Paraoxonase 2 (PON2) Deficiency Reproduces Lipid Alterations of Diabetic and Inflammatory Glomerular Disease and Affects TRPC6 Signaling.

Diabetes and inflammatory diseases are associated with an altered cellular lipid composition due to lipid peroxidation. The pathogenic potential of these lipid alterations in glomerular kidney diseases remains largely obscure as suitable cell culture and animal models are lacking. In glomerular disease, a loss of terminally differentiated glomerular epithelial cells called podocytes refers to irreversible damage. Podocytes are characterized by a complex ramified cellular architecture and highly active transmembrane signaling. Alterations in lipid composition in states of disease have been described in podocytes but the pathophysiologic mechanisms mediating podocyte damage are unclear. In this study, we employ a genetic deletion of the anti-oxidative, lipid-modifying paraoxonase 2 enzyme (PON2) as a model to study altered cellular lipid composition and its effects on cellular signaling in glomerular disease. PON2 deficiency reproduces features of an altered lipid composition of glomerular disease, characterized by an increase in ceramides and cholesterol. PON2 knockout mice are more susceptible to glomerular damage in models of aggravated oxidative stress such as adriamycin-induced nephropathy. Voltage clamp experiments in cultured podocytes reveal a largely increased TRPC6 conductance after a membrane stretch in PON2 deficiency. Correspondingly, a concomitant knockout of TRPC6 and PON2 partially rescues the aggravated glomerular phenotype of a PON2 knockout in the adriamycin model. This study establishes PON2 deficiency as a model to investigate the pathophysiologic mechanisms of podocyte dysfunction related to alterations in the lipid composition, as seen in diabetic and inflammatory glomerular disease. Expanding the knowledge on these routes and options of intervention could lead to novel treatment strategies for glomerular disease.

Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis.

BACKGROUND: Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. METHODS: We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. RESULTS: Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of purified podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homolog Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. CONCLUSIONS: We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype-to-protein correlation.

Anaerobic Glycolysis Maintains the Glomerular Filtration Barrier Independent of Mitochondrial Metabolism and Dynamics.

The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1α, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).

Metabolism and homeostasis in the kidney: metabolic regulation through insulin signaling in the kidney.

Metabolic signaling pathways orchestrate the dynamic turnover between catabolic and anabolic processes. Thereby, they ensure the viability of the cell and assure proper function of the tissue in changing environments regarding the availability of nutrients. Yet, renal cells are not considered to be prime targets of metabolic signaling. Research of the last decade has proposed new roles of specifically altered metabolic signaling pathways. In particular, the insulin signaling cascade, a potent regulator of cellular metabolism and energy homeostasis, seems to be implicated in the progression of diabetic and non-diabetic kidney disease. The aim of this review is to summarize the current knowledge on metabolic signaling events in different renal compartments in states of health and disease. We will focus on the role of insulin signaling events and highlight recent advances in the understanding of the regulatory interplay between insulin signaling and mitochondrial function contributing to the pathogenesis of kidney disease.

Single and Transient Ca2+ Peaks in Podocytes do not induce Changes in Glomerular Filtration and Perfusion.

Chronic alterations in calcium (Ca2+) signalling in podocytes have been shown to cause proteinuria and progressive glomerular diseases. However, it is unclear whether short Ca2+ peaks influence glomerular biology and cause podocyte injury. Here we generated a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) knock-in mouse line to manipulate intracellular Ca2+ levels. By mating to a podocyte-specific Cre driver we are able to investigate the impact of Ca2+ peaks on podocyte biology in living animals. Activation of the engineered G-protein coupled receptor with the synthetic compound clozapine-N-oxide (CNO) evoked a short and transient Ca2+ peak in podocytes immediately after CNO administration in vivo. Interestingly, this Ca2+ peak did neither affect glomerular perfusion nor filtration in the animals. Moreover, no obvious alterations in the glomerular morphology could be observed. Taken together, these in vivo findings suggest that chronic alterations and calcium overload rather than an induction of transient Ca2+ peaks contribute to podocyte disease.

PPARγ agonist-induced fluid retention depends on αENaC expression in connecting tubules.

BACKGROUND/AIMS: Thiazolidinediones (TZDs, like rosiglitazone (RGZ)) are peroxisome proliferator-activated receptor γ (PPARγ) agonists used to treat type 2 diabetes. Clinical limitations include TZD-induced fluid retention and body weight (BW) increase, which are inhibited by amiloride, an epithelial-sodium channel (ENaC) blocker. RGZ-induced fluid retention is maintained in mice with αENaC knockdown in the collecting duct (CD). Since ENaC in the connecting tubule (CNT) rather than in CD appears to be critical for normal NaCl retention, we aimed to further explore the role of ENaC in CNT in RGZ-induced fluid retention. METHODS: Mice with conditional inactivation of αENaC in both CNT and CD were used (αENaC lox/lox AQP2-Cre; 'αENaC-CNT/CD-KO') and compared with littermate controls (αENaC lox/lox mice; 'WT'). BW was monitored and total body water (TBW) and extracellular fluid volume (ECF) were determined by bioelectrical impedance spectroscopy (BIS) before and after RGZ (320 mg/kg diet for 10 days). RESULTS: On regular NaCl diet, αENaC-CNT/CD-KO had normal BW, TBW, ECF, hematocrit, and plasma Na(+), K(+), and creatinine, associated with an increase in plasma aldosterone compared with WT. Challenging αENaC-CNT/CD-KO with a low NaCl diet unmasked impaired NaCl and K homeostasis, consistent with effective knockdown of αENaC. In WT, RGZ increased BW (+6.1%), TBW (+8.4%) and ECF (+10%), consistent with fluid retention. These changes were significantly attenuated in αENaC-CNT/CD-KO (+3.4, 1.3, and 4.3%). CONCLUSION: Together with the previous studies, the current results are consistent with a role of αENaC in CNT in RGZ-induced fluid retention, which dovetails with the physiological relevance of ENaC in this segment.

Breaking the chain at the membrane: paraoxonase 2 counteracts lipid peroxidation at the plasma membrane.

Lipid peroxidation through electrophilic molecules of extracellular origin is involved in the pathogenesis of many inflammatory conditions. To counteract free radical actions at the plasma membrane, cells host a variety of antioxidative enzymes. Here we analyzed localization, membrane topology, and trafficking of PON2 a member of the paraoxonase family of 3 enzymatically active proteins (PON1-3) found to have antiatherogenic properties. Immunohistochemistry localized PON2 to the villous tip of human intestinal epithelial cells. Employing membrane preparations, surface biotinylation experiments, and mutational analyses in HEK 293T and HeLa cells, we demonstrate that PON2 is a type II transmembrane protein. A hydrophobic stretch in the N terminus was identified as single transmembrane domain of PON2. The enzymatically active domain faced the extracellular compartment, where it suppressed lipid peroxidation (P<0.05) and regulated the glucosylceramide content, as demonstrated by mass spectrometry (P<0.05). PON2 translocation to the plasma membrane was dependent on intracellular calcium responses and could be induced to >10-fold as compared to baseline (P=0.0001) by oxidative stress. Taken together, these data identify the paraoxonase protein PON2 as a type II transmembrane protein, which is dynamically translocated to the plasma membrane in response to oxidative stress to counteract lipid peroxidation.

Dipeptidyl peptidase IV inhibitor lowers PPARγ agonist-induced body weight gain by affecting food intake, fat mass, and beige/brown fat but not fluid retention.

Peroxisome proliferator-activated receptor-γ (PPARγ) agonists like pioglitazone (PGZ) are effective antidiabetic drugs, but they induce fluid retention and body weight (BW) gain. Dipeptidyl peptidase IV (DPP IV) inhibitors are antidiabetic drugs that enhance renal Na(+) and fluid excretion. Therefore, we examined whether the DPP IV inhibitor alogliptin (ALG) ameliorates PGZ-induced BW gain. Male Sv129 mice were treated with vehicle (repelleted diet), PGZ (220 mg/kg diet), ALG (300 mg/kg diet), or a combination of PGZ and ALG (PGZ + ALG) for 14 days. PGZ + ALG prevented the increase in BW observed with PGZ but did not attenuate the increase in body fluid content determined by bioimpedance spectroscopy (BIS). BIS revealed that ALG alone had no effect on fat mass (FM) but enhanced the FM-lowering effect of PGZ; MRI analysis confirmed the latter and showed reductions in visceral and inguinal subcutaneous (sc) white adipose tissue (WAT). ALG but not PGZ decreased food intake and plasma free fatty acid concentrations. Conversely, PGZ but not ALG increased mRNA expression of thermogenesis mediator uncoupling protein 1 in epididymal WAT. Adding ALG to PGZ treatment increased the abundance of multilocular cell islets in sc WAT, and PGZ + ALG increased the expression of brown-fat-like "beige" cell marker TMEM26 in sc WAT and interscapular brown adipose tissue and increased rectal temperature vs. vehicle. In summary, DPP IV inhibition did not attenuate PPARγ agonist-induced fluid retention but prevented BW gain by reducing FM. This involved ALG inhibition of food intake and was associated with food intake-independent synergistic effects of PPARγ agonism and DPP-IV inhibition on beige/brown fat cells and thermogenesis.