Martin H. de Borst

Cross Talk Between the Renin-Angiotensin-Aldosterone System and Vitamin D-FGF-23-klotho in Chronic Kidney Disease

There is increasingly evidence that the interactions between vitamin D, fibroblast growth factor 23 (FGF-23), and klotho form an endocrine axis for calcium and phosphate metabolism, and derangement of this axis contributes to the progression of renal disease. Several recent studies also demonstrate negative regulation of the renin gene by vitamin D. In chronic kidney disease (CKD), low levels of calcitriol, due to the loss of 1-alpha hydroxylase, increase renal renin production. Activation of the renin-angiotensin-aldosterone system (RAAS), in turn, reduces renal expression of klotho, a crucial factor for proper FGF-23 signaling. The resulting high FGF-23 levels suppress 1-alpha hydroxylase, further lowering calcitriol. This feedback loop results in vitamin D deficiency, RAAS activation, high FGF-23 levels, and renal klotho deficiency, all of which associate with progression of renal damage. Here we examine current evidence for an interaction between the RAAS and the vitamin D-FGF-23-klotho axis as well as its possible implications for progression of CKD.

Active Vitamin D Treatment for Reduction of Residual Proteinuria: A Systematic Review

Despite renin-angiotensin-aldosterone system blockade, which retards progression of CKD by reducing proteinuria, many patients with CKD have residual proteinuria, an independent risk factor for disease progression. We aimed to address whether active vitamin D analogs reduce residual proteinuria. We systematically searched for trials published between 1950 and September of 2012 in the Medline, Embase, and Cochrane Library databases. All randomized controlled trials of vitamin D analogs in patients with CKD that reported an effect on proteinuria with sample size≥50 were selected. Mean differences of proteinuria change over time and odds ratios for reaching ≥15% proteinuria decrease from baseline to last measurement were synthesized under a random effects model. From 907 citations retrieved, six studies (four studies with paricalcitol and two studies with calcitriol) providing data for 688 patients were included in the meta-analysis. Most patients (84%) used an angiotensin-converting enzyme inhibitor or angiotensin receptor blocker throughout the study. Active vitamin D analogs reduced proteinuria (weighted mean difference from baseline to last measurement was -16% [95% CI, -13% to -18%]) compared with controls (+6% [95% CI, 0% to +12%]; P<0.001). Proteinuria reduction was achieved more commonly in patients treated with an active vitamin D analog (204/390 patients) than control patients (86/298 patients; OR, 2.72 [95% CI, 1.82 to 4.07]; P<0.001). Thus, active vitamin D analogs may further reduce proteinuria in CKD patients in addition to current regimens. Future studies should address whether vitamin D therapy also retards progressive renal functional decline.

Possible renoprotection by vitamin D in chronic renal disease: beyond mineral metabolism

Vitamin D is typically viewed as a key player in the regulation of calcium and phosphate levels and the control of bone metabolism; however, growing evidence suggests that vitamin D deficiency may also have an important role in the progressive loss of renal function. Vitamin D deficiency is particularly frequent in patients with chronic kidney disease, in whom it is associated with increased mortality. Studies indicate that treatment with vitamin D analogues reduces proteinuria, suppresses the renin–angiotensin–aldosterone system (RAAS), and exerts anti-inflammatory and immunomodulatory effects. These pleiotropic effects render vitamin D a potentially interesting treatment modality for renoprotection in patients with chronic kidney disease. Whether vitamin D has clinically relevant renoprotective effects in addition to RAAS blockade is currently under investigation.

Inhibition of Renal Rho Kinase Attenuates Ischemia/Reperfusion-Induced Injury

The Rho kinase pathway plays an important role in dedifferentiation of epithelial cells and infiltration of inflammatory cells. For testing of the hypothesis that blockade of this cascade within the kidneys might be beneficial in the treatment of renal injury the Rho kinase inhibitor, Y27632 was coupled to lysozyme, a low molecular weight protein that is filtered through the glomerulus and is reabsorbed in proximal tubular cells. Pharmacokinetic studies with Y27632-lysozyme confirmed that the conjugate rapidly and extensively accumulated in the kidney. Treatment with Y27632-lysozyme substantially inhibited ischemia/reperfusion-induced tubular damage, indicated by reduced staining of the dedifferentiation markers kidney injury molecule 1 and vimentin, and increased E-cadherin relative to controls. Rho kinase activation was inhibited by Y27632-lysozyme within tubular cells and the interstitium. Y27632-lysozyme also inhibited inflammation and fibrogenesis, indicated by a reduction in gene expression of monocyte chemoattractant protein 1, procollagen Ialpha1, TGF-beta1, tissue inhibitor of metalloproteinase 1, and alpha-smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of alpha-smooth muscle actin, collagen I, collagen III, and fibronectin. In contrast, unconjugated Y27632 did not have these beneficial effects but instead caused systemic adverse effects, such as leukopenia. Neither treatment improved renal function in the bilateral ischemia/reperfusion model. In conclusion, the renally targeted Y27632-lysozyme conjugate strongly inhibits tubular damage, inflammation, and fibrogenesis induced by ischemia/reperfusion injury.

Fibroblast Growth Factor 23 and Cardiovascular Mortality after Kidney Transplantation

BACKGROUND AND OBJECTIVES Circulating fibroblast growth factor 23 (FGF23) is associated with adverse cardiovascular outcomes in CKD. Whether FGF23 predicts cardiovascular mortality after kidney transplantation, independent of measures of mineral metabolism and cardiovascular risk factors, is unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The association between plasma C-terminal FGF23 and cardiovascular mortality was analyzed in a single-center prospective cohort of 593 stable kidney transplant recipients (mean age ± SD, 52 ± 12 years; 54% male; estimated GFR, 47 ± 16 ml/min per 1.73 m(2)), at a median of 6.1 (interquartile range, 2.7-11.7) years after transplantation. Multivariate Cox regression models were built, adjusting for measures of renal function and mineral metabolism; Framingham risk factors; the left ventricular wall strain markers midregional fragment of pro-A-type natriuretic peptide (MR-proANP) and N-terminal-pro brain natriuretic peptide (NT-proBNP); and copeptin, the stable C-terminal portion of the precursor of vasopressin. RESULTS In multivariate linear regression analysis, MR-proANP (β=0.20, P<0.001), NT-proBNP (β=0.18, P<0.001), and copeptin (β=0.26, P<0.001) were independently associated with FGF23. During follow-up for 7.0 (interquartile range, 6.2-7.5) years, 128 patients (22%) died, of whom 66 (11%) died due to cardiovascular disease; 54 (9%) had graft failure. FGF23 was associated with an higher risk of cardiovascular mortality in a fully adjusted multivariate Cox regression model (hazard ratio [HR], 1.88 [95% confidence interval (CI), 1.11 to 3.19]; P=0.02). FGF23 was also independently associated with all-cause mortality (full model HR, 1.86 [95% CI, 1.27 to 2.73]; P=0.001). Net reclassification improved for both cardiovascular mortality (HR, 0.07 [95% CI, 0.01 to 0.14]; P<0.05) and all-cause mortality (HR, 0.11 [95% CI, 0.05 to 0.18]; P<0.001). CONCLUSIONS Plasma FGF23 is independently associated with cardiovascular and all-cause mortality after kidney transplantation. The association remained significant after adjustment for measures of mineral metabolism and cardiovascular risk factors.

Intracellular delivery of the p38 mitogen-activated protein kinase inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] in renal tubular cells : A novel strategy to treat renal fibrosis

During renal injury, activation of p38 mitogen-activated protein kinase (MAPK) in proximal tubular cells plays an important role in the inflammatory events that eventually lead to renal fibrosis. We hypothesized that local inhibition of p38 within these cells may be an interesting approach for the treatment of renal fibrosis. To effectuate this, we developed a renal-specific conjugate of the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] and the carrier lysozyme. First, we demonstrated that SB202190 inhibited the expression of albumin-induced proinflammatory (monocyte chemoattractant protein-1) and transforming growth factor (TGF)-β1-induced profibrotic (procollagen-Iα1) genes over 50% in renal tubular cells (normal rat kidney-52E). Next, we conjugated SB202190 via a carbamate linkage to lysozyme. However, this conjugate rapidly released the drug upon incubation in serum. Therefore, we applied a new platinum(II)-based linker approach, the so-called universal linkage system (ULS), which forms a coordinative bond with SB202190. The SB202190-ULS-lysozyme remained stable in serum but released the drug in kidney homogenates. SB202190-ULS-lysozyme accumulated efficiently in renal tubular cells and provided a local drug reservoir during a period of 3 days after a single intravenous injection. Treatment with SB202190-ULS-lysozyme inhibited TGF-β1-induced gene expression for procollagen-Iα1 by 64% in HK-2 cells. Lastly, we evaluated the efficacy of a single dose of the conjugate in the unilateral renal ischemia-reperfusion rat model. A reduction of intrarenal p38 phosphorylation and α-smooth muscle actin protein expression was observed 4 days after the ischemia-reperfusion injury. In conclusion, we have developed a novel strategy for local delivery of the p38 MAPK inhibitor SB202190, which may be of use in the treatment of renal fibrosis.

Calcification Propensity and Survival among Renal Transplant Recipients

Calciprotein particle maturation time (T50) in serum is a novel measure of individual blood calcification propensity. To determine the clinical relevance of T50 in renal transplantation, baseline serum T50 was measured in a longitudinal cohort of 699 stable renal transplant recipients and the associations of T50 with mortality and graft failure were analyzed over a median follow-up of 3.1 years. Predictive value of T50 was assessed for patient survival with reference to traditional (Framingham) risk factors and the calcium-phosphate product. Serum magnesium, bicarbonate, albumin, and phosphate levels were the main determinants of T50, which was independent of renal function and dialysis vintage before transplant. During follow-up, 81 (12%) patients died, of which 38 (47%) died from cardiovascular causes. Furthermore, 45 (6%) patients developed graft failure. In fully adjusted models, lower T50 values were independently associated with increased all-cause mortality (hazard ratio, 1.43; 95% confidence interval, 1.11 to 1.85; P=0.006 per SD decrease) and increased cardiovascular mortality (hazard ratio, 1.55; 95% confidence interval, 1.04 to 2.29; P=0.03 per SD decrease). In addition to age, sex, and eGFR, T50 improved prognostication for all-cause mortality, whereas traditional risk factors or calcium-phosphate product did not. Lower T50 was also associated with increased graft failure risk. The associations of T50 with mortality and graft failure were confirmed in an independent replication cohort. In conclusion, reduced serum T50 was associated with increased risk of all-cause mortality, cardiovascular mortality, and graft failure and, of all tested parameters, displayed the strongest association with all-cause mortality in these transplant recipients.

Antiproteinuric treatment reduces urinary loss of vitamin D-binding protein but does not affect vitamin D status in patients with chronic kidney disease

Vitamin D deficiency is common in chronic kidney disease (CKD). Increased urinary loss of vitamin D binding protein (VDBP), the main transporter of 25-hydroxyvitamin D(3) in the circulation, has been postulated to contribute to vitamin D deficiency in proteinuria. To test this hypothesis we analyzed urinary and plasma levels of VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) from proteinuric patients, before and after antiproteinuric interventions. We performed a post-hoc analysis of a clinical trial in CKD patients (n=13, creatinine clearance median 60 (range 25-177) ml/min) subjected to the following study periods: washout (no antiproteinuric treatment, 4 weeks), lisinopril 40mg QD (ACEi, 6 weeks), or indomethacin 75mg BID (NSAID, 4 weeks) in randomized sequence. Healthy subjects screened for donation (n=10) served as controls. Plasma and urine VDBP levels were measured by ELISA, 25-hydroxyvitamin D(3) levels by LC-MS and 1,25-dihydroxyvitamin D(3) levels by radioimmunoassay. In CKD patients urinary VDBP excretion was strongly increased (median (range) 5413 (155-211,027) μg/24h) as compared to healthy controls (64 (23-111) μg/24h, p<0.001). Both NSAID and ACEi significantly decreased urinary VDBP excretion, in proportion to proteinuria reduction. Plasma VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) levels, however, were similar between patients and controls and not affected by antiproteinuric intervention. Urinary VDBP excretion is markedly increased in proteinuria and responds to antiproteinuric treatment. Urinary VDBP loss is not associated with plasma VDBP or vitamin D(3) levels, suggesting that urinary loss of VDBP does not affect vitamin D status.

c-Jun NH2-terminal kinase is crucially involved in renal tubulo-interstitial inflammation.

Chronic inflammation is a major outcome determinant in several renal disorders. Induction of monocyte chemoattractant protein (MCP)-1 expression in tubular epithelial cells contributes importantly to the recruitment of inflammatory cells from the circulation toward the damaged tubulo-interstitium. Because the MCP-1 gene contains several c-Jun binding sites, we hypothesized that the c-Jun NH2-terminal kinase (JNK) pathway regulates MCP-1 expression and subsequently tubulo-interstitial inflammation. This was investigated in cultured rat tubular epithelial cells (NRK-52E) and in the rat unilateral ischemia/reperfusion (I/R) model. In NRK-52E cells, the JNK inhibitor anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125) reduced interleukin-1β-, transforming growth factor-β-, or bovine serum albumin-induced MCP-1 expression in a potent manner (up to 150-fold). In the rat I/R model, JNK activation was low in controls but induced in tubular cells from 30 min after I/R. The extent of JNK activation correlated with interstitial macrophage accumulation. Treatment with SP600125 (30 mg/kg/day i.p. for 4 days) reduced renal c-Jun activation; MCP-1, osteopontin, and vimentin expression; and interstitial macrophage and T-cell accumulation (all p < 0.05). In human renal disease, we also found induction of JNK activation, which correlated strongly with interstitial macrophage accumulation, tubulointerstitial fibrosis, and renal function loss. In conclusion, these data indicate that the JNK pathway plays an important role in renal inflammation, at least in part through induction of MCP-1 gene expression in tubular epithelial cells.

Laboratory aspects of circulating α-Klotho

BACKGROUND α-Klotho is a protein mainly produced in the kidney. Its circulating form has been suggested to link renal damage and distant tissue pathology. As three assays to measure α-Klotho became commercially available, we performed an evaluation of these commercially available Klotho assays. METHODS We studied within-run variation, between-run variation, matrix effects, linearity, and recovery of added recombinant human Klotho in the α-Klotho assays of IBL (IBL International GmbH, Hamburg, Germany), Cusabio (Cusabio Biotech, Wuhan, China) and USCN (USCN life Science, Inc., Wuhan, China) using both serum and ethylenediaminetetraacetic acid plasma. RESULTS Within run variation was 4, 13 and 32% for the IBL, Cusabio and USCN assay, respectively. Agreement between serum and EDTA plasma was good in the IBL assay, but poor in the USCN and Cusabio assays however improved after modifications in the Cusabio assay. Standardization and agreement between assays was poor. CONCLUSIONS The commercially available methods for the measurement of α-Klotho differ in quality. Some of the manufacturers should improve their assays in order to produce accurate results so that reliable conclusions can be drawn from studies in which these assays are used.