Henry Quiñones

Klotho Deficiency Causes Vascular Calcification in Chronic Kidney Disease

Soft-tissue calcification is a prominent feature in both chronic kidney disease (CKD) and experimental Klotho deficiency, but whether Klotho deficiency is responsible for the calcification in CKD is unknown. Here, wild-type mice with CKD had very low renal, plasma, and urinary levels of Klotho. In humans, we observed a graded reduction in urinary Klotho starting at an early stage of CKD and progressing with loss of renal function. Despite induction of CKD, transgenic mice that overexpressed Klotho had preserved levels of Klotho, enhanced phosphaturia, better renal function, and much less calcification compared with wild-type mice with CKD. Conversely, Klotho-haploinsufficient mice with CKD had undetectable levels of Klotho, worse renal function, and severe calcification. The beneficial effect of Klotho on vascular calcification was a result of more than its effect on renal function and phosphatemia, suggesting a direct effect of Klotho on the vasculature. In vitro, Klotho suppressed Na(+)-dependent uptake of phosphate and mineralization induced by high phosphate and preserved differentiation in vascular smooth muscle cells. In summary, Klotho is an early biomarker for CKD, and Klotho deficiency contributes to soft-tissue calcification in CKD. Klotho ameliorates vascular calcification by enhancing phosphaturia, preserving glomerular filtration, and directly inhibiting phosphate uptake by vascular smooth muscle. Replacement of Klotho may have therapeutic potential for CKD.

Klotho deficiency is an early biomarker of renal ischemia–reperfusion injury and its replacement is protective

Klotho is an antiaging substance with pleiotropic actions including regulation of mineral metabolism. It is highly expressed in the kidney and is present in the circulation and urine but its role in acute kidney injury (AKI) is unknown. We found that ischemia-reperfusion injury (IRI) in rodents reduced Klotho in the kidneys, urine, and blood, all of which were restored upon recovery. Reduction in kidney and plasma Klotho levels were earlier than that of neutrophil gelatinase-associated lipocalin (NGAL), a known biomarker of kidney injury. Patients with AKI were found to have drastic reductions in urinary Klotho. To examine whether Klotho has a pathogenic role, we induced IRI in mice with different endogenous Klotho levels ranging from heterozygous Klotho haploinsufficient, to wild-type (WT), to transgenic mice overexpressing Klotho. Klotho levels in AKI were lower in haploinsufficient and higher in transgenic compared with WT mice. The haploinsufficient mice had more extensive functional and histological alterations compared with WT mice, whereas these changes were milder in overexpressing transgenic mice, implying that Klotho is renoprotective. Rats with AKI given recombinant Klotho had higher Klotho protein, less kidney damage, and lower NGAL than rats with AKI given vehicle. Hence, AKI is a state of acute reversible Klotho deficiency, low Klotho exacerbates kidney injury and its restoration attenuates renal damage and promotes recovery from AKI. Thus, endogenous Klotho not only serves as an early biomarker for AKI but also functions as a renoprotective factor with therapeutic potential.

Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors Is Mediated by Calcineurin Homologous Protein

Adenosine is an autacoid that regulates renal Na+ transport. Activation of adenosine A1 receptor (A1R) by N6-cyclopentidyladenosine (CPA) inhibits the Na+/H+ exchanger 3 (NHE3) via phospholipase C/Ca2+/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A1R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10-6 M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3C terminus; prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3C terminus (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3Δ552) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3462-552) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3Full-length interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A1R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.

The demonstration of αKlotho deficiency in human chronic kidney disease with a novel synthetic antibody

BACKGROUND αKlotho is the prototypic member of the Klotho family and is most highly expressed in the kidney. αKlotho has pleiotropic biologic effects, and in the kidney, its actions include regulation of ion transport, cytoprotection, anti-oxidation and anti-fibrosis. In rodent models of chronic kidney disease (CKD), αKlotho deficiency has been shown to be an early biomarker as well as a pathogenic factor. The database for αKlotho in human CKD remains controversial even after years of study. METHODS We used a synthetic antibody library to identify a high-affinity human antigen-binding fragment that recognizes human, rat and mouse αKlotho primarily in its native, rather than denatured, form. RESULTS Using an immunoprecipitation-immunoblot (IP-IB) assay, we measured both serum and urinary levels of full-length soluble αKlotho in humans and established that human CKD is associated with αKlotho deficiency in serum and urine. αKlotho levels were detectably lower in early CKD preceding disturbances in other parameters of mineral metabolism and progressively declined with CKD stages. We also found that exogenously added αKlotho is inherently unstable in the CKD milieu suggesting that decreased production may not be the sole reason for αKlotho deficiency. CONCLUSION Synthetic antibody libraries harbor tremendous potential for a variety of biomedical and clinical applications. Using such a reagent, we furnish data in support of αKlotho deficiency in human CKD, and we set the foundation for the development of diagnostic and therapeutic applications of anti-αKlotho antibodies.

Acute regulation of renal Na+/H+ exchanger NHE3 by dopamine: role of protein phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56δ (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, coimmunoprecipitation, blot overlay, and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small T antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition.

Catheter-Related Bloodstream Infections in Patients on Emergent Hemodialysis.

OBJECTIVE This study had 2 objectives: (1) to describe the epidemiology of catheter-related bloodstream infections (CRBSI) in patients with end-stage renal disease (ESRD) who have no access to scheduled dialysis and (2) to evaluate whether a positive culture of the heparin-lock solution is associated with subsequent development of bacteremia. DESIGN Retrospective observational cohort design for objective 1; and prospective cohort design for objective 2. SETTING AND PARTICIPANTS The study was conducted in a 770-bed public academic tertiary hospital in Dallas, Texas. The participants were patients with ESRD undergoing scheduled or emergent hemodialysis. METHODS We reviewed the records of 147 patients who received hemodialysis between January 2011 and May 2011 and evaluated the rate of CRBSI in the previous 5 years. For the prospective study, we cultured the catheter heparin-lock solution in 62 consecutive patients between June 2012 and August 2012 and evaluated the incidence of CRBSI at 6 months. RESULTS Of the 147 patients on emergent hemodialysis, 125 had a tunneled catheter, with a CRBSI rate of 2.61 per 1,000 catheter days. The predominant organisms were Gram-negative rods (GNR). In the prospective study, we found that the dialysis catheter was colonized more frequently in patients on emergent hemodialysis than in those on scheduled hemodialysis. Colonization with GNR or Staphylococcus aureus was associated with subsequent CRBSI at 6 months follow-up. CONCLUSIONS Patients undergoing emergent hemodialysis via tunneled catheter are predisposed to Gram-negative CRBSI. Culturing the heparin-lock solution may predict subsequent episodes of CRBSI if it shows colonization with GNR or Staphylococcus aureus. Prevention approaches in this population need to be studied further.

CKD and Cardiovascular Events: Unraveling the Disparities Among Minorities

Chronic kidney disease (CKD) is a national public health issue that has been well recognized as an important cause of premature morbidity and mortality. The prevalence of CKD in the United States increased from 10% in 1988-1994 to 13% in 19992004; even so, the general public’s awareness of kidney disease continues to be very low. By 2030, the burden of kidney disease is expected to increase further and it has been estimated that approximately 2 million individuals will then require dialysis. Racial/ethnic disparities in advanced CKD have been well described, with rates of end-stage renal disease (ESRD) among minorities ranging from 1.4 to 3.0 times higher compared with non-Hispanic whites. Despite the higher prevalence of cardiovascular (CV) risk factors, barriers to health care, and lower socioeconomic status among minorities, previous studies have shown that as dialysis patients, they experience a survival advantage compared with non-Hispanic whites, leading researchers to conclude that a “survival paradox” potentially exists. However, this association between earlier stages of CKD and CV outcomes remains elusive, reinforcing the need for prospective longitudinal studies in multiethnic cohorts. In this issue of AJKD, Lash et al analyzed prospectively collected data from the Chronic Renal Insufficiency Cohort (CRIC) and Hispanic CRIC (HCRIC) studies to examine racial/ethnic differences in CV outcomes (atherosclerotic events, heart failure, and all-cause death) among 3,785 adults aged 21 to 74 years with mild to moderate CKD (estimated glomerular filtration rate [eGFR], 20-70 mL/min/ 1.73 m) recruited from 7 clinical centers in the United States from 2003 to 2007. After a mean follow-up of 6.6 years, no significant racial or ethnic differences were observed when assessing atherosclerotic events (myocardial infarction, stroke, or peripheral vascular disease) and heart failure compared with non-Hispanic whites. No association was found even after adjusting for numerous covariates, including sociodemographic characteristics, baseline kidney function, proteinuria,

Dopamine reduces cell surface Na+/H+ exchanger-3 protein by decreasing NHE3 exocytosis and cell membrane recycling

The intrarenal autocrine-paracrine dopamine (DA) system mediates a significant fraction of the natriuresis in response to a salt load. DA inhibits a number of Na+ transporters to effect sodium excretion, including the proximal tubule Na+/H+ exchanger-3 (NHE3). DA represent a single hormone that regulates NHE3 at multiple levels, including translation, degradation, endocytosis, and protein phosphorylation. Because cell surface NHE3 protein is determined by the balance between exocytotic insertion and endocytotic retrieval, we examined whether DA acutely affects the rate of NHE3 exocytosis in a cell culture model. DA inhibited NHE3 exocytosis at a dose-dependent manner with a half maximal around 10-6 M. The DA effect on NHE3 exocytosis was blocked by inhibition of protein kinase A and by brefeldin A, which inhibits endoplasmic reticulum-to-Golgi transport. NHE3 directly interacts with the ε-subunit of coatomer protein based on yeast-two-hybrid and coimmunoprecipitation. Because NHE3 has been shown to be recycled back to the cell membrane after endocytosis, we measured NHE3 recycling using a biochemical reinsertion assay and showed that reinsertion of NHE3 back to the membrane is also inhibited by DA. In conclusion, among the many mechanisms by which DA reduces apical membrane NHE3 and induces proximal tubule natriuresis, one additional mechanism is inhibition of exocytotic insertion and reinsertion of NHE3 in the apical cell surface.