Nephrotic syndrome-associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors.

BACKGROUND: Thrombosis is a potentially life-threatening nephrotic syndrome (NS) complication. We have previously demonstrated that hypercoagulopathy is proportional to NS severity in rat models and that pioglitazone (Pio) reduces proteinuria both independently and in combination with methylprednisolone (MP), a glucocorticoid (GC). However, the effect of these treatments on NS-associated hypercoagulopathy remains unknown. We thus sought to determine the ability of Pio and GC to alleviate NS-associated hypercoagulopathy. METHODS: Puromycin aminonucleoside-induced rat NS was treated with sham, Low- or High-dose MP, Pio, or combination (Pio + Low-MP) and plasma was collected at day 11. Plasma samples were collected from children with steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) upon presentation and after 7 weeks of GC therapy. Plasma endogenous thrombin potential (ETP), antithrombin (AT) activity, and albumin (Alb) were measured using thrombin generation, amidolytic, and colorimetric assays, respectively. RESULTS: In a rat model of NS, both High-MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (p < .05). Proteinuria (p = .005) and hypoalbuminemia (p < .001) were correlated with ETP. In childhood NS, while ETP was not different at presentation, GC therapy improved proteinuria, hypoalbuminemia, and ETP in children with SSNS (p < .001) but not SRNS (p = .330). CONCLUSIONS: Both Pio and GC diminish proteinuria and significantly alleviate hypercoagulopathy. Both Pio and MP improved hypercoagulopathy in rats, and successful GC therapy (SSNS) also improved hypercoagulopathy in childhood NS. These data suggest that even a partial reduction in proteinuria may reduce NS-associated thrombotic risk.

Publisher Correction: Whole Transcriptome Analysis of Renal Intercalated Cells Predicts Lipopolysaccharide Mediated Inhibition of Retinoid X Receptor alpha Function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Predicting and Defining Steroid Resistance in Pediatric Nephrotic Syndrome Using Plasma Proteomics.

INTRODUCTION: Nephrotic syndrome (NS) is a characterized by massive proteinuria, edema, hypoalbuminemia, and dyslipidemia. Glucocorticoids (GCs), the primary therapy for >60 years, are ineffective in approximately 50% of adults and approximately 20% of children. Unfortunately, there are no validated biomarkers able to predict steroid-resistant NS (SRNS) or to define the pathways regulating SRNS. METHODS: We performed proteomic analyses on paired pediatric NS patient plasma samples obtained both at disease presentation before glucocorticoid initiation and after approximately 7 weeks of GC therapy to identify candidate biomarkers able to either predict steroid resistance before treatment or define critical molecular pathways/targets regulating steroid resistance. RESULTS: Proteomic analyses of 15 paired NS patient samples identified 215 prevalent proteins, including 13 candidate biomarkers that predicted SRNS before GC treatment, and 66 candidate biomarkers that mechanistically differentiated steroid-sensitive NS (SSNS) from SRNS. Ingenuity Pathway Analyses and protein networking pathways approaches further identified proteins and pathways associated with SRNS. Validation using 37 NS patient samples (24 SSNS/13 SRNS) confirmed vitamin D binding protein (VDB) and APOL1 as strong predictive candidate biomarkers for SRNS, and VDB, hemopexin (HPX), adiponectin (ADIPOQ), sex hormone-binding globulin (SHBG), and APOL1 as strong candidate biomarkers to mechanistically distinguish SRNS from SSNS. Logistic regression analysis identified a candidate biomarker panel (VDB, ADIPOQ, and matrix metalloproteinase 2 [MMP-2]) with significant ability to predict SRNS at disease presentation (P = 0.003; area under the receiver operating characteristic curve = 0.78). CONCLUSION: Plasma proteomic analyses and immunoblotting of serial samples in childhood NS identified a candidate biomarker panel able to predict SRNS at disease presentation, as well as candidate molecular targets/pathways associated with clinical steroid resistance.

Whole Transcriptome Analysis of Renal Intercalated Cells Predicts Lipopolysaccharide Mediated Inhibition of Retinoid X Receptor alpha Function.

The renal collecting duct consists of intercalated cells (ICs) and principal cells (PCs). We have previously demonstrated that collecting ducts have a role in the innate immune defense of the kidney. Transcriptomics is an important tool used to enhance systems-level understanding of cell biology. However, transcriptomics performed on whole kidneys provides limited insight of collecting duct cell gene expression, because these cells comprise a small fraction of total kidney cells. Recently we generated reporter mouse models to enrich collecting duct specific PC and ICs and reported targeted gene expression of anti-microbial peptide genes. Here we report transcriptomics on enriched ICs and PCs and performed a pilot study sequencing four single ICs. We identified 3,645 genes with increased relative expression in ICs compared to non-ICs. In comparison to non-PCs, 2,088 genes had higher relative expression in PCs. IC associated genes included the innate interleukin 1 receptor, type 1 and the antimicrobial peptide(AMP) adrenomedullin. The top predicted canonical pathway for enriched ICs was lipopolysaccharide/Interleukin 1 mediated inhibition of Retinoid X Receptor alpha function and decreased Retinoid X Receptor expression was confirmed to occur 1-hour post experimental murine UTI in ICs but not in non-ICs.

Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct.

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+ mice to fluorescently label ICs and AQP2-cre+tdT+ mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome.

Nie X, Chanley MA, Pengal R, Thomas DB, Agrawal S, Smoyer WE. Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome. Am J Physiol Renal Physiol 314: F602-F613, 2018. First published November 29, 2017; doi: 10.1152/ajprenal.00207.2017 .-The p38 MAPK pathway plays a crucial role in various glomerulopathies, with activation being associated with disease and inhibition being associated with disease amelioration. We hypothesized that the downstream targets of p38 MAPK, MAPK-activated protein kinase 2 and/or 3 (MK2 and/or MK3), play an important role in mediating injury in experimental nephrotic syndrome via their actions on their downstream substrates heat shock protein B1 (HSPB1) and cyclooxygenase-2 (COX-2). To test this hypothesis, the effects of both pharmacological and genetic inhibition of MK2 and MK3 were examined in mouse adriamycin (ADR) and rat puromycin aminonucleoside (PAN) nephropathy models. MK2-/-, MK3-/-, and MK2-/-MK3-/- mice were generated in the Sv129 background and subjected to ADR-induced nephropathy. MK2 and MK3 protein expression was completely abrogated in the respective knockout genotypes, and massive proteinuria and renal histopathological changes developed after ADR treatment. Furthermore, renal cortical HSPB1 was induced in all four genotypes by day 21, but HSPB1 was activated only in the wild-type and MK3-/- mice. Expression of the stress proteins HSPB8 and glucose-regulated protein 78 (GRP78) remained unaltered across all genotypes. Finally, while MK2 and/or MK3-knockout downregulated the proinflammatory enzyme COX-2, ADR significantly induced renal cortical COX-2 only in MK2-/- mice. Additionally, pharmacological MK2 inhibition with PF-318 during PAN-induced nephropathy did not result in significant proteinuria reduction in rats. Together, these data suggest that while the inhibition of MK2 and/or MK3 regulates the renal stress response, our currently available approaches are not yet able to safely and effectively reduce proteinuria in experimental nephrotic syndrome and that other p38MAPK downstream targets should also be considered to improve the future treatment of glomerular disease.

Disease Severity Correlates with Thrombotic Capacity in Experimental Nephrotic Syndrome.

Thrombotic disease, a major life-threatening complication of nephrotic syndrome, has been associated with proteinuria and hypoalbuminemia severity. However, it is not fully understood how disease severity correlates with severity of the acquired hypercoagulopathy of nephrotic syndrome. Without this knowledge, the utility of proteinuria and/or hypoalbuminemia as biomarkers of thrombotic risk remains limited. Here, we show that two well established ex vivo hypercoagulopathy assays, thrombin generation and rotational thromboelastometry, are highly correlated with proteinuria and hypoalbuminemia in the puromycin aminonucleoside and adriamycin rat models of nephrotic syndrome. Notably, in the puromycin aminonucleoside model, hyperfibrinogenemia and antithrombin deficiency were also correlated with proteinuria severity, consistent with reports in human nephrotic syndrome. Importantly, although coagulation was not spontaneously activated in vivo with increasing proteinuria, vascular injury induced a more robust thrombotic response in nephrotic animals. In conclusion, hypercoagulopathy is highly correlated with nephrotic disease severity, but overt thrombosis may require an initiating insult, such as vascular injury. Our results suggest that proteinuria and/or hypoalbuminemia could be developed as clinically meaningful surrogate biomarkers of hypercoagulopathy to identify patients with nephrotic syndrome at highest risk for thrombotic disease and potentially target them for anticoagulant pharmacoprophylaxis.

Albumin-induced podocyte injury and protection are associated with regulation of COX-2.

Albuminuria is both a hallmark and a risk factor for progressive glomerular disease, and results in increased exposure of podocytes to serum albumin with its associated factors. Here in vivo and in vitro models of serum albumin-overload were used to test the hypothesis that albumin-induced proteinuria and podocyte injury directly correlate with COX-2 induction. Albumin induced COX-2, MCP-1, CXCL1, and the stress protein HSP25 in both rat glomeruli and cultured podocytes, whereas B7-1 and HSP70i were also induced in podocytes. Podocyte exposure to albumin induced both mRNA and protein and enhanced the mRNA stability of COX-2, a key regulator of renal hemodynamics and inflammation, which renders podocytes susceptible to injury. Podocyte exposure to albumin also stimulated several kinases (p38 MAPK, MK2, JNK/SAPK, and ERK1/2), inhibitors of which (except JNK/SAPK) downregulated albumin-induced COX-2. Inhibition of AMPK, PKC, and NFκB also downregulated albumin-induced COX-2. Critically, albumin-induced COX-2 was also inhibited by glucocorticoids and thiazolidinediones, both of which directly protect podocytes against injury. Furthermore, specific albumin-associated fatty acids were identified as important contributors to COX-2 induction, podocyte injury, and proteinuria. Thus, COX-2 is associated with podocyte injury during albuminuria, as well as with the known podocyte protection imparted by glucocorticoids and thiazolidinediones. Moreover, COX-2 induction, podocyte damage, and albuminuria appear mediated largely by serum albumin-associated fatty acids.

Crucial roles of the protein kinases MK2 and MK3 in a mouse model of glomerulonephritis.

Elevated mitogen-activated protein kinase p38 (p38 MAPK) signaling has been implicated in various experimental and human glomerulopathies, and its inhibition has proven beneficial in animal models of these diseases. p38 MAPK signaling is partially mediated through MK2 and MK3, two phylogenetically related protein kinases that are its direct substrates. The current study was designed to determine the specific roles of MK2 and MK3 in a mouse model of acute proliferative glomerulonephritis, using mice with disrupted MK2 and/or MK3 genes. We found that the absence of MK3 alone worsened the disease course and increased mortality slightly compared to wild-type mice, whereas the absence of MK2 alone exhibited no significant effect. However, in an MK3-free background, the disease course depended on the presence of MK2 in a gene dosage-dependent manner, with double knock-out mice being most susceptible to disease induction. Histological and renal functional analyses confirmed kidney damage following disease induction. Because the renal stress response plays a crucial role in kidney physiology and disease, we analyzed the stress response pattern in this disease model. We found that renal cortices of diseased mice exhibited a pronounced and specific pattern of expression and/or phosphorylation of stress proteins and other indicators of the stress response (HSPB1, HSPB6, HSPB8, CHOP, eIF2α), partially in a MK2/MK3 genotype-specific manner, and without induction of a general stress response. Similarly, the expression and activation patterns of other protein kinases downstream of p38 MAPK (MNK1, MSK1) depended partially on the MK2/MK3 genotype in this disease model. In conclusion, MK2 and MK3 together play crucial roles in the regulation of the renal stress response and in the development of glomerulonephritis, which can potentially be exploited to develop novel therapeutic approaches to treat glomerular disease.