Greg Eigner Jablonski

Surgically Induced Uremia in Rats I: Effect on Bone Strength and Metabolism

During the course of chronic renal failure (CRF) in man, renal osteodystrophy (osteitis fibrosa and/or osteomalacia) gradually develops. The present study aimed to establish a similar type of CRF leading to renal osteodystrophy in rats.During progressive CRF development over 225 days after 5/6 nephrectomy, the following serum variables were measured: creatinine, immunoreactive parathryoid hormone (iPTH), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a25-hydroxyvitamin D3, (25(OH)D3), alkaline phosphatase, albumin, phosphate, urea nitrogen, total calcium, and other blood electrolytes. Subsequent to sacrifice, mechanical properties of the rat femur, bone histomorphometry (osteoid and eroded surfaces) and bone contents of calcium, phosphate and hydroxyproline were also examined.Serum creatinine in rats with CRF gradually escalated by some 70%, while circulating 1,25(OH)2D3 was reduced beneath detection level. Total plasma calcium and phosphate concentrations were, however, almost unchanged indicating that PTH-induced bone remodeling due to moderate hyperparathyroidism sustained calcium homeostasis. Alkaline phosphatase levels were reduced by some 50%, which reflects chronically impeded bone formation. Bone histomorphometry assessment revealed substantial elevation of resorption with moderate accompanying fibrosis in about 70% of afflicted animals. Bone calcium, phosphate and hydroxpyroline contents remained unaltered. However, hydroxoproline/calcium ratio was marginally reduced. These results, together with altered mechanical bending stress characteristics and diminished diaphysis cross section area, confirm development of mixed bone lesions in the uremic animals.Our results are compatible with the early development of CRF in man. The established rat model is therefore useful in elucidating the precipitation and early treatment of renal osteodystrophy in humans.

STUB1 mutations in autosomal recessive ataxias – evidence for mutation-specific clinical heterogeneity

BackgroundA subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism.Methods and resultsWith a combination of homozygozity mapping and exome sequencing, we identified three mutations in STUB1 in two families with ARCA and cognitive impairment; a homozygous missense variant (c.194A > G, p.Asn65Ser) that segregated in three affected siblings, and a missense change (c.82G > A, p.Glu28Lys) which was inherited in trans with a nonsense mutation (c.430A > T, p.Lys144Ter) in another patient. STUB1 encodes CHIP (C-terminus of Heat shock protein 70 – Interacting Protein), a dual function protein with a role in ubiquitination as a co-chaperone with heat shock proteins, and as an E3 ligase. We show that the p.Asn65Ser substitution impairs CHIP’s ability to ubiquitinate HSC70 in vitro, despite being able to self-ubiquitinate. These results are consistent with previous studies highlighting this as a critical residue for the interaction between CHIP and its co-chaperones. Furthermore, we show that the levels of CHIP are strongly reduced in vivo in patients’ fibroblasts compared to controls.ConclusionsThese results suggest that STUB1 mutations might cause disease by impacting not only the E3 ligase function, but also its protein interaction properties and protein amount. Whether the clinical heterogeneity seen in STUB1 ARCA can be related to the location of the mutations remains to be understood, but interestingly, all siblings with the p.Asn65Ser substitution showed a marked appearance of accelerated aging not previously described in STUB1 related ARCA, none display hormonal aberrations/clinical hypogonadism while some affected family members had diabetes, alopecia, uveitis and ulcerative colitis, further refining the spectrum of STUB1 related disease.

Vitamin D3 analogs and salmon calcitonin partially reverse the development of renal osteodystrophy in rats

We have previously established an uremic rat model which is suitable for investigating the effect of various treatment modalities on the progression of renal osteodystrophy [1]. Four months subsequent to 5/6 nephrectomy, animals were treated three times a week for 3 months with either vehicle, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 1,25(OH)2D3+24,25-dihydroxyvitamin D3 [24,25(OH)2D3], 1,25(OH)2D3+calcitonin (CT), or 1,25(OH)2D3+ 24,25(OH)2D3+CT. At termination of the study, clinical chemistry, chemical composition, and mechanical properties of femurs, calvarial parathyroid hormone (PTH)-elicited adenylate cyclase (AC), and phospholipase C (PL-C) activities, femoral cross-sectional area, and bone histomorphometry were analyzed. The main findings were that 1,25(OH)2D3±24,25(OH)2D3 treatment enhanced elasticity as well as time to fracture at the femoral metaphysis. CT potentiated the increase in elasticity obtained by 1,25(OH)2D3±24,25(OH)2D3 treatment. Only 24,25(OH)2D3 administration rectified the supernormal PTH-stimulated uremic bone AC, and only 1,25(OH)2D3 medication normalized the diminished CT-elicited AC. The obliterated uremic bone PTH-sensitive PL-C was fully normalized by all drug regimens. Femoral shaft inner zone diameter was enhanced by uremia, however, all drug treatments normalized it. Ditto effect was registered with either drug treatment on the subnormal outer and inner zone widths. Histomorphometrical analyses showed that 1,25(OH)2D3 administration reduced both eroded and osteoid surfaces. Most prominently, adjuvant 24,25(OH)2D3 or CT administration potentiated the beneficial effect of 1,25(OH)2D3 on fibrosis and osteomalacia. We assert that vitamin D3 treatment markedly reverses the development of renal osteodystrophy, and CT potentiates the effect of vitamin D3.

Aluminium-induced bone disease in uremic Rats: Effect of deferoxamine

We have previously established a rat model of chronic uremia, which is suitable to investigate the effect of various treatment modalities on renal osteodystrophy [1]. After four months subsequent to 5/6 nephrectomy, some animals were treated by gavage for 9 weeks with tap water (controls), or with aluminium (Al-citrate) 3 × 25 mg/week/kg b.wt ± subsequent deferoxamine (DFO) 3 × 50 mg/ week/kg b.wt. for 4 weeks. At termination of the study, serum clinical chemistry, femoral chemical composition and mechanical properties, calvarial parathyroid hormone (PTH)-elicited adenylate cyclase (AC) and phospholipase C (PLC) activities, cross-sectional femoral area, as well as bone histomorphometry, were analyzed. Animals given Al displayed moderately enhanced serum Al and bone Al accumulation, however, DFO-treatment did not fully alleviate bone Al retainment. A small increase in serum PTH was seen in all animals rendered uremic. Furthermore, a marked fall in serum alkaline phosphatase (ALP) below normal controls was observed in Al ± DFO-treated animals compared with uremic controls. The uremic condition led to reduced femoral ratios of hydroxyproline (HYP) over Ca2+ and phosphate (Pi), while Al-intoxication alone enhanced femoral Hyp contents above values seen for normal controls. The protracted ureamia caused a deterioration of long bone resilience and brittleness, however, Al ± DFO-treatment seemed to normalize the latter. Contrastingly, Al ± DFO-gavage enhanced time to fracture. Uremic rats intoxicated with Al showed a complete loss of calvarial PTH-sensitive AC and PLC activities. DFO-treatment normalized PTH-elicited PLC, while PTH-susceptible AC remained super-normal. Al apparently exerts a long term down-regulation of both PTH-sensitive signaling systems as evidenced by studies of rat UMR 106 osteosarcoma cells in culture. The uremic condition enhanced endosteal bone resorption as shown by femoral shaft dimension analysis, while AI ± DFO-treatment insignificantly reversed the condition. Finally, histomorphometrical analyses showed that DFO-administration tended to normalize aberrant trabecular bone volume, while rectifying both bone resorption and degree of mineralization. In conclusion, we assert that Al-intoxication hampers both processes (i.e. formation and resorption) of bone turnover, and that DFO-treatment to a certain extent prevents the uremia- and Al-induced bone disease in rats.

1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) - sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.

Surgically induced uremia in rats II: Osseous PTH-susceptible signaling systems as predictors of bone resorption

Predicting the course of parathormone (PTH)-elicited bone turnover in both humans and experimental rat models with moderate chronic uremia, using only standard clinical chemistry analyses, is often difficult. Consequently, rat bone from 1+2/3 nephrectomized animals, after 230 days of progressive renal failure, was examined for PTH-stimulated adenylate cyclase (AC) and phospholipase C (PLC) activities. Correlations to biological parameters related to the function of bone and kidney were made. Reduced renal function was demonstrated by increased serum creatinine; circulating 1,25 dihydroxyvitamin D3 below detection level; diminished renal PTH-elicited AC activity; and decreased urinary cAMP excretion. PTH-activated renal PL-C was also reduced. However, no significant differences were seen in urine creatinine, calcium, phosphate, and hydroxyproline, nor in serum PTH, alkaline phosphatase, calcium, and phosphate. Notwithstanding, renal osteodystrophy developed as estimated by increased plasticity of the long bones, as well as reduction of the diaphyseal (Dd) and inner femoral midshaft (Di) diameters. Femoral cancellous bone exhibited a substantial elevation of both eroded surface (ES) and osteoid surface (OS) as well as a marked reduction in trabecular bone volume (TBV). Calvarial PTH-activated AC was enhanced, whereas corresponding PL-C was markedly reduced. PTH-enhanced AC correlated positively with ES and negatively with Di, respectively. PTH-enhanced PL-C, however, correlated positively with bone calcium content and negatively with ES. Our results indicate that bone modeling and remodeling are to a large extent related to PTH-elicited signaling systems, and cannot easily be predicted by standard clinical chemistry analyses.

Insertion Depth in Cochlear Implantation and Outcome in Residual Hearing and Vestibular Function.

Objectives: It has long been known that cochlear implantation may cause loss of residual hearing and vestibular function. Different insertion depths may cause varying degrees of intracochlear trauma in the apical region of the cochlea. The present study investigated the correlation between the insertion depth and postoperative loss of residual hearing and vestibular function. Design: Thirty-nine adults underwent unilateral cochlear implantation. One group received a Med-El +Flex24 electrode array (24 mm; n = 4), 1 group received a Med-El +Flex28 electrode array (28 mm; n = 18), and 1 group received a Med-El +FlexSOFT electrode array (31.5 mm; n = 17). Residual hearing, cervical vestibular-evoked myogenic potentials, videonystagmography, and subjective visual vertical/horizontal were explored before and after surgery. The electrode insertion depth and scalar position were examined with high-resolution rotational tomography after implantation in 29 subjects. Results: There was no observed relationship between the angular insertion depth (405° to 708°) and loss of low-frequency pure-tone average. Frequency-specific analysis revealed a weak relationship between the angular insertion depth and loss of hearing at 250 Hz (R2= 0.20; p = 0.02). There was no statistically significant difference in the residual hearing and vestibular function between the +Flex28 and the +FlexSOFT electrode array. Eight percent of the cases had vertigo after surgery. The electrode arrays were positioned inside the scala tympani and not scala vestibuli in all subjects. In 18% of the cases, the +FlexSOFT electrode array was not fully inserted. Conclusions: The final outcome in residual hearing correlates very weakly with the angular insertion depth for depths above 405°. Postoperative loss of vestibular function did not correlate with the angular insertion depth or age at implantation. The surgical protocol used in this study seems to minimize the risk of postoperative vertigo symptoms.

Scalar position in cochlear implant surgery and outcome in residual hearing and the vestibular system

Abstract Objective: To evaluate the effect of the intracochlear electrode position on the residual hearing and VNG- and cVEMP responses. Design: Prospective pilot study. Study sample: Thirteen adult patients who underwent unilateral cochlear implant surgery were examined with high-resolution rotational tomography after cochlear implantation. All subjects were also tested with VNG, and 12 of the subjects were tested with cVEMP and audiometry before and after surgery. Results: We found that although the electrode was originally planned to be positioned inside the scala tympani, only 8 of 13 had full insertion into the scala tympani. Loss of cVEMP response occurred to the same extent in the group with full scala tympani positioning and the group with scala vestibuli involvement. There was a non-significant difference in the loss of caloric response and residual hearing between the two groups. Interscalar dislocation of the electrode inside the cochlea was observed in two patients. A higher loss of residual hearing could be seen in the group with electrode dislocation between the scalae. Conclusions: Our findings indicate that intracochlear electrode dislocation is a possible cause to loss of residual hearing during cochlear implantation but cannot be the sole cause of postoperative vestibular loss.

Cochlear implantees: Analysis of behavioral and objective measures for a clinical population of various age groups

Abstract Introduction As of 2014 more than 1200 patients have received a cochlear implant (CI) at Oslo University Hospital (OUS) and approximately half of them have been children. The data obtained from these patients have been used to develop a comprehensive database for a systematic analysis of several objective measurements and programming measurements. During the past 10 years, we have used an objective measurements protocol for our CI surgeries. Our intra-operative protocol includes: Evoked Compound Action Potentials (ECAP), visually observed Electrically evoked Stapedius Reflex Threshold (ESRT), and electrode impedances. Post-operative (Post-OP) programming sessions typically begin 4–6 weeks after surgery and continue on a scheduled basis. The initial programming data include threshold levels (T-levels) and comfortable levels (C-levels) for the different patient age groups. In this study, we compared initial stimulation levels and stimulation levels after at least 1 year of CI with objective measurements obtained intra-operatively. Method This study focused on the development of a comprehensive database of detailed intra-operative objective measures and post-OP programming measurements from a group of 296 CI patients who received the same type of CI and electrode configuration (Cochlear® Corporation CI with Contour® electrode). This group included 92 bilateral CI patients. Measurements from 388 CI devices were studied. Patients were divided into 5 different age groups at the age of implantation: 0–2, 2–5, 5–10, 10–20, and above 20 years in order to investigate age-related differences in programming levels and objective measurements. For the comparison analysis we used T- and C-levels obtained after the last day of initial programming and also after at least 1 year implant use. These programming levels were then correlated with some of the intra-operative objective measurements. Results T-levels were found to be the lowest for the youngest patient group and increased with age. C-levels varied within age groups and frequency range. Patients above 20 years of age had the highest comfort levels in the low to mid-frequencies (electrodes 22–8) and the lowest comfort levels in the high-frequency range (electrodes 1–7). Correlation coefficients between intra-operative objective measurements and programming levels were found to be in the range of no correlation to moderate correlation. Adult patients had the most significant correlation coefficients between ECAP thresholds and T-levels in the low frequencies. The younger patients aged 10–20 years and 5–10 years had more significant correlations in the higher frequency channels compared to the other age groups. Intra-operative visually observed ESRTs and electrode impedances were not significantly correlated with initial or stable programming levels for the children or adults. Conclusion Analyzing initial and follow-up mapping levels from previous patients is very important for a CI Center in terms of quality control. The mean T/C-levels reported in this study can provide guidance to our programming audiologists and help them determine the initial programming levels to be stored in the speech processor, especially for very young patients. Unfortunately intra-operative objective measures in our study, such as ECAP, ESRT, and electrode impedances did not provide statistically significant correlations that may help to predict the programming T- and C-levels for all patients. However, we have observed cases where the intra-operative objective measures of ESRT and TECAP profiles were very similar to an individuals MAP profile. It was not possible, however, to determine why some patients did not have an objective measures profile that was similar to their programming levels profile.

Diverse expression of G‐proteins in human sarcoma cell lines with different osteogenic potential: Evidence for the involvement of Gi2 in cell proliferation

Previously, it has been shown that the GTP‐binding protein Gi2 is implicated in cellular growth [1,2] and differentiation [2,3]. In the present paper we demonstrate that this is also the case for human sarcoma cells.