R. Todd Alexander

Kidney stones and kidney function loss: a cohort study

Objective To investigate whether the presence of kidney stones increase the risk of end stage renal disease (ESRD) or other adverse renal outcomes. Design A registry cohort study using validated algorithms based on claims and facility utilisation data. Median follow-up of 11 years. Setting Alberta, Canada, between 1997 and 2009. Participants 3 089 194 adult patients without ESRD at baseline or a history of pyelonephritis. Of these, 1 954 836 had outpatient serum creatinine measurements and were included in analyses of chronic kidney disease and doubling of serum creatinine level. Exposure One or more kidney stones during follow-up. Main outcome measures Incident ESRD, development of stage 3b–5 chronic kidney disease (estimated glomerular filtration rate <45 mL/min/1.73 m2), and sustained doubling of serum creatinine concentration from baseline. Results 23 706 (0.8%) patients had at least one kidney stone, 5333 (0.2%) developed ESRD, 68 525 (4%) developed stage 3b–5 chronic kidney disease, and 6581 (0.3%) experienced sustained doubling of serum creatinine. Overall, one or more stone episodes during follow-up was associated with increased risk of ESRD (adjusted hazard ratio 2.16 (95% CI 1.79 to 2.62)), new stage 3b–5 chronic kidney disease (hazard ratio 1.74 (1.61 to 1.88)), and doubling of serum creatinine (hazard ratio 1.94 (1.56 to 2.43)), all compared with those without kidney stones during follow-up. The excess risk of adverse outcomes associated with at least one episode of stones seemed greater in women than in men, and in people aged <50 years than in those aged ≥50. However, the risks of all three adverse outcomes in those with at least one episode of stones were significantly higher than in those without stones in both sexes and age strata. The absolute increase in the rate of adverse renal outcomes associated with stones was small: the unadjusted rate of ESRD was 2.48 per million person days in people with one or more episodes of stones versus 0.52 per million person days in people without stones. Conclusion Even a single kidney stone episode during follow-up was associated with a significant increase in the likelihood of adverse renal outcomes including ESRD. However, the increases were small in absolute terms.

Traditional and emerging roles for the SLC9 Na(+)/H (+) exchangers

The SLC9 gene family encodes Na+/H+ exchangers (NHEs). These transmembrane proteins transport ions across lipid bilayers in a diverse array of species from prokaryotes to eukaryotes, including plants, fungi, and animals. They utilize the electrochemical gradient of one ion to transport another ion against its electrochemical gradient. Currently, 13 evolutionarily conserved NHE isoforms are known in mammals [22, 46, 128]. The SLC9 gene family (solute carrier classification of transporters: www.bioparadigms.org) is divided into three subgroups [46]. The SLC9A subgroup encompasses plasmalemmal isoforms NHE1-5 (SLC9A1-5) and the predominantly intracellular isoforms NHE6-9 (SLC9A6-9). The SLC9B subgroup consists of two recently cloned isoforms, NHA1 and NHA2 (SLC9B1 and SLC9B2, respectively). The SLC9C subgroup consist of a sperm specific plasmalemmal NHE (SLC9C1) and a putative NHE, SLC9C2, for which there is currently no functional data [46]. NHEs participate in the regulation of cytosolic and organellar pH as well as cell volume. In the intestine and kidney, NHEs are critical for transepithelial movement of Na+ and HCO3− and thus for whole body volume and acid–base homeostasis [46]. Mutations in the NHE6 or NHE9 genes cause neurological disease in humans and are currently the only NHEs directly linked to human disease. However, it is becoming increasingly apparent that members of this gene family contribute to the pathophysiology of multiple human diseases.

Activation of the Ca2+-sensing receptor increases renal claudin-14 expression and urinary Ca2+ excretion

Kidney stones are a prevalent clinical condition imposing a large economic burden on the healthcare system. Hypercalciuria remains the major risk factor for development of a Ca(2+)-containing stone. The kidneys ability to alter Ca(2+) excretion in response to changes in serum Ca(2+) is in part mediated by the Ca(2+)-sensing receptor (CaSR). Recent studies revealed renal claudin-14 (Cldn14) expression localized to the thick ascending limb (TAL) and its expression to be regulated via the CaSR. We find that Cldn14 expression is increased by high dietary Ca(2+) intake and by elevated serum Ca(2+) levels induced by prolonged 1,25-dihydroxyvitamin D3 administration. Consistent with this, activation of the CaSR in vivo via administration of the calcimimetic cinacalcet hydrochloride led to a 40-fold increase in Cldn14 mRNA. Moreover, overexpression of Cldn14 in two separate cell culture models decreased paracellular Ca(2+) flux by preferentially decreasing cation permeability, thereby increasing transepithelial resistance. These data support the existence of a mechanism whereby activation of the CaSR in the TAL increases Cldn14 expression, which in turn blocks the paracellular reabsorption of Ca(2+). This molecular mechanism likely facilitates renal Ca(2+) losses in response to elevated serum Ca(2+). Moreover, dysregulation of the newly described CaSR-Cldn14 axis likely contributes to the development of hypercalciuria and kidney stones.

Kidney Stones and Cardiovascular Events: A Cohort Study

BACKGROUND AND OBJECTIVES Kidney stones are common in general clinical practice, and their prevalence is increasing. Kidney stone formers often have risk factors associated with atherosclerosis, but it is uncertain whether having a kidney stone is associated with higher risk of cardiovascular events. This study sought to assess the association between one or more kidney stones and the subsequent risk of cardiovascular events. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Cohort study of 3,195,452 people aged≥18 years registered in the universal health care system in Alberta, Canada, between 1997 and 2009 (median follow-up of 11 years). People undergoing dialysis or with a kidney transplant at baseline were excluded. The primary outcome was the first acute myocardial infarction (AMI) during follow-up. We also considered other cardiovascular events, including death due to coronary heart disease, percutaneous transluminal coronary angioplasty (PTCA), coronary artery bypass grafting (CABG), and stroke. RESULTS In total, 25,532 (0.8%) participants had at least one kidney stone, and 91,465 (3%) individuals had at least one cardiovascular event during follow-up. Compared with people without kidney stones and after adjustment for cardiovascular risk factors and other potential confounders, people who had at least one kidney stone had a higher risk of subsequent AMI (adjusted hazard ratio [HR], 1.40; 95% confidence interval [95% CI], 1.30 to 1.51), PTCA/CABG (HR, 1.63; 95% CI, 1.51 to 1.76), and stroke (HR, 1.26; 95% CI, 1.12 to 1.42). The magnitude of the excess risk associated with a kidney stone appeared more pronounced for younger people than for older people (P<0.001) and for women than men (P=0.01). CONCLUSIONS The occurrence of a kidney stone is associated with a higher risk of cardiovascular events, including AMI, PTCA/CABG, and stroke.

Survival in pediatric dialysis and transplant patients.

BACKGROUND AND OBJECTIVES Long-term follow-up data are few in children with ESRD. We sought to describe long-term survival, assess risk factors for death, and compare survival between two time periods in pediatric ESRD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We used a population-based retrospective cohort utilizing data from a national organ failure registry and from Canadas universal healthcare system. We included 843 children (ages, 0 to 18) initiating renal replacement therapy from 1992 to 2007 and followed them until death or date of last contact (median follow-up, 6.8 years; interquartile range, 3.0 to 10.6). We assessed risk factors for death and examined cause-specific mortality. RESULTS During 5991 patient-years of follow-up, 107 (12.7%) patients died. Unadjusted cumulative survival for the cohort was: 91.7% (95% CI, 89.8 to 93.7%) at 5 years and 85.8% (95% CI, 82.8 to 88.8%) at 10 years. Among patients commencing dialysis, overall adjusted survival was poorest among those who started dialysis at age <1 year. No secular trends in survival were noted for either dialysis or transplant patients. The proportion of incident patients receiving pre-emptive transplantation increased over time. Pre-emptively transplanted patients did not demonstrate superior adjusted survival compared with those who spent >2 years on dialysis before transplant (hazard ratio, 1.53; 95% CI, 0.63 to 3.67). CONCLUSIONS No significant improvements in survival were observed among ESRD patients over the study period. Time with transplant function had the strongest association with survival. Pre-emptive transplantation was not associated with improved survival in adjusted models.

Graft failure and adaptation period to adult healthcare centers in pediatric renal transplant patients.

Background. Transfer from pediatric to adult care may require a period of adaptation to the new healthcare environment. We sought to determine whether this adaptation period was associated with an increased risk of graft failure. Methods. Children (age, 0–18 years) recorded in the Canadian Organ Replacement Register who received a first kidney transplant in a pediatric health center between 1992 and 2007, and who had more than or equal to 3 months of graft function, were followed up until death, loss to follow-up, or December 31, 2007. Cox proportional hazards models were used to estimate the excess risk associated with a period of adaptation to adult-oriented care, defined as the interval 0.5 years before to 2.5 years after the first recorded adult care visit. Models were adjusted for age, gender, donor source, and ethnicity. Results. Of the 413 patients evaluated, 149 were transferred to adult care during study period. In total, 78 (18.9%) patients experienced graft failure—23 during the adaptation period. Compared with the period before adaptation, the adjusted hazard ratio for graft loss within the adaptation period was 2.24 (95% confidence interval [CI]: 1.19–4.20). The adjusted graft failure rate was 2.26 (1.04–4.93) times higher after 18 years of age than between 0 and 13 years. Aboriginal ethnicity and deceased donor source were also associated with a significantly higher risk of graft failure. Conclusions. The period of adaptation to adult-oriented care is associated with a high risk of graft failure in pediatric renal transplant patients.

Membrane Curvature Alters the Activation Kinetics of the Epithelial Na+/H+ Exchanger, NHE3

The epithelial Na+/H+ exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them.

Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System

ATPase H+-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H+-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na+-K+-2Cl- cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity.

Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells

The chemokine CX3CL1 plays a key role in glomerulonephritis and can act as both chemoattractant and adhesion molecule. CX3CL1 also is upregulated in tubulointerstitial injury, but little is known about the subcellular distribution and function of CX3CL1 in renal tubular epithelial cells (RTEC). Unexpectedly, it was found that CX3CL1 is expressed predominantly on the apical surface of tubular epithelium in human renal transplant biopsy specimens with acute rejection or acute tubular necrosis. For studying the targeting of CX3CL1 in polarized RTEC, MDCK cells that expressed untagged or green fluorescent protein-tagged CX3CL1 were generated. The chemokine was present on the apical membrane and in subapical vesicles. Apical targeting of CX3CL1 was not due to signals that were conferred by its intracellular domain, to associations with lipid rafts, or to O-glycosylation but, rather, depended on N-linked glycosylation of the protein. With the use of fluorescence recovery after photobleaching, it was found that CX3CL1 is immobile in the apical membrane. However, CX3CL1 partitioned with the triton-soluble rather than -insoluble cellular fraction, indicating that it is not associated directly with the actin cytoskeleton or with lipid rafts. Accordingly, disruption of rafts through cholesterol depletion did not render CX3CL1 mobile. For exploration of potential functions of apical CX3CL1, binding of CX3CR1-expressing leukocytes to polarized RTEC was examined. Leukocyte adhesion to the luminal surface was enhanced significantly when CX3CL1 was present. These data demonstrate that CX3CL1 is expressed preferentially on the apical membrane of RTEC and suggest a novel function for the chemokine in recruitment and retention of leukocytes in tubulointerstitial inflammation.

The tumor suppressor gene, RASSF1A, is essential for protection against inflammation -induced injury.

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a+/−, Rassf1a−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.