The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics.

Calbindin-D28k (CBD-28k) is a calcium binding protein located in the distal convoluted tubule (DCT) and plays an important role in active calcium transport in the kidney. Loop and thiazide diuretics affect renal Ca and Mg handling: both cause Mg wasting, but have opposite effects on Ca excretion as loop diuretics increase, but thiazides decrease, Ca excretion. To understand the role of CBD-28k in renal Ca and Mg handling in response to diuretics treatment, we investigated renal Ca and Mg excretion and gene expression of DCT Ca and Mg transport molecules in wild-type (WT) and CBD-28k knockout (KO) mice. Mice were treated with chlorothiazide (CTZ; 50 mg · kg(-1) · day(-1)) or furosemide (FSM; 30 mg · kg(-1) · day(-1)) for 3 days. To avoid volume depletion, salt was supplemented in the drinking water. Urine Ca excretion was reduced in WT, but not in KO mice, by CTZ. FSM induced similar hypercalciuria in both groups. DCT Ca transport molecules, including transient receptor potential vanilloid 5 (TRPV5), TRPV6, and CBD-9k, were upregulated by CTZ and FSM in WT, but not in KO mice. Urine Mg excretion was increased and transient receptor potential subfamily M, member 6 (TRPM6) was upregulated by both CTZ and FSM in WT and KO mice. In conclusion, CBD-28k plays an important role in gene expression of DCT Ca, but not Mg, transport molecules, which may be related to its being a Ca, but not a Mg, intracellular sensor. The lack of upregulation of DCT Ca transport molecules by thiazides in the KO mice indicates that the DCT Ca transport system is critical for Ca conservation by thiazides.

Pharmacologic recruitment of regulatory T cells as a therapy for ischemic acute kidney injury.

Regulatory T cells (Tregs) are key components of the peripheral tolerance system and have become an immunotherapeutic agent for treating inflammatory processes. This therapeutic option, however, is hampered by problems arising from isolating and expanding desirable Tregs. Here we used an alternative approach with a pharmacologic agent to stimulate Tregs to achieve immunosuppressive effects. Pretreatment of mice with the naturally occurring sphingosine N,N-dimethylsphingosine (DMS) was found to increase both tissue-infiltrating T effectors (Teffs, CD4(+)Foxp3(-)) and Tregs (CD4(+)Foxp3(+)) in the early phase of bilateral renal ischemia/reperfusion injury. DMS itself had no effects on renal function or histopathology, but rapidly and transiently increased both Teffs and Tregs and increased the expression of chemokines CXCL9, CCL5, and CXCL10 in non-ischemic kidneys (sham operation). This renoprotection was abolished by administration of the Treg suppressing agents, anti-CTLA-4 or anti-CD25 monoclonal antibodies, suggesting that Tregs play a key role in DMS-induced renoprotection. Thus, Tregs recruited to the kidney by DMS ameliorate acute kidney injury and provide a new approach to control inflammatory diseases.

Renal adaptation to gentamicin-induced mineral loss.

BACKGROUND: Gentamicin, a well-known nephrotoxic drug, affects calcium and magnesium homeostasis. Although gentamicin induces urinary calcium and magnesium wasting immediately, it rarely causes significant hypocalcemia or hypomagnesemia clinically. METHODS: We conducted an animal study to investigate the renal adaptation in calcium and magnesium handling after gentamicin treatment and effects on the expression of calcium and magnesium transport molecules in distal tubule. Gentamicin (40 mg/kg) was injected daily in male Sprague-Dawley rats (220-250 g) for up to 7 days. RESULTS: This treatment did not affect serum creatinine, calcium, or magnesium levels. Gentamicin induced significant hypercalciuria (14-fold) and hypermagnesiuria (10-fold) in 6 h, which was associated with upregulation of TRPV5 (175 ± 3%), TRPV6 (170 ± 4%), TRPM6 (156 ± 4%) and calbindin-D28k (174 ± 3%; all p < 0.05 vs. control). This gene upregulation was maintained with daily injection of gentamicin for 7 days. The gentamicin-induced urinary calcium loss was reduced by 80% at days 3 and 7, while magnesium loss was reduced by 52 and 57% at days 3 and 7, respectively. On the other hand, urinary loss of potassium became worse on day 7 (2-fold), and phosphorus loss worse from day 3 to day 7 (3-fold). CONCLUSION: There is a rapid adaptation to gentamicin-induced hypercalciuria and hypermagnesiuria. The upregulation of distal tubule transport molecules, TRPV5, TRPV6, TRPM6 and calbindin-D28k occurs within 6 h of gentamicin treatment. This renal adaptation prevents further mineral loss due to gentamicin treatment.

Variations of dietary salt and fluid modulate calcium and magnesium transport in the renal distal tubule.

BACKGROUND: The renal distal tubule fine-tunes renal epithelial calcium transport. Dietary intake of salt and fluid varies day-to-day and the kidney adapts accordingly to maintain homeostasis. The alternations in salt and fluid balance affect calcium and magnesium transport in the distal tubule, but the mechanisms are not fully understood. METHODS: Sprague-Dawley rats were grouped into high-salt, low-salt and dehydration treatment. Daily intake, water consumption and urine output were recorded. At the end of the experiment, blood and urine samples were collected for hormonal and biochemical tests. Genetic analysis, immunoblotting and immunofluorescence studies were then performed to assess the alterations of calcium and magnesium transport-related molecules. RESULTS: High-salt treatment increased urinary sodium, calcium and magnesium excretion. Low-salt treatment and dehydration were associated with decreased urinary excretion of all electrolytes. High-salt treatment was associated with increased intact parathyroid hormone levels. A significant increase in gene expression of TRPV5, TRPV6, calbindin-D28k and TRPM6 was found during high-salt treatment, while low salt and dehydration diminished expression. These findings were confirmed with immunofluorescence studies. High-salt and low-salt intake or dehydration did not cause any significant changes in WNK1, WNK3 and WNK4. CONCLUSIONS: Alternations in salt and water intake affect renal calcium and magnesium handling. High-salt intake increases the distal delivery of the divalent cations which upregulates distal tubule calcium and magnesium transport molecules, while the opposite effects are associated with low-salt intake or dehydration.

Site-specific expression of IQGAP1, a key mediator of cytoskeleton, in mouse renal tubules.

IQGAP1 is a multifunctional junction molecule that is involved in cell migration, proliferation, differentiation, cell polarity, and cell-cell adhesion. It is highly expressed in the kidney and has recently been identified in the glomerular basement membrane as a nephrin-associated protein. However, the distribution of IQGAP1 in renal tubular epithelial cells is unknown. We performed confocal microscopic studies to localize IQGAP1 in each nephron segment using dual immunofluorescence staining with various antibodies against segment-specific markers. We found that IQGAP1 was strongly expressed in the distal convoluted tubule (DCT), collecting duct, and macula densa and moderately in the thick ascending limb and proximal tubule. In the DCT, the IQGAP1-F-actin complex forms a comb-like structure with multiple parallel spikes sitting on the basal membrane. In the macula densa cells, IQGAP1 is strongly expressed in the apical membrane, whereas in type A intercalated cells, IQGAP1 is expressed in the basolateral membrane, where it colocalizes with anion exchanger 1, and in principal cells, it is diffusely expressed. In conclusion, we showed the expression and subcellular localization of IQGAP1 in various nephron segments. The site-specific expression pattern of this potent modulator of multiple biological pathways in the renal tubules suggests that IQGAP1 may have multiple important roles in various renal functions.

Is bowel preparation before colonoscopy a risky business for the kidney?

Acute phosphate nephropathy after bowel preparation with oral sodium phosphate (OSP) for colonoscopy has emerged as an important clinical entity. In 2004, five cases of nephrocalcinosis and irreversible renal failure after bowel preparation with OSP were reported. More recently, several retrospective studies have shown that the incidence of acute kidney injury after OSP use is in the range of 1-4%, similar to the incidence of contrast nephropathy in the general population. The degree of renal failure is not generally as severe as in the first reported cases, but irreversible damage can still occur. Millions of people worldwide undergo screening colonoscopies for colon and rectal cancer after the age of 50, so careful patient selection and monitoring for possible complications is essential when OSP is used. In addition to educating patients about the possibility of renal damage, physicians should routinely watch for considerable weight loss during bowel preparation and correct the fluid deficit as needed. Carrying out a renal function panel, which includes serum phosphorus level, is prudent after colonoscopy. Alternative bowel cleansing agents are needed because calcium phosphate precipitation is inevitable after OSP use even in the normal kidney.

Functionally improved bone in calbindin-D28k knockout mice.

In vitro studies indicate that Calbindin-D28k, a calcium binding protein, is important in regulating the life span of osteoblasts as well as the mineralization of bone extracellular matrix. The recent creation of a Calbindin-D28k knockout mouse has provided the opportunity to study the physiological effects of the Calbindin-D28k protein on bone remodeling in vivo. In this experiment, histomorphometry, microCT, and bend testing were used to characterize bones in Calbindin-D28k KO (knockout) mice. The femora of Calbindin-D28k KO mice had significantly increased cortical bone volume (60.4% +/- 3.1) compared to wild-type (WT) mice (45.4% +/- 4.6). The increased bone volume was due to a decrease in marrow cavity area, and significantly decreased endosteal perimeters (3.397 mm +/- 0.278 in Calbindin-D28k KO mice, and 4.046 mm +/- 0.450 in WT mice). Similar changes were noted in the analysis of the tibias in both mice. The bone formation rates were similar in the femoral and tibial cortical bones of both mice. microCT analysis of the trabecular bone in the tibial plateau indicated that Calbindin-D28k KO mice had an increased bone volume (35.2% +/- 3.1) compared to WT mice (24.7% +/- 4.9) which was primarily due to increased trabecular number (8.99 mm(-1) +/- 0.94 in Calbindin-D28k KO mice compared to 6.75 mm(-1) +/- 0.85 in WT mice). Bone mineral content analysis of the tibias indicated that there is no difference in the calcium or phosphorus content between the Calbindin-D28k KO and WT mice. Cantilever bend testing of the femora demonstrated significantly lower strains in the bones of Calbindin-D28k KO mice (4135 micro strain/kg +/- 1266) compared to WT mice (6973 micro strain/kg +/- 998) indicating that the KO mice had stiffer bones. Three-point bending demonstrated increased failure loads in bones of Calbindin-D28k KO mice (31.6 N +/- 2.1) compared to WT mice (15.0 N +/- 1.7). In conclusion, Calbindin-D28k KO mice had increased bone volume and stiffness indicating that Calbindin-D28k plays an important role in bone remodeling.

Top 10 Things Primary Care Physicians Should Know About Maintenance Immunosuppression for Transplant Recipients.

The success of organ transplantation allows many transplant recipients to return to life similar to nontransplant patients. Their need for regular health care, including preventive medicine, has switched the majority of responsibilities for their health care from transplant specialists to primary care physicians. To take care of transplant recipients, it is critical for primary care physicians to be familiar with immunosuppressive medications, their side effects, and common complications in transplant recipients. Ten subjects are reviewed here in order to assist primary care physicians in providing optimal care for transplant recipients.

Pharmacokinetics of single, oral-dose voriconazole in peritoneal dialysis patients.

BACKGROUND: Voriconazole is a new triazole antifungal drug. Its pharmacokinetics and transfer to peritoneal dialysate in peritoneal dialysis (PD) patients have not been studied. METHODS: Five patients with end-stage renal disease requiring support by PD were administered a single dose of 200 mg of voriconazole orally. Plasma and peritoneal dialysate were collected for measurement of voriconazole concentrations at times 1, 2, 4, and 24 hours. RESULTS: Voriconazole was absorbed and achieved maximum concentration (Cmax) in plasma at mean time 2.4 +/- 0.7 (SE) hours. Time to Cmax in dialysate was 2.8 +/- 0.5 hours. Mean Cmax for plasma was 0.55 +/- 0.20 microg/mL, and for dialysate, approximately half that of plasma (0.25 +/- 0.09 mug/mL). The dialysate to plasma ratio of voriconazole was 0.66 +/- 0.11. Less than 1% of the administered voriconazole dose (1.3 +/- 0.2 mg) was recovered in dialysate 24 hours after dosing. CONCLUSION: Voriconazole penetrates well into peritoneal fluid. There is minimal peritoneal clearance of voriconazole; therefore, no dosage adjustment is needed for patients on PD therapy.

Bilateral femoral head and distal tibial osteonecrosis in a patient with Fabry disease.

Fabry disease is a lysosomal storage disease caused by alpha-galactosidase A deficiency. The classic presentation of Fabry disease involves multiple organs, including kidneys, heart, skin, eyes, and nervous system. Osteonecrosis is rarely reported in patients with Fabry disease. In this article, we describe the case of a 37-year-old white man who had Fabry disease and no risk factors for osteonecrosis but who developed osteonecrosis in both femoral heads and in an unusual site, bilateral distal tibiae. Results of mutation analysis showed a nonsense mutation (R227X) in the alpha-galactosidase A gene. This case suggests that Fabry disease may be a risk factor for development of osteonecrosis. The enzyme replacement therapy currently available may be an effective method of preventing this complication.

Effect and Tolerability of Agalsidase Alfa in Patients with Fabry Disease Who Were Treatment Naïve or Formerly Treated with Agalsidase Beta or Agalsidase Alfa.

OBJECTIVES: In a multicenter, open-label, treatment protocol (HGT-REP-059; NCT01031173), clinical effects and tolerability of agalsidase alfa (agalα; 0.2 mg/kg every other week) were evaluated in patients with Fabry disease who were treatment naïve or switched from agalsidase beta (switch). Over 24 months, data were collected on the safety profile; renal and cardiac parameters were assessed using estimated glomerular filtration rate (eGFR), left ventricular mass index (LVMI), and midwall fractional shortening (MFS). RESULTS: Enrolled patients included 71 switch (median [range] age, 46.6 [5-84] years; male to female [M:F], 40:31) and 29 treatment naïve (38.7 [12-74] years; M:F, 14:15). Adverse events (AEs) were consistent with the known safety profile of agalα. Two switch patients had hospitalization due to possibly/probably drug-related serious AEs (one with transient ischemic attack, one with infusion-related AEs). One switch and two treatment-naïve patients discontinued treatment because of AEs. Three patients (one each switch, treatment naïve, and previous agalα) died; no deaths were considered drug-related. There was no significant change from baseline in LVMI or MFS in either group. Similarly, eGFR remained stable; mean ± standard error annualized change in eGFR (mL/min/1.73 m(2)) was -2.40 ± 1.04 in switch and -1.68 ± 2.21 in treatment-naïve patients. CONCLUSIONS: This is the largest cohort of patients with Fabry disease who were started on or switched to agalα in an FDA-accepted protocol during a worldwide supply shortage of agalsidase beta. Because this protocol was primarily designed to provide access to agalα, there were limitations, including not having stringent selection criteria and the lack of a placebo group.

Chimeric RNA/DNA oligonucleotide-based gene therapy.

BACKGROUND: Chimeric RNA/DNA oligonucleotides, emerging as a potential strategy for gene therapy, have been shown to induce site-specific correction of point mutations in several genetic disease models. METHODS: Six recent studies of chimeric RNA/DNA oligonucleotide-based gene therapy in genetic disease models are reviewed. Chimeric RNA/DNA oligonucleotides, complementary to 25 to 30 residues of genomic DNA flanking the mutation site with the exception of a mismatch in the center, were delivered via different routes and delivery vehicles to target different tissues and organs. Corrections of the mutation at genotypic and phenotypic levels were assessed using various methods, including allele-specific polymerase chain reaction assay, restriction enzyme digestion, colony-lifting assays, sequencing, Northern and Western blot analyses, enzyme activity assay, immunohistochemical staining, and functional studies. RESULTS: The gene correction frequency varied, ranging from less than 1% to more than 40%. This represented several magnitudes higher conversion rate compared with homologous recombination frequency, which is in the range of 10(-5) to 10(-6). The resulting phenotype changes lasted longer than one year in some studies. CONCLUSION: Chimeric RNA/DNA oligonucleotide-based gene therapy has the potential to develop into powerful therapeutic modality for genetic diseases. It can offer permanent expression and normal regulation of corrected genes in appropriate cells or tissues. Further efforts to elucidate the mechanisms of chimeric RNA/DNA oligonucleotide-based gene therapy are warranted in order to increase the efficacy and safety of this method.

Multicentric papillary renal carcinoma in renal allograft.

A renal transplant recipient with 13 years of excellent allograft function was found incidentally to have a malignant mass in his transplanted kidney. After resection, pathological analysis showed 29 separate lesions of renal cell carcinoma. All tumors were confined within the renal capsule. The majority of tumors (21 of 29 tumors) were chromophil basophilic carcinoma with papillary architecture, 5 tumors were clear cell, 2 tumors were mixed cell type, and 1 tumor was chromophil eosinophilic papillary carcinoma. These histological findings are similar to those reported in hereditary papillary renal carcinoma. To our knowledge, this is the first case of multicentric papillary renal carcinoma occurring in the renal allograft. We speculate that the allograft in this case is predisposed to malignant changes because of preexisting genetic mutations, as well as prolonged immunosuppression.

A novel alpha-galactosidase a mutant (M42L) identified in a renal variant of Fabry disease.

A 65-year-old man presented to our institution for workup of proteinuria. His serum creatinine level was 1.7 mg/dL (130 micromol/L), and he had proteinuria with protein of almost 5 g/24 h. Fabry disease was diagnosed by means of kidney biopsy and low serum and leukocyte levels of alpha-galactosidase A. Review of his history, family history, physical examinations, and diagnostic studies did not show other findings typical of this disease. His renal function continued to decline, and he eventually underwent a living unrelated renal transplantation 5 years later. Three years after transplantation, his creatinine level is 1.7 mg/dL (130 micromol/L), and corrected iothalamate clearance is 53 mL/min/1.73 m2 . Genetic studies showed that he has a novel missense mutation (M42L) in exon 1. Methionine at codon 42 is highly conserved in eukaryotic alpha-galactosidase A orthologues. This genotype predicts a minor misfolding of alpha-galactosidase A because of a small difference in hydrophobicity between methionine and leucine. His mutation resulted in a very low, but detectable, serum level of alpha-galactosidase A (0.002 U/L; normal range, 0.016 to 0.2 U/L). Cases of Fabry disease that present with predominantly renal manifestations are rare and require a high index of suspicion for diagnosis. Because treatment for Fabry disease recently has become available, it is important for clinicians to be aware of this disease and pursue the diagnosis in cases of otherwise unexplained renal dysfunction.

Gene therapy for renal disorders.

During the last 20 years there have been major improvements in renal replacement therapy, including dialysis and kidney transplantation; however, the treatment options for renal diseases are still limited. Gene therapy is a potential modality for many renal diseases for which we are as yet unable to offer specific treatment. This article reviews the recent data on gene therapy in animal models applicable to human renal diseases and evaluates its efficacy, safety and clinical relevance. Several approaches appear to be promising, including adeno-associated viral vectors for long-term gene expression, electroporation for muscular gene delivery, ultrasound/microbubble-mediated gene targeting, macrophage-based gene therapy and small interfering RNAs.