A novel frameshift mutation (2436insT) produces an immediate stop codon in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein. METHODS: We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family.

[Autosomal dominant polycystic kidney disease: detection of a new mutation in the PKD1 gene]. / Poliquistosis renal autosómica dominante. Detección de una nueva mutación en el gen PKD1.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder characterized by genetic heterogeneity. Up to three loci are involved in this disease, PKD1 on chromosome 16p13.3, PKD2 on 4q21, and a third locus of unknown location. Since the identification of the PKD1 gene, the interest was centered in the characterization of the mutations responsible for the disease. Most mutations found were diverse and situated throughout the gene with no phenotypic correlation. Here we describe a new mutation in exon 44 from PKD1 gene in a family previously characterized as PKD1 by linkage analysis. The mutation is a single base substitution from a C to a T at position 12220 originating a stop codon at the mutation site. This would lead to premature termination and the formation of a truncated protein lacking part of the carboxi-terminus.

Isolated polycystic liver disease not linked to polycystic kidney disease 1 and 2.

Autosomal dominant polycystic liver disease occurs commonly in association with autosomal dominant polycystic kidney disease, types 1 and 2. It may also exist as a separate entity, genetically distinct from autosomal dominant polycystic kidney disease types 1 and 2, as has been recently established to exist in a Belgian family. We report here a large Argentinian family of Spanish-Belgian ancestry with autosomal dominant polycystic liver disease, where proximal and distal markers for both polycystic kidney disease 1 and 2 failed to demonstrate genetic linkage. The data support the notion that polycystic liver disease and autosomal dominant polycystic kidney disease may have separate chromosomal loci.

Poliquistosis renal autosómica dominante. Detección de una nueva mutación en el gen PKD1. / [Autosomal dominant polycystic kidney disease: detection of a new mutation in the PKD1 gene]

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder characterized by genetic heterogeneity. Up to three loci are involved in this disease, PKD1 on chromosome 16p13.3, PKD2 on 4q21, and a third locus of unknown location. Since the identification of the PKD1 gene, the interest was centered in the characterization of the mutations responsible for the disease. Most mutations found were diverse and situated throughout the gene with no phenotypic correlation. Here we describe a new mutation in exon 44 from PKD1 gene in a family previously characterized as PKD1 by linkage analysis. The mutation is a single base substitution from a C to a T at position 12220 originating a stop codon at the mutation site. This would lead to premature termination and the formation of a truncated protein lacking part of the carboxi-terminus.

Regulation of cell cyclic AMP in medullary thick ascending limb of Henle in a rat model of chronic renal failure.

Chronic renal failure (CRF) is accompanied by adaptive changes in electrolyte reabsorption in the thick ascending limb of Henle of surviving nephrons. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in micro-dissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF an increase of basal cAMP from 25.6 +/- 10.0 in controls to 65.8 +/- 11.3 fmol mm-1 tubule in CRF (P < 0.05). Vasopressin and calcitonin stimulated mTAL adenylate-cyclase in a dose-dependent manner in controls but failed to stimulate in CRF. Likewise, maximal stimulation with 10(-3) M 3-isobutyl-1-methylxanthine (IBMX) plus 10(-5) M forskolin increased cAMP in controls to 63.0 +/- 16.0 but not in CRF, where maximal stimulated values remained at 63.1 +/- 18.8 fmol mm-1 tubule (P NS). Alpha2-adrenoreceptor activation with clonidine at concentrations ranging from 10(-8) to 10(-6) M diminished cAMP production by 37% in CRF (P < 0.05), whereas no differences were found in controls. Thus, the basal intracellular cAMP is increased in rat mTAL in CRF. The finding that neither forskolin nor vasopressin were able to further augment intracellular cAMP would suggest that stimulatory pathways of the adenylate-cyclase system are activated in the basal state. However, mTAL cells in CRF seem to retain the response of normal epithelium to inhibitory pathways such as the one mediated by alpha2-adrenoreceptors.

Pre-transplant recipient-donor interaction: a prognostic indicator in living related kidney transplantation?

Based on the hypothesis that not only genetically determined immune characteristics, but also psychosocial and especially interpersonal factors may influence the outcome in living related kidney transplantation, we investigated the type of relationship between recipient and donor, and its association with graft prognosis. The study group consisted of 154 kidney transplant candidates and their selected donors. Donor and recipient were assessed prospectively prior to transplantation using an interactional task (Usandivaras Marbles Test) and assigned to one of four groups, according to their pattern of contact. Kidney survival was calculated for each test group, and results compared by life table methods and logistic regression. The group that showed progression from initial contact avoidance or enmeshment to contact with boundaries had a significantly better outcome than the other groups (no change or loss of contact with boundaries). Differences could not be related to other variables such as age, sex, sex difference, relationship, HLA-matching, and treatment.

Genetic heterogeneity of autosomal dominant polycystic kidney disease in Argentina.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder with genetic heterogeneity. Up to three loci are involved in this disease, PKD1 on chromosome 16p13.3, PKD2 on 4q21, and a third locus of unknown location. Here we report the existence of locus heterogeneity for this disease in the Argentinian population by performing linkage analysis on 12 families of Caucasian origin. Eleven families showed linkage to PKD 1 and one family showed linkage to PKD2. Two recombinants in the latter family placed the locus PKD2 proximal to D4S1563, in agreement with data recently published on the cloning of this gene. Analysis of clinical data suggests a milder ADPKD phenotype for the PKD2 family.

Cellular adaptation of the rat medullary thick ascending limb to chronic renal failure.

Chronic renal failure (CRF) is accompanied by adaptive changes in renal and extrarrenal epithelial ionic transport. Fluid reabsorption in the thick ascending limb of Henle is increased and the capacity to lower the urine osmolality in water diuresis is preserved. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in microdissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF nephrons an increase of basal cAMP from 6.0 +/- 1.5 in controls to 47.0 +/- 10.3 fmol. mm-1 tubule in CRF (P < 0.05). Maximally stimulated cAMP levels (10(-3) M IBMX plus 10(-5) M Forskolin) were different from basal levels in controls (6.0 +/- 1.5 vs 63.1 +/- 18.8, P < 0.05) but not from basal levels in CRF (47.0 +/- 10.3 vs 63.0 +/- 16.0, P = N.S.). Preincubation with the adenylate cyclase inhibitor 2'5'-dideoxyadenosine (DDA) 10(-4) M produced no changes in cAMP in controls (93.7 +/- 10.3% of DDA untreated samples) whereas it decreased to 76.2 +/- 8.8% (24% inhibition) in CRF (P < 0.05). No differences between controls and CRF groups were found in basal and stimulated cAMP in red blood cells and distal colon. The data would suggest that the cAMP pathway is an intracellular signal for mTAL adaptation in epithelial transport and that the adenylate-cyclase system is specifically activated in CRF.

Cellular adaptation of the rat medullary thick ascending limb to chronic renal failure.

Chronic renal failure (CRF) is accompanied by adaptive changes in renal and extrarrenal epithelial ionic transport. Fluid reabsorption in the thick ascending limb of Henle is increased and the capacity to lower the urine osmolality in water diuresis is preserved. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in microdissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF nephrons an increase of basal cAMP from 6.0 +/- 1.5 in controls to 47.0 +/- 10.3 fmol. mm-1 tubule in CRF (P < 0.05). Maximally stimulated cAMP levels (10(-3) M IBMX plus 10(-5) M Forskolin) were different from basal levels in controls (6.0 +/- 1.5 vs 63.1 +/- 18.8, P < 0.05) but not from basal levels in CRF (47.0 +/- 10.3 vs 63.0 +/- 16.0, P = N.S.). Preincubation with the adenylate cyclase inhibitor 25-dideoxyadenosine (DDA) 10(-4) M produced no changes in cAMP in controls (93.7 +/- 10.3

of DDA untreated samples) whereas it decreased to 76.2 +/- 8.8

(24

inhibition) in CRF (P < 0.05). No differences between controls and CRF groups were found in basal and stimulated cAMP in red blood cells and distal colon. The data would suggest that the cAMP pathway is an intracellular signal for mTAL adaptation in epithelial transport and that the adenylate-cyclase system is specifically activated in CRF.

Effect of NPPB on chloride (Cl-) transport in distal colon of potassium (K+) adapted rats.

Secondary hyperaldosteronism enhances the rate of K secretion in distal colon, at least in part, through the stimulation of Na(+)-K(+)-Cl- cotransport across the basolateral membrane. To maintain a constant intracellular Cl- activity an increase in Cl- transport out of the cell must be assumed. We explored, under amiloride 10(-4) M and short circuited conditions, conductive pathways for Cl- exit in the distal colon of K(+)-adapted rats by means of a putative Cl- channel blocker, NPPB (5-nitro-2(3-phenyl-propylamino-benzoate. Results prior to NPPB showed an increase in JClms after K+ loading from 5.84 +/- 0.66 to 8.33 +/- 0.86 and JClsm from 4.77 +/- 0.55 to 8.16 +/- 0.96 microEq h-1 cm-2 (P < 0.001), when compared with controls. Net fluxes were not different between groups. Luminal NPPB in K+ adaptation resulted in a decrease of JClsm, from 7.85 +/- 1.5 to 6.69 +/- 1.5 microEq h-1 cm-2 (P < 0.05). There were no changes in both unidirectional Cl- fluxes in controls under luminal NPPB and in potential difference (V) and short-circuit current (Isc) under any condition. Finally, K+ adaptation resulted in an increase of luminal cyclic AMP (cAMP) concentration (0.09 +/- 0.02 to 0.20 +/- 0.03 pmol 100 microliters -1, P < 0.005), when compared with control rats. The data may suggest a transcellular recycling of Cl- and an activated NPPB inhibitable serosal to mucosal Cl- pathway on luminal membrane in the K+ adapted state, possibly mediated by an increase in cAMP production.

Chronic indomethacin administration and its relation with the renal kallikrein kinin system during rat pregnancy.

The main purpose of the present study was to elucidate the potential role of vasodilator prostaglandins and the kallikrein kinin system in renal hemodynamic changes observed during rat gestation. Nineteen pregnant rats, un-treated and treated with Indomethacin (3 mg/kg body/wt) for 4 days during peak glomerular hyperfiltration, were studied before and during pregnancy. Twenty-two non-pregnant rats were also included as controls. Daily urinary volume, electrolytes and kallikrein excretion and creatinine clearance were measured along the experiment. Baseline creatinine clearance increased by 43% at the beginning of the third week of pregnancy to decline thereafter. Urinary kallikrein rose earlier, at the second week of pregnancy, and decreased near term while at the same time sodium excretion dropped by 30%. Indomethacin treatment prevented both the maximum increment in glomerular filtration rate occurring in normal pregnancy between days 14 to 18 and the physiological near term decline in kallikrein excretion. Furthermore, it induced an increase in sodium excretion in late pregnancy. These results suggest that vasodilator prostaglandins and the kallikrein kinin system may well participate in gestational hyperfiltration and sodium homeostasis of pregnant rats.

Water and ion coupling in the rat cecum during dietary potassium loading.

The transepithelial net water movement (Jw) was minute by minute recorded in the cecum of Wistar rats adapted to a high potassium diet (HKD). The potential difference (PD), short circuit current (SCC) and unidirectional 22Na, 36Cl, and 89Rb fluxes were also measured. The hydrostatic and osmotic permeabilities were not modified by potassium adaptation when a standard bicarbonate buffer was employed. Potassium adaptation implicated the development of a secretory, transport-associated Jw (Jwt), associated to an increase in transepithelial PD, SCC and net sodium transport. Contrary to the case of control rats, no net chloride transport was observed in HKD rats. The secretory Jwt compensated, in the presence of sodium, chloride and bicarbonate, an absorptive Jwt. Water secretion was coupled to net potassium secretion. Replacement of chloride by sulphate ions was paralleled by the development of a net absorptive Jw and by increases in the transepithelial Pd and hydrostatic permeability. Replacement of the bicarbonate buffer by a tris-hepes buffer made drop both the observed absorptive Jw and PD amiloride sensitive Na+ entry was observed.

Water and ion coupling in the rat cecum during dietary potassium loading

Se midió a minuto el movimiento neto de agua transepitelial (Jw) en el ciego de ratas Wistar adaptadas a una dieta con alto potasio (HKD). También se midió la diferencia de potencial (PD), la corriente de cortocircuito (SCC) y los flujos unidireccionales de 22Na, 36Cl 89Rb. Cuando se utilizó un buffer bicarbonato, las permeabilidades hidrostática y osmótica no se modificaron con la adaptación al potasio. La adaptación al potasio implicó el desarrollo de un Jw secretorio asociado al transporte (Jw), relacionado con un incremento en la PD transepitelial, la SCC y el transporte neto de sodio. Contrariamente al caso de las ratas controles, no se observó ningún transporte neto de cloruro en las ratas HKD. En la presencia de sodio, cloruro y bicarbonato, el Jwt secretorio compensó un Jwt absortivo. La secreción de agua se acopló a una secreción neta de potasio. El reemplazo de iones cloruro por iones sulfato estuvo acompañado por el desarrollo de un Jw neto absortivo y por incrementos en la PD transepitelial y la permeabilidad hidrostática. El reemplazo del buffer bicarbonato por un buffer tris-hepes hizo decaer el Jw absortivo observado y la PD. Por último, se observó el cambio de una reabsorción de NaCI no electrogénica a una entrada electrogénica de Na+ sensible al amiloride