Phosphate Restriction Prevents Metabolic Acidosis and Curbs Rise in FGF23 and Mortality in Murine Folic Acid-Induced AKI.

SIGNIFICANCE STATEMENT: Patients with AKI suffer a staggering mortality rate of approximately 30%. Fibroblast growth factor 23 (FGF23) and phosphate (P i ) rise rapidly after the onset of AKI and have both been independently associated with ensuing morbidity and mortality. This study demonstrates that dietary P i restriction markedly diminished the early rise in plasma FGF23 and prevented the rise in plasma P i , parathyroid hormone, and calcitriol in mice with folic acid-induced AKI (FA-AKI). Furthermore, the study provides evidence for P i -sensitive osseous Fgf23 mRNA expression and reveals that P i restriction mitigated calciprotein particles (CPPs) formation, inflammation, acidosis, cardiac electrical disturbances, and mortality in mice with FA-AKI. These findings suggest that P i restriction may have a prophylactic potential in patients at risk for AKI. BACKGROUND: In AKI, plasma FGF23 and P i rise rapidly and are independently associated with disease severity and outcome. METHODS: The effects of normal (NP) and low (LP) dietary P i were investigated in mice with FA-AKI after 3, 24, and 48 hours and 14 days. RESULTS: After 24 hours of AKI, the LP diet curbed the rise in plasma FGF23 and prevented that of parathyroid hormone and calcitriol as well as of osseous but not splenic or thymic Fgf23 mRNA expression. The absence of Pth prevented the rise in calcitriol and reduced the elevation of FGF23 in FA-AKI with the NP diet. Furthermore, the LP diet attenuated the rise in renal and plasma IL-6 and mitigated the decline in renal α -Klotho. After 48 hours, the LP diet further dampened renal IL-6 expression and resulted in lower urinary neutrophil gelatinase-associated lipocalin. In addition, the LP diet prevented the increased formation of CPPs. Fourteen days after AKI induction, the LP diet group maintained less elevated plasma FGF23 levels and had greater survival than the NP diet group. This was associated with prevention of metabolic acidosis, hypocalcemia, hyperkalemia, and cardiac electrical disturbances. CONCLUSIONS: This study reveals P i -sensitive FGF23 expression in the bone but not in the thymus or spleen in FA-AKI and demonstrates that P i restriction mitigates CPP formation, inflammation, acidosis, and mortality in this model. These results suggest that dietary P i restriction could have prophylactic potential in patients at risk for AKI.

Skin regulation of salt and blood pressure and potential clinical implications.

Sodium chloride, as salt, gives rise to hypertension. Nevertheless, individual susceptibility to the ramifications of sodium chloride is heterogeneous. The conventional nephron-centric regulation of sodium with neurohormonal inputs and responses is now expanded to include an intricate extrarenal pathway including the endothelium, skin, lymphatics, and immune cells. An overabundance of sodium is buffered and regulated by the skin interstitium. Excess sodium passes through (and damages) the vascular endothelium and can be dynamically stored in the skin, modulated by skin immune cells and lymphatics. This excess interstitially stored sodium is implicated in hypertension, cardiovascular dysfunction, metabolic disruption, and inflammatory dysregulation. This extrarenal pathway of regulating sodium represents a novel target for better blood pressure management, rebalancing disturbed inflammation, and hence addressing cardiovascular and metabolic disease.

A Metabolic Reprogramming of Glycolysis and Glutamine Metabolism Is a Requisite for Renal Fibrogenesis-Why and How?

Chronic Kidney Disease (CKD) is characterized by organ remodeling and fibrosis due to failed wound repair after on-going or severe injury. Key to this process is the continued activation and presence of matrix-producing renal fibroblasts. In cancer, metabolic alterations help cells to acquire and maintain a malignant phenotype. More recent evidence suggests that something similar occurs in the fibroblast during activation. To support these functions, pro-fibrotic signals released in response to injury induce metabolic reprograming to meet the high bioenergetic and biosynthetic demands of the (myo)fibroblastic phenotype. Fibrogenic signals such as TGF-ß1 trigger a rewiring of cellular metabolism with a shift toward glycolysis, uncoupling from mitochondrial oxidative phosphorylation, and enhanced glutamine metabolism. These adaptations may also have more widespread implications with redirection of acetyl-CoA directly linking changes in cellular metabolism and regulatory protein acetylation. Evidence also suggests that injury primes cells to these metabolic responses. In this review we discuss the key metabolic events that have led to a reappraisal of the regulation of fibroblast differentiation and function in CKD.

HBEGF: an EGF-like growth factor with FGF23-like activity?

Renal signaling networks downstream of FGF23 are not well delineated, but elucidating them may offer an opportunity to control target genes independent of FGF23 in states of dysregulated mineral metabolism. Ni et al. identify HBEGF as a paracrine/autocrine factor in the proximal tubules of mice that mimics the inductive effect of FGF23 on the vitamin D-catabolizing enzyme 24-hydroxylase through a common mitogen-activated protein kinase-dependent pathway. An understanding of how these findings relate to human disease is eagerly anticipated.

TGF-ß1 is a regulator of the pyruvate dehydrogenase complex in fibroblasts.

TGF-ß1 reprograms metabolism in renal fibroblasts, inducing a switch from oxidative phosphorylation to aerobic glycolysis. However, molecular events underpinning this are unknown. Here we identify that TGF-ß1 downregulates acetyl-CoA biosynthesis via regulation of the pyruvate dehydrogenase complex (PDC). Flow cytometry showed that TGF-ß1 reduced the PDC subunit PDH-E1α in fibroblasts derived from injured, but not normal kidneys. An increase in expression of PDH kinase 1 (PDK1), and reduction in the phosphatase PDP1, were commensurate with net phosphorylation and inactivation of PDC. Over-expression of mutant PDH-E1α, resistant to phosphorylation, ameliorated effects of TGF-ß1, while inhibition of PDC activity with CPI-613 was sufficient to induce αSMA and pro-collagen I expression, markers of myofibroblast differentiation and fibroblast activation. The effect of TGF-ß1 on PDC activity, acetyl-CoA, αSMA and pro-collagen I was also ameliorated by sodium dichloroacetate, a small molecule inhibitor of PDK. A reduction in acetyl-CoA, and therefore acetylation substrate, also resulted in a generalised loss of protein acetylation with TGF-ß1. In conclusion, TGF-ß1 in part regulates fibroblast activation via effects on PDC activity.

Profiling histone modifications in the normal mouse kidney and after unilateral ureteric obstruction.

Posttranslational modification of nucleosomal histones is a major determinant of chromatin structure and gene activity. In the present study, we hypothesized that unilateral ureteric obstruction (UUO), a widely used model of tubulointerstitial injury, would be associated with a distinct pattern of histone modifications (marks) in the kidney. Mass spectrometry was used to profile 63 different histone marks in normal mouse kidneys and those after 10 days of UUO. A subsequent histochemical analysis further examined examples of specific marks that changed significantly after UUO for which antisera are available. Histone marks were much more widely distributed and abundant in the normal kidney than is usually appreciated. Although aggregate analysis of the mass spectrometry results revealed net differences between control and UUO groups, residue-specific variations were subtle. Of the 16/63 significant changes (P < 0.05), only 8 changes were quantitatively different by >5%. Nevertheless, we identified several that are not usually examined in the kidney, including marks in the globular domain of core histones (H3:K79), linker histones (H1.4), and histone variants (H3.1:K27 and H3.3:K27). In several cases, there were complementary changes in different marks on the same amino acid. Using H3:K79ME2 as an example, mark enrichment was heterogeneous but largely colocalized with active transcription in a subset of tubular pathology. In conclusion, our study highlights the importance of unbiased screening in examining histone marks. Simultaneous changes in multiple marks on the same amino acid indicate a coordinated histone mark signature. The heterogeneous enrichment of marks, even within the same tubule, highlights the importance of regulatory context.

Calciprotein Particle Formation in Peritoneal Dialysis Effluent Is Dependent on Dialysate Calcium Concentration.

BACKGROUND: The accumulation of fetuin-A-containing calciprotein particles (CPP) in the serum of patients with renal disease and those with chronic inflammation may be involved in driving sterile inflammation and extraosseous mineral deposition. We previously showed that both fetuin-A and CPP were present in the peritoneal dialysis (PD) effluent of stable PD patients. It is unknown whether different PD fluids might affect the formation of CPP in vivo. METHOD: Peritoneal effluent from 12 patients was collected after a 6-hour dwell with 7 different commercial PD fluids. Calciprotein particles and inflammatory cytokines were measured by flow cytometry. RESULTS: High inter-subject variability in CPP concentration was observed. Peritoneal dialysis fluids containing 1.75 mmol/L calcium were associated with enhanced formation of CPP in vivo, compared with fluids containing 1.25 mmol/L calcium. Osmotic agent, fluid pH, and glucose concentration did not affect CPP formation. Peritoneal dialysis effluent CPP levels were not associated with changes in inflammatory cytokines. CONCLUSION: High calcium-containing PD fluids favor intraperitoneal CPP formation. This finding may have relevance for future PD fluid design.

The Role of Secondary Calciprotein Particles in the Mineralisation Paradox of Chronic Kidney Disease.

Mineralisation paradox is prevalent in chronic kidney disease and ageing where increased vascular calcification is accompanied by reduced bone mineralisation and osteopenia. Secondary calciprotein particles (CPP2), colloidal nanoparticles containing hydroxyapatite crystal stabilised by a protein shell, have been implicated in vascular calcification in chronic kidney disease. Here, we describe the effect of CPP2 on osteoblasts and vascular smooth muscle cells (VSMC) mineralisation in an in vitro model system. The mineralisation paradox can be simulated in vitro by the addition of phosphate ions (Pi, 3 mM) and CPP2 (10 µg/ml of Ca equivalent). Pi alone induced osteoblast mineralisation but had no effect on VSMC mineralisation. CPP2 alone had no effect on mineralisation in either cell line, but when combined with elevated Pi, reduced osteoblast-like mineralisation (P < 0.001) whilst induced VSMC mineralisation (P < 0.001). These results suggest that in an in vitro system the synergistic interaction between Pi and CPP2 could mimic the mineralisation paradox, and may provide a potential mechanistic link to explain these clinical observations.

Progression of Tubulointerstitial Fibrosis and the Chronic Kidney Disease Phenotype - Role of Risk Factors and Epigenetics.

Although the kidney has capacity to repair after mild injury, ongoing or severe damage results in scarring (fibrosis) and an associated progressive loss of kidney function. However, despite its universal significance, evidence highlights a population based heterogeneity in the trajectory of chronic kidney disease (CKD) in these patients. To explain the heterogeneity of the CKD phenotype requires an understanding of the relevant risk factors for fibrosis. These factors include both the extrinsic nature of injury, and intrinsic factors such as age, gender, genetics, and perpetual activation of fibroblasts through priming. In many cases an additional level of regulation is provided by epigenetic mechanisms which integrate the various pro-fibrotic and anti-fibrotic triggers in fibrogenesis. In this review we therefore examine the various molecular and structural changes of fibrosis, and how they are influenced by extrinsic and intrinsic factors. Our aim is to provide a unifying hypothesis to help explain the transition from acute to CKD.

Epigenetic Modifications to H3K9 in Renal Tubulointerstitial Cells after Unilateral Ureteric Obstruction and TGF-ß1 Stimulation.

Introduction: Epigenetic regulation of fibrogenesis through post-translational histone modifications (marks) may be a key determinant of progression in renal disease. In this study, we examined the distribution and acquisition of histone 3 Lysine 9 (H3K9) marks after injury and stimulation with the pro-fibrotic cytokine TGF-ß1. Our focus was on their presence in activated fibroblasts (myofibroblasts) and epithelial cells (epithelial-mesenchymal transition). Methods and Results: Immunofluorescent microscopy was used to examine global H3K9 acetylation (H3K9Ac) and tri-methylation (H3K9Me3) after unilateral ureteric obstruction (UUO) in mice. Confocal, super resolution microscopy and flow cytometry were used to determine the in vitro effect of TGF-ß1 on structural arrangement of these marks, and their relationship with α-smooth muscle actin (αSMA) expression, a marker of myofibroblasts and early EMT. The number of individual histone marks was increased 10 days after UUO (p < 0.05 vs. control), with both marks clearly seen in various cell types including proximal tubules and myofibroblasts. Sub-nuclear microscopy in primary rat renal fibroblasts and a proximal tubule cell line (NRK-52e) showed that H3K9Ac was co-localized with phosphorylated-Ser2 RNA polymerase II (pRNAPol II), while H3K9Me3 was not, consistent with permissive and repressive effects on gene expression respectively. In both cell types H3K9Ac was diffusely distributed throughout the nucleus, while H3K9Me3 was found in compartments resembling the nucleolus, and in the case of the fibroblast, also juxtapositioned with the nuclear membrane. TGF-ß1 had no effect on H3K9Ac marks in either cell, but resulted in a redistribution of H3K9Me3 within the fibroblast nucleus. This was unrelated to any change in mitogenesis, but was associated with increased αSMA expression. Conclusion: These findings highlight why it is important to consider the epigenetics of each cell individually, because whilst no overall enrichment occurred, renal myofibroblast differentiation was accompanied by distinct changes in histone mark arrangements.

Anakinra reduces blood pressure and renal fibrosis in one kidney/DOCA/salt-induced hypertension.

OBJECTIVE: To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. METHODS: Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. RESULTS: By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P<0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by ∼20mmHg (n=16, P<0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced (∼30%) renal expression of some (CCL5, CCL2; n=7-8; P<0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n=7-8, P>0.05). Anakinra reduced renal collagen content (n=6, P<0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n=8-9, P<0.001) that accompanied 1K/DOCA/salt-induced hypertension. CONCLUSION: Despite its anti-hypertensive and renal anti-fibrotic actions, anakinra had minimal effects on inflammation and leukocyte infiltration in mice with 1K/DOCA/salt-induced hypertension. Future studies will assess whether the anti-hypertensive actions of anakinra are mediated by protective actions in other BP-regulating or salt-handling organs such as the arteries, skin and brain.

Relative abundance of fetuin- A in peritoneal dialysis effluent and its association with in situ formation of calciprotein particles: an observational pilot study.

AIM: In patients with renal failure or chronic inflammation, the accumulation of fetuin-A-containing calciprotein particles (CPP) in the extracellular fluid has been implicated in driving inflammatory pathways and extraosseous mineral deposition. We aimed to discover whether CPP are present in the peritoneal dialysis fluid effluent (PDF) of stable peritoneal dialysis (PD) patients, and if so, how these particles might be formed. METHODS: Serum and PDF were sampled from 20 stable PD patients. CPP were quantified by the reduction in fetuin-A concentration after high speed centrifugation. 8-iso-PGF2α in PDF was measured as a marker of oxidative stress. Fetuin-A and phosphate were added to commercially available dialysis fluids to assess their ability to support CPP formation ex vivo. RESULTS: We report that the major protein component of these mineral-containing nanoparticles, fetuin-A, is relatively abundant in PDF and that CPP were present in the PDF of 17/20 PD patients. PDF CPP levels were strongly correlated with 8-iso-PGF2α concentrations. In vitro experiments suggested that commonly used peritoneal dialysate fluids, irrespective of composition, could not sustain appreciable de novo CPP formation ex vivo. CONCLUSION: Fetuin-A is either actively transported or locally produced by the peritoneal membrane in PD patients. The association between fetuin-A-containing CPP and markers of oxidative stress warrants further mechanistic studies.

Hypoxia and hypoxia signaling in tissue repair and fibrosis.

Following injury, vascular damage results in the loss of perfusion and consequent low oxygen tension (hypoxia) which may be exacerbated by a rapid influx of inflammatory and mesenchymal cells with high metabolic demands for oxygen. Changes in systemic and cellular oxygen concentrations induce tightly regulated response pathways that attempt to restore oxygen supply to cells and modulate cell function in hypoxic conditions. Most of these responses occur through the induction of the transcription factor hypoxia-inducible factor-1 (HIF-1) which regulates many processes needed for tissue repair during ischemia in the damaged tissue. HIF-1 transcriptionally upregulates expression of metabolic proteins (GLUT-1), adhesion proteins (integrins), soluble growth factors (TGF-ß and VEGF), and extracellular matrix components (type I collagen and fibronectin), which enhance the repair process. For these reasons, HIF-1 is viewed as a positive regulator of wound healing and a potential regulator of organ repair and tissue fibrosis. Understanding the complex role of hypoxia in the loss of function in scarring tissues and biology of chronic wound, and organ repair will aid in the development of pharmaceutical agents that can redress the detrimental outcomes often seen in repair and scarring.

Parathyroid hormone has a prosclerotic effect on vascular smooth muscle cells.

Although accelerated atherosclerosis and arteriosclerosis are common in patients with renal failure, the pathogenesis of these changes is poorly understood. Parathyroid hormone (PTH) levels are elevated in renal failure, and have been linked to uraemic vascular changes in some studies. We examined the in vitro effects of increasing doses of the 1-34 fragment of PTH on human aortic vascular smooth muscle cells (VSMCs). Factors examined were: (1) collagen production using tritiated hydroxyproline incorporation and transcription of procollagen alpha(1)(I) mRNA; (2) change in the surface area of collagen I lattices; (3) mRNA transcription of the collagen binding protein beta1 integrin; (4) proliferation using tritiated thymidine incorporation, and (5) methyl tetrazolium salt conversion to estimate live cell number after 5 days' exposure to PTH. PTH at a concentration of 200 pmol/l increased total collagen synthesis (188 +/- 25% of control, p < 0.01) as well as transcription of procollagen alpha(1)(I) mRNA (136 +/- 11% of control, p < 0.005). PTH also increased reorganisation of collagen I lattices (surface area 47 +/- 8% of well for control vs. 35.7 +/- 2.5 and 34.3 +/- 3.0% for PTH 100 and 200 pmol/l, respectively, p = 0.02) and upregulated beta1 integrin mRNA expression (160 +/- 20% of control at PTH concentration of 200 pmol/l, p < 0.05). PTH had no effect on VSMC proliferation or number at doses up to 200 pmol/l. In conclusion, PTH increases production and reorganisation of collagen by VSMCs in vitro. It is possible that more aggressive control of hyperparathyroidism in patients with renal failure may help to reduce the burden of cardiovascular disease in this patient population.