Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.

Natural history of hepatitis C virus infection in adult renal graft recipients.

AIM: To study the natural history of hepatitis C virus infection in renal transplantation, 464 HbsAg negative patients were prospectively studied from 1989. METHODS: AntiHCV was tested by ELISA II and HCVRNA by Amplicor HCV RNA tests. RESULTS: Two hundred nine patients were antiHCV positive (C+). HCVRNA was confirmed in 89% of C+ patients. Compared with the 255 anti-HCV negative (C-), C+ had undergone longer periods of dialysis (P = .0001), were more transfused (P = .01), and included more retransplants (P = .002). Immunosuppression was azathioprine (AZA) plus steroids in 133 and cyclosporine (CsA) in 331 patients. Liver biopsy showed chronic active hepatitis in 50, cirrhosis in 8, and fibrosing cholestatic hepatitis in 2 patients. Histologic progression of liver disease was confirmed in 18 of 26 patients. The causes of death in 84 patients (51 C+ vs 33 C-) were cardiovascular disease in 49%, sepsis in 13%, liver failure in 14%, neoplasia in 21%, and hepatocarcinoma in 2%. The 14-year patient survival was 75% in C+ and 86% in C- (P = .002). By multivariate analysis, age (>40) (P = .001) and C+ (P = .019) correlated with a worse patient survival. If patients were stratified according to age (<40 vs > or =40), younger C+ patients had a lower survival probability (P = .03). The 14-year graft survival was 44% in C+ vs 60% in C- patients (P = .001) but pure graft survival was similar (68% in C+ vs 72% in C-) (P = .13). CONCLUSION: The presence of C+ significantly reduced both patient and graft survival in the long-term with liver failure being the second most frequent cause of death.

Prevention of calcium nephrolithiasis with low-dose thiazide, amiloride and allopurinol.

We report 5 years' experience with low-dose hydrochlorothiazide, 50 mg/day and amiloride, 5 mg/day, in 519 patients with recurrent calcium nephrolithiasis. Additional treatment with allopurinol, 100 mg/day was prescribed for approximately 50 percent of the patients. All patients had active stone formation, having 3,464 stones in 3,126 patient-years (6.67 stones per patient, 1.10 stones per year). Hypercalciuria was present in 65 percent of the patients and hyperuricosuria in 24 percent. The administration of low-dose hydrochlorothiazide was effective in reducing urinary calcium excretion in most patients. It is possible that the hypocalciuric effect of hydrochlorothiazide were enhanced by amiloride, an agent which has been shown to cause hypocalciuria when given alone. Significant side effects requiring discontinuation of the drug were observed in only 5 percent of the patients. During 872.8 patient-years of treatment, only 53 new stones were formed (0.10 stones per patient, 0.06 stones per year) in contrast with the 916 predicted ones. The difference (chi-square) is statistically significant (p less than 0.001). These results show that the administration of low-dose hydrochlorothiazide and amiloride, either alone or in association with allopurinol, is clinically effective in reducing the rate of recurrence of calcium nephrolithiasis.

A randomized prospective trial comparing cyclosporine monotherapy with triple-drug therapy in renal transplantation.

In a prospective trial 151 recipients of renal transplants were randomly assigned to treatment with CsA alone (74 patients) and to low dose of AZA, prednisolone, and CsA (77 patients). At two years, graft survival was 84% for the monotherapy and 90% for the triple therapy. This difference was not statistically significant. The number of rejection episodes was similar in the two groups, but the severity of rejection was significantly worse among the patients on monotherapy. More kidneys were lost because of rejection (6 versus 3), and a higher number of methylprednisolone pulses was used for treating rejection (5.2 +/- 2.3 versus 4.3 +/- 2.9; P = 0.0077). CsA nephrotoxicity episodes were more frequent among patients on monotherapy (23 versus 7; P less than 0.02). Infectious episodes were equally distributed between the two groups. Creatinine clearance was poorer in the monotherapy-treated patients at the third month (42 +/- 16 ml/min versus 48 +/- 15 ml/min; P = 0.02), but no differences were observed between the two groups since the sixth month after transplantation. Many patients on monotherapy required changes in maintenance therapy. In fact, one patient was switched to conventional immunosuppression because of Cremophor-induced anaphylaxis. Another patient who developed Kaposi's sarcoma 4 months after surgery was switched to steroids alone. Excluding 5 patients who lost their grafts a few days after transplantation, only 30 of 74 patients (40%) could be kept without steroids. We conclude that both the therapeutic protocols can give good results in renal allotransplantation; however, monotherapy could create some problems in keeping the balance between drug toxicity and significant immunosuppression. On the contrary, triple therapy is easier to handle, especially in the early posttransplant period when the differential diagnosis between acute rejection and CsA-related nephrotoxicity can be difficult even for a skilled clinician.

Effects of three immunosuppressive regimens on vertebral bone density in renal transplant recipients: a prospective study.

The influence of three different immunosuppressive regimens with cyclosporine (CsA) on the development of osteopenia in renal transplant patients was assessed. Fifty-three adults with first kidney transplants participated in a randomized trial to analyze the efficacy of three different immunosuppressive regimens: CsA alone (group 1), CsA plus steroids (group 2), and CsA plus steroids plus azathioprine (group 3). Lumbar spine bone mineral density was assessed by dual energy x-ray absorptiometry every 6 months for 18 months. The values for trabecular mass were expressed as bone mineral density and as a fraction of the standard deviation of the mean of the normal value for patient's sex and decade of age (Z-score). Statistical analysis was performed on Z-score and "Z-score change" (value after 6 months minus the basal value at transplantation). At the 18th month, the Z-score increased significantly in treatment group 1 without steroids (P=0.006) and decreased significantly in steroid-treated groups 2 (P<0.001) and 3 (P<0.001). Comparing the two genders, Z-score decreased less in premenopausal women than in men (P=0.018). "Z-score change" did not correlate with steroid dosage, was high in patients with high basal bone mineral density, and was directly associated with the duration of dialysis (P=0.008). In conclusion, premenopausal transplant recipients showed a lower decrease of lumbar bone mineral density than men. In transplant recipients given CsA with steroids, lumbar bone mineral density decreased significantly, while it increased significantly in patients given CsA alone.

A randomized trial comparing triple-drug and double-drug therapy in renal transplantation. Analysis at 7 years.

This is the 7-year update of a randomized trial comparing triple (TT) and double (DT) immunosuppressive therapy in renal transplantation. At 7 years, patient survival rate was 85% in DT vs. 87% in TT (P = NS); graft survival rate was 73% in DT and 68% in TT (P = NS); pure graft survival was 86% in DT vs. 77% in TT (P = 0.096). The 7-year graft survival rate was 67% for cadaver graft recipients vs. 92% for living-related graft recipients (P = 0.044). No difference in the slopes of plasma creatinine between the two groups was observed. Ten DT and 13 TT patients changed their original therapy: statistical analysis, however, was carried out according to intention to treat. Both CsA levels and doses were significantly higher in DT than in TT group (P < 0.001) at any time point up to the 7th year. At univariate analysis, a living-related donor kidney (P = 0.044) and immediate recovery of renal function (P < 0.001) were the only two parameters associated with graft survival at 7 years. At multivariate analysis, only early graft function recovery was correlated with late graft survival (RR = 10.480). Thus, even in the longterm, there is no difference between DT and TT, either in patient or in graft survival: at the doses we used, TT had a lower prevalence of late side effects than DT, however, long-term pure graft survival was better, although not significantly, in DT than in TT. The possibility of a safe shift from one regimen to the other one makes the two treatments complementary rather than alternatives.

The impact of azathioprine and cyclosporine on long-term function in kidney transplantation.

To assess the impact of cyclosporine on long-term kidney function in transplant patients, we retrospectively analyzed 273 patients on azathioprine and 308 on CsA with graft functioning at 1 year. To balance the length of follow-ups, the observation of patients was cut at 5 years. Actual graft survival rate at 5 years was similar in Aza and CsA (88% vs. 90%). Multivariate analysis in Aza pts showed that proteinuria (P = 0.006) and hypertension at 1 year (P = 0.002) increased the probability of irreversible graft failure by 2.47 and 2.85, respectively. In CsA patients, proteinuria (P = 0.007) and plasma creatinine higher than 2.5 mg/dl (P = 0.006) increased the probability of graft failure by 5.12 and 6.48, respectively. In both Aza and CsA patients with a follow-up of at least 5 years, plasma creatinine levels were significantly worse at 5 years vs. 1 year (P = 0.004). The slopes of plasma creatinine values plotted vs time were not different between the two groups. Chronic graft dysfunction (CGD) was defined as a stable increase of plasma creatinine of at least 50% above stable values at 1 year. The probability of remaining without CGD at 5 years was 75% for CsA and 80% for Aza patients (P = N.S.). Multivariate analysis of factors influencing the development of CGD showed that hypertension (P = 0.003) and proteinuria at 1 year (P = 0.081) increased the probability of developing CGD by 2.19 and 1.76, respectively, in Aza, while in CsA patients proteinuria only (P = 0.063) increased the probability of developing CGD by 2.29. Graft survival at 5 years after development of CGD was 34% in Aza and 53% in CsA-treated patients. These data confirm that in the long-term CsA does not cause a higher prevalence of CGD and show that, in the presence of CGD, CsA has a superior protective effect than Aza.

A randomized trial comparing triple-drug and double-drug therapy in renal transplantation.

A controlled trial was carried out in 86 cadaveric and 14 living haploidentical renal transplant recipients to compare the effects of low doses of cyclosporine (CsA), azathioprine (Aza) and steroids with those of higher doses of CsA plus steroids. Patients were followed for 12-26 months after transplantation. The actuarial 2-year patient and graft survival rate was 100% for living-donor transplants. In cadaver renal transplants the 2-year patient survival rate was 100% for patients assigned to the triple regimen and 93% for those allocated to the double regimen. The actuarial 2-year cadaver graft survival rates were 86% and 90.6%, respectively. There were significantly more patients who had severe infections (P less than 0.05), particularly interstitial pneumonia (P less than 0.005), in the double-therapy group. On the other hand, there were more patients who rejected and more patients with severe rejections; more pulses of steroids were also required for patients on the triple regimen, although these differences were not significant. The mean trough blood levels of cyclosporine at the various times were about half as high in patients on triple therapy. There were no differences between the two groups in creatinine clearance at any time. A control renal biopsy, taken from patients with stable renal function after 6-12 months, showed only mild abnormalities. The lesions were semiquantitatively assessed. There was a higher score for interstitial infiltrate in patients on triple therapy (P less than 0.05). On the other hand, the incidence and the mean score of interstitial fibrosis were greater in patients on double therapy, although these differences were not significant. Thus, although similar results were obtained with both regimens, at the doses we used double therapy seems to have more powerful immunosuppressive effects and may prevent rejection, either acute or chronic, better. However, it might expose the patient to a greater risk of infection and of cyclosporine-related nephrotoxicity than triple therapy.

High prevalence, low pathogenicity of hepatitis G virus in kidney transplant recipients.

BACKGROUND: Prevalence and pathogenicity of hepatitis G virus infection in long-term renal transplant recipients, are not fully known. AIM: To evaluate long-term impact of HGV infection on liver disease of renal transplanted patients. PATIENTS AND METHODS: A total of 155 hepatitis B surface antigen negative kidney transplant recipients, followed for a mean of 11 years after renal transplantation, were studied. Of these 48 (31%) patients had persistently elevated serum aminotransferase values. Frozen serum samples were tested for HGV-RNA and HCV-RNA by nested reverse transcribed polymerase chain reaction, and for anti-hepatitis G virus and anti-hepatitis C virus by enzyme-linked immunosorbent assay Hepatitis C virus-RNA was typed by a line probe assay and quantified by a branched DNA signal amplification assay RESULTS: Hepatitis G virus-RNA was detected in 37 (24%) patients and anti-hepatitis G virus in another 26 (17%). Seventy (45%) patients had serum anti-hepatitis C virus and 63 of these (90%) had serum hepatitis C virus-RNA. Hepatitis G virus-RNA positive and negative patients were similar in terms of age, sex, duration of dialysis, rate of transfusion, chronic liver disease, rate of hepatitis C virus infection and immunosuppressive therapy. Fifteen (41%) hepatitis G virus-RNA seropositive patients were hepatitis C virus co-infected. Hepatitis C virus-RNA levels were significantly lower in the 15 hepatitis C virus/hepatitis G virus co-infected patients than in the 48 patients with hepatitis C virus infection only (2.2 vs 10.8 MEq/ml, p = 0.02). Only 3 hepatitis G virus carriers had persistently elevated alanine aminotransferase compared to 29 hepatitis C virus carriers (14% vs 60%, p < 0.001), 10 patients co-infected with both hepatitis G virus and hepatitis C virus, and in 6 patients with neither infection (67% vs 8%, p < 0.001). CONCLUSIONS: Hepatitis G virus infection is common among kidney transplant patients, it carries a low risk of chronic liver disease even in long-term follow-up. Low levels of hepatitis C virus-RNA found in hepatitis G virus carriers suggest an interaction between these two viruses in immunosuppressed patients.

From cyclosporine to the future.

Most of the experience acquired in our unit with cyclosporine (CsA) comes from randomized trials. A first trial demonstrated that CsA-treated patients had a better 10-year graft survival than azathioprine-treated patients. A second trial showed equivalence between double therapy with CsA plus steroids and triple therapy with CsA, steroids, and azatioprine. A third trial showed similar 2-year graft survival with CsA monotherapy and triple therapy. A larger multicenter study that compared three different CsA-based regimens showed similar long-term graft survival with monotherapy, double therapy, and triple therapy. However, patients given monotherapy had less frequent steroid-related side-effects. Finally a more recent multicenter international trial showed that the rate of acute rejection can be reduced without increasing side effects by adding the monoclonal antibody basiliximab to the triple therapy. By reviewing our cumulative experience with CsA we found a mean graft half-life of 18.7 years for cadaver renal transplant recipients and 31.9 for the living transplant recipients. No significant attrition of graft function was found for patients with grafts functioning at 15 years. Two important issues with the present immunosuppression concern the long-term nephrotoxicity of calcineurin inhibitors and the cardiovascular disease, which is at least in part related to the use of steroids. To face these problems, we are currently involved in two multicenter trials, one comparing sirolimus plus mycophenolate mofetil to sirolimus plus low-dose CsA, while the other trial compares certican plus CsA to certican plus CsA plus corticosteroids.

Design of a trial comparing sirolimus plus mycophenolate mofetil versus sirolimus plus cyclosporine.

We present the study design of a prospective, multicenter, randomized trial aimed at comparing the effects of two different combinations of sirolimus. Renal transplant recipients will be allocated to receive either sirolimus and mycophenolate mofetil (group A) or sirolimus and cyclosporine (group B). The primary endpoint will be the graft function at 3, 6, 12, 24, 36, 48, and 60 months. A number of secondary endpoints will also be considered. To obtain a significant difference in the primary endpoint 180 patients will be enrolled.

[Bone complications of renal transplantation. How to identify and prevent them]. / Le complicanze ossee del trapianto renale. Come identificarle e prevenirle.

Bone disease is a very frequent complication after renal transplantation (RTx). The main features of transplant bone disease include the osteopenic-osteoporotic syndrome, often complicated by fractures, avascular osteonecrosis of bones, bone pain syndrome and growth retardation in the children. The bone loss is greater during the first 12 months after RTx and can reach the osteoporotic range in above 40% of patients, with a fracture rate of 2-3% per year. The story of bone disease over the long pre-uremic and uremic period is one of the main causal factors. After RTx, glucocorticoid therapy seems to play the major causal role. Much more disputed is the role of the other immunosuppressive drugs, of persistent secondary hyperparathyroidism, and age. Hypophosphatemia and some genetic factors could also affect bone loss after RTx. Diabetic patients are particularly prone to develop bone disease after RTx. The main prophylactic interventions consist in the optimal control of hyperparathyroidism during the pre-transplant period, prescribing parathyroidectomy for autonomous hyperparathyroidism, not-responding to medical therapy. After RTx, both bisphosphonate and vitamin D metabolites, variably associated with calcium supplementation, have been demonstrated to have some beneficial effect on bone loss, at least in the first year after RTx. However, there are no data about the possible efficacy of these treatments on fracture rate.

Risk factors associated to kidney stones in primary hyperparathyroidism.

Nephrolithiasis is the most important clinical manifestation of primary hyperparathyroidism (PHPT), although nowadays this disorder is often asymptomatic. Clinical or biochemical differences between PHPT patients with and without nephrolithiasis have not been clearly identified in most of the previous studies. The aim of the study was to investigate clinical and biochemical parameters in kidney stone former (SF) and non-stone former (NSF) patients with PHPT in order to identify potential risk factors. Serum and plasma samples from 55 consecutive patients (43 females, 12 males) with PHPT were collected after overnight fasting; 24-h urine collection and a fresh sample of urine for sediment analysis were obtained from all patients. Clinical data were recorded in all. Out of 55 patients, 22 had kidney stones, which were symptomatic in 73%. SFs showed circulating PTH, total and ionized calcium, 1,25 dihydroxyvitamin D3, urinary calcium excretion and 24-h urine oxalate levels significantly higher than NSFs. Hypercalciuria was often concomitant with massive quantities of calcium oxalate crystals in urine sediment. Hypercalciuria and relatively high oxaluria were associated with stone formation with an odds ratio (OR) of 4.0 and 7.0, respectively, which rose to 33.5 when they coexisted. Hypomagnesuria and hypocitraturia were common in at least one third of all PHPT patients, but they were not associated to an increased OR. As expected, they were positively correlated with urine calcium excretion, suggesting that calcium, magnesium and citrate are commonly regulated at renal level. In conclusion, hypercalciuria, higher oxalate excretion and severe PHPT are associated with kidney stones in PHPT.