Alexander H. Kirsch

Potential role of regulatory T cells in reversing obesity-linked insulin resistance and diabetic nephropathy

OBJECTIVE To assess the potential role of FoxP3-expressing regulatory T cells (Tregs) in reversing obesity-linked insulin resistance and diabetic nephropathy in rodent models and humans. RESEARCH DESIGN AND METHODS To characterize the role of Tregs in insulin resistance, human visceral adipose tissue was first evaluated for Treg infiltration and second, the db/db mouse model was evaluated. RESULTS Obese patients with insulin resistance displayed significantly decreased natural Tregs but an increase in adaptive Tregs in their visceral adipose tissue as compared with lean control subjects. To further evaluate the pathogenic role of Tregs in insulin resistance, the db/db mouse model was used. Treg depletion using an anti-CD25 monoclonal antibody enhanced insulin resistance as shown by increased fasting blood glucose levels as well as an impaired insulin sensitivity. Moreover, Treg-depleted db/db mice developed increased signs of diabetic nephropathy, such as albuminuria and glomerular hyperfiltration. This was paralleled by a proinflammatory milieu in both murine visceral adipose tissue and the kidney. Conversely, adoptive transfer of CD4+FoxP3+ Tregs significantly improved insulin sensitivity and diabetic nephropathy. Accordingly, there was increased mRNA expression of FoxP3 as well as less abundant proinflammatory CD8+CD69+ T cells in visceral adipose tissue and kidneys of Treg-treated animals. CONCLUSIONS Data suggest a potential therapeutic value of Tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the proinflammatory milieu.

Cardioprotection and lifespan extension by the natural polyamine spermidine

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.

Performance of hemodialysis with novel medium cut-off dialyzers

Background. Compared to high-flux dialysis membranes, novel medium cut-off (MCO) membranes show greater permeability for larger middle molecules. Methods. In two prospective, open-label, controlled, randomized, crossover pilot studies, 39 prevalent hemodialysis (HD) patients were studied in four dialysis treatments as follows: study 1, three MCO prototype dialyzers (AA, BB and CC with increasing permeability) and one high-flux dialyzer in HD; and study 2, two MCO prototype dialyzers (AA and BB) in HD and high-flux dialyzers in HD and hemodiafiltration (HDF). Primary outcome was lambda free light chain (&lgr;FLC) overall clearance. Secondary outcomes included overall clearances and pre-to-post-reduction ratios of middle and small molecules, and safety of MCO HD treatments. Results. MCO HD provided greater &lgr;FLC overall clearance [least square mean (standard error)] as follows: study 1: MCO AA 8.5 (0.54), MCO BB 11.3 (0.51), MCO CC 15.0 (0.53) versus high-flux HD 3.6 (0.51) mL/min; study 2: MCO AA 10.0 (0.58), MCO BB 12.5 (0.57) versus high-flux HD 4.4 (0.57) and HDF 6.2 (0.58) mL/min. Differences between MCO and high-flux dialyzers were consistently significant in mixed model analysis (each P < 0.001). Reduction ratios of &lgr;FLC were greater for MCO. Clearances of &agr;1-microglobulin, complement factor D, kappa FLC (&kgr;FLC) and myoglobin were generally greater with MCO than with high-flux HD and similar to or greater than clearances with HDF. Albumin loss was moderate with MCO, but greater than with high-flux HD and HDF. Conclusions. MCO HD removes a wide range of middle molecules more effectively than high-flux HD and even exceeds the performance of high-volume HDF for large solutes, particularly &lgr;FLC.

A porcine model of hypertensive cardiomyopathy: implications for heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFPEF) evolves with the accumulation of risk factors. Relevant animal models to identify potential therapeutic targets and to test novel therapies for HFPEF are missing. We induced hypertension and hyperlipidemia in landrace pigs (n = 8) by deoxycorticosteroneacetate (DOCA, 100 mg/kg, 90-day-release subcutaneous depot) and a Western diet (WD) containing high amounts of salt, fat, cholesterol, and sugar for 12 wk. Compared with weight-matched controls (n = 8), DOCA/WD-treated pigs showed left ventricular (LV) concentric hypertrophy and left atrial dilatation in the absence of significant changes in LV ejection fraction or symptoms of heart failure at rest. The LV end-diastolic pressure-volume relationship was markedly shifted leftward. During simultaneous right atrial pacing and dobutamine infusion, cardiac output reserve and LV peak inflow velocities were lower in DOCA/WD-treated pigs at higher LV end-diastolic pressures. In LV biopsies, we observed myocyte hypertrophy, a shift toward the stiffer titin isoform N2B, and reduced total titin phosphorylation. LV superoxide production was increased, in part attributable to nitric oxide synthase (NOS) uncoupling, whereas AKT and NOS isoform expression and phosphorylation were unchanged. In conclusion, we developed a large-animal model in which loss of LV capacitance was associated with a titin isoform shift and dysfunctional NOS, in the presence of preserved LV ejection fraction. Our findings identify potential targets for the treatment of HFPEF in a relevant large-animal model.

The mTOR-inhibitor rapamycin mediates proteinuria in nephrotoxic serum nephritis by activating the innate immune response

Rapamycin (Rapa) is an immunosuppressant used to prevent rejection in recipients of renal transplants. Its clinical use is limited by de novo onset or exacerbation of preexisting proteinuria. In the present study, Rapa administration was started 14 days after induction of murine nephrotoxic serum nephritis (NTS) to study glomerular effects of this mammalian target of rapamycin (mTOR) inhibitor. Glomeruli were laser-microdissected, and real-time PCR was performed to assess effects on glomerular cells and the expression of inflammatory cytokines. Immunohistochemical stainings were performed to confirm mRNA data on the protein level. Compared with nephritic control animals, Rapa-treated mice developed significantly increased albuminuria. This was accompanied by a more prominent glomerular infiltration by CD4(+) T cells and macrophages. Glomerular mRNA expression profiling revealed increased levels of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, and the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1β and their cognate macrophage-associated receptors CCR2 and CCR5 in the Rapa-treated animals. Furthermore, there were elevated glomerular transcription levels of the regulatory T cell phenotype transcription factor Foxp3. No differences in the glomerular expression of the podocyte marker nephrin or the endothelial cell marker CD31 were observed on the mRNA or protein level. In conclusion, our data indicate that Rapa-induced proteinuria in NTS is a result of the activation of the innate immune system rather than a direct toxicity to podocytes or glomerular endothelial cells.

Lipocalin-2 Expressed in Innate Immune Cells Is an Endogenous Inhibitor of Inflammation in Murine Nephrotoxic Serum Nephritis

Lipocalin-2 (Lcn-2) is involved in divergent processes such as acute kidney injury or bacterial host defence. Our study was designed to evaluate the functional role of Lcn-2 in nephrotoxic serum nephritis (NTS). Since Lcn-2 is expressed in tubular epithelial cells as well as in cells of innate immunity such as macrophages and polymorphonuclear neutrophils (PMN), we induced NTS in wild-type (WT), Lcn-2 knock-out (KO) mice and WT/Lcn-2 KO chimeras. Mice lacking Lcn-2 exhibited more glomerular damage with increased proteinuria and interstitial leukocyte accumulation compared to WT mice. Chimeras able to express Lcn-2 in macrophages and PMN but not in epithelial cells were found to develop NTS comparable to wild-type controls. In contrast, chimeras expressing Lcn-2 in tubular epithelial cells with no expression in innate immune cells developed increased NTS due to decreased concerted apoptosis but increased necrosis and formation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB-1) in the kidney. In vivo blockade of HMGB-1, a toll-like receptor (TLR)-2 agonist, significantly reduced inflammation and NTS in Lcn-2 knock-out mice. In parallel, TLR-2 signalling was found to drive Lcn-2 transcription in vitro. Taken together, Lcn-2 expressed in innate immune cells is protective in NTS by inducing concerted apoptosis and inhibiting the formation of HMGB-1 thereby limiting cytokine production via TLR-2 signalling. In parallel, TLR-2 dependent transcription of Lcn-2 is an endogenous inhibitor of inflammation in NTS.

A Murine Model of Phosphate Nephropathy

We established a murine model of phosphate nephropathy with secondary hyperparathyroidism. db/db mice, which develop obesity and type 2 diabetes mellitus, were uninephrectomized at the age of 6 weeks and were fed either standard chow or a phosphorus-rich diet during the next 8 weeks. Thereafter, renal cryosections showed abundant tubular casts with a strong histochemical von Kossa reaction in all db/db mice on the phosphorus-rich diet but none in the controls. X-ray diffraction and Raman spectroscopy proved that these tubular casts consist mostly of hydroxyapatite Ca₅(PO₄)₃(OH). These intraluminal precipitations were located in distal tubuli and collecting ducts and were associated with degenerative tubular changes and peritubular infiltration of T cells and macrophages. In line, kidneys of db/db mice on the phosphorus-rich diet displayed significantly increased mRNA expression of the T(H)1 cytokines interferon γ, IL-6, and tumor necrosis factor α. In addition, mice developed signs of secondary hyperparathyroidism as shown by elevated serum phosphate, decreased serum calcium, and increased parathyroid hormone, osteopontin, and fibroblast growth factor 23 levels. db/db mice on the phosphorus-rich diet also presented with significantly lower body weight, lower homeostasis model assessment of insulin resistance index, and hypertrophic cardiomyopathy. Thus, we provide a murine model of phosphate nephropathy and secondary hyperparathyroidism, which can be used for future pharmacologic and pathophysiologic studies to analyze the effect of hyperphosphatemia on renal, metabolic, and cardiovascular phenotypes.

MicroRNAs 223-3p and 93-5p in patients with chronic kidney disease before and after renal transplantation.

Chronic kidney disease (CKD) is associated with a multifactorial dysregulation of bone and vascular calcification and closely linked to increased cardiovascular mortality and concomitant bone disease. We aimed to investigate specific microRNA (miRNA) signatures in CKD patients to find indicators for vascular calcification and/or bone mineralization changes during CKD and after kidney transplantation (KT). A miRNA array was used to investigate serum miRNA profiles in CKD patients, then selected miRNAs were quantified in a validation cohort comprising 73 patients in CKD stages 3 to 5, 67 CKD patients after KT, and 36 healthy controls. A spectrum of biochemical parameters including markers for kidney function, inflammation, glucose, and mineral metabolism was determined. The relative expression of miR-223-3p and miR-93-5p was down-regulated in patients with CKD stage 4 and 5 compared to healthy controls. This down-regulation disappeared after kidney transplantation even when lower glomerular filtration rates (eGFR) persisted. MiR-223-3p and miR-93-5p were associated with interleukin-6 (IL-6) and eGFR levels, and by trend with interleukin-8 (IL-8), C-peptide, hematocrit, and parathyroid hormone (PTH). This study contributes new knowledge of serum miRNA expression profiles in CKD, potentially reflecting pathophysiological changes of bone and calcification pathways associated with inflammation, vascular calcification, mineral and glucose metabolism. Identified miRNA signatures can contribute to future risk markers or future therapeutic targets in bone and kidney disease.

Heterogeneous susceptibility for uraemic media calcification and concomitant inflammation within the arterial tree

Background End-stage renal disease (ESRD) is strongly associated with arterial calcification of the tunica media, decreased vascular compliance and sudden cardiac death. Here, we analysed the distribution pattern of uraemic media calcification and concomitant inflammation in mice and men. Methods Uraemia was induced in DBA/2 mice with high-phosphate diet. Subsequently, we analysed arterial medial calcification using histology, mass spectrometry, and wire myography. Gene expression was quantified using a whole transcriptome array and quantitative PCR. In a cohort of 36 consecutive patients with CKD stage 4–5, we measured the calcium score of the coronary arteries, the ascending thoracic aorta and the infrarenal abdominal aorta using computed tomography scans. Results Uraemic DBA/2 mice showed only minor calcifications in thoracic aortas, whereas there was overt media calcification in abdominal aortas. The transcriptional profile and immunohistochemistry revealed induction of Vcam1 expression by vascular smooth muscle cells in uraemic abdominal aortas. Macrophages infiltrated the tunica media of the abdominal aorta. Anti-inflammatory treatment did not improve uraemic media calcification in our animal model. Arterial calcifications in ESRD patients showed a similar distribution pattern in computed tomography scans, with higher calcium scores of the abdominal aorta when compared with the thoracic aorta. Conclusion Taken together, there was a similar heterogeneous pattern of calcification in both mice and humans, where the abdominal aorta was more prone to media calcification when compared with the thoracic aorta. In uraemia, smooth muscle cells of the abdominal aorta showed a phenotypic switch to an inflammatory and osteoblastic phenotype.

Innate and adaptive immunity in experimental glomerulonephritis: a pathfinder tale

The role of innate and adaptive immune cells in the experimental model of nephrotoxic serum nephritis (NTS) has been rigorously studied in recent years. The model is dependent on kidney-infiltrating T helper (TH) 17 and TH1 cells, which recruit neutrophils and macrophages, respectively, and cause sustained kidney inflammation. In a later phase of disease, regulatory T cells (Tregs) infiltrate the kidney in an attempt to limit disease activity. In the early stage of NTS, lymph node drainage plays an important role in disease initiation since dendritic cells present the antigen to T cells in the T cell zones of the draining lymph nodes. This results in the differentiation and proliferation of TH17 and TH1 cells. In this setting, immune regulatory cells (Tregs), namely, CCR7-expressing Tregs and mast cells (MCs), which are recruited by Tregs via the production of interleukin-9, exert their immunosuppressive capacity. Together, these two cell populations inhibit T cell differentiation and proliferation, thereby limiting disease activity by as yet unknown mechanisms. In contrast, the spleen plays no role in immune activation in NTS, but constitutes a place of extramedullary haematopoiesis. The complex interactions of immune cells in NTS are still under investigation and might ultimately lead to targeted therapies in glomerulonephritis.