tubular atrophy and interstitial fibrosis and secondary glomerular damage .
the c.340c > t ( p.r114 * ) mutation is a nonsense mutation located in exon 2 , which most likely leads to a premature stop codon and a complete loss of claudin-16 function .
this mutation is located in the first extracellular loop of the protein , similar to the large majority of previously reported mutations . the c.427 + 5g > a mutation affects an intronic nucleotide located close to the donor splicing site of cldn16 exon 2 .
still , given the severity of the disease and the absence of claudin-16 expression in the patient 's tal tubules , we speculate that this mutation also results in a complete loss of claudin-16 function . of note ,
a mutation with residual expression and function of the protein would predict a milder clinical course [ 9 , 10 ] . in summary
in addition to the identification of novel mutations of cldn16 gene , the present case illustrates a late diagnosis of fhhnc in early adulthood and emphasizes the progressive nephron loss associated with this tubulopathy , as well as the glomerular consequences .
conservative management of ckd and oral supplementation of mg remain the cornerstones of fhhnc treatment .
the efficiency of thiazides , which decrease urinary ca excretion , on gfr decline remains controversial .
 familial hypomagnesaemia with hypercalciuria and nephrocalcinosis is an autosomal - recessive disease caused by mutations in the cldn16 or cldn19 genes , which encode tight junction - associated proteins , claudin-16 and -19 . 
 the resultant tubulopathy leads to urinary loss of mg2 + and ca2 + , with subsequent nephrocalcinosis and end - stage renal disease ( esrd ) . 
 an 18-year - old boy presented with chronic kidney disease and proteinuria , as well as hypomagnesaemia , hypercalciuria and nephrocalcinosis . 
 a kidney biopsy revealed tubular atrophy , interstitial fibrosis and segmental sclerosis of some glomeruli . 
 two novel mutations in the cldn16 gene were identified : c.340c > t ( nonsense ) and c.427 + 5g > a ( splice site ) . 
 the patient reached esrd at 23 and benefited from kidney transplantation . 
Background
Case report
Discussion
Conflict of interest statement