American journal of physiology. Renal physiology最新文献

We previously published that hypertensive males have greater renal necrosis and a more pro-inflammatory immune profile than females. Hypertension causes the release of damage-associated molecular patterns (DAMPs), which stimulate inflammation. The goal of the current study was to determine if high-mobility group box 1 (HMGB1), a well-characterized DAMP, contributes to greater T-cell activation in hypertensive males versus females and normotensive controls of both sexes. To test this hypothesis, initial studies measured renal and plasma HMGB1 levels in 13-wk-old male and female spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats by Western blot and ELISA. Total renal CD3⁺ T-cells and IL-6⁺ cells were measured by flow cytometry. The contribution of HMGB1 to T-cell activation was measured in isolated renal T-cells via mixed lymphocyte reaction (MLR) in the presence of control IgG or anti-HMGB1 neutralizing antibody. Plasma HMGB1 levels were greater in male SHRs compared with those in female SHR and WKY rats of both sexes. Renal HMGB1 levels were higher in SHR than in WKY and tended to be greater in males versus females in both strains. Consistent with this finding, T-cell activation and renal interleukin (IL)-6 were highest in male SHR. Interestingly, anti-HMGB1 antibody treatment decreased T-cell activation to the same extent in male and female SHRs, with negligible effects on WKY. These results indicate a role for HMGB1 in T-cell activation in SHR. However, despite male SHR having greater levels of HMGB1 than females, HMGB1 does not account for sex differences in T-cell activation.NEW & NOTEWORTHY There is growing evidence that T-cells contribute to both the development of hypertension and sex differences in blood pressure control. Our work establishes the damage-associated molecular pattern HMGB1 as an important contributor to T-cell activation in hypertension.

We previously demonstrated that TNF inhibits NKCC2 phosphorylation in the thick ascending limb (TAL); however, the underlying mechanism remains unclear. We tested the hypothesis that the induction of calcineurin (CN) activity and the expression of CN isoforms contribute to the mechanism by which TNF inhibits phospho-NKCC2 (pNKCC2) expression. CN activity increased by approximately 2-fold in primary cultures of medullary (m)TAL cells challenged with mouse recombinant TNF. In contrast, silencing TNF production in mTAL cells using lentivirus U6-TNF-ex4 reduced CN activity. pNKCC2 expression decreased in mTAL cells challenged with TNF whereas inhibition of CN activity with cyclosporine A (CsA) increased pNKCC2 expression. Although mTAL cells express both the calcineurin A subunit (CNA) a and b isoforms, only CNA b isoform mRNA increased after mTAL cells were challenged with TNF. In vivo, both TNF and CNA b expression increased in outer medulla (OM) from mice given 1% NaCl in the drinking water for 7 days and intrarenal lentivirus silencing of TNF selectively reduced expression of CNA b. Intrarenal injection of a lentivirus that specifically silenced CNA b (U6-CNAb-ex6) increased pNKCC2 expression and attenuated the inhibitory effects of TNF on pNKCC2 expression in freshly isolated TAL tubules. Collectively, the study is the first to demonstrate that TNF increases CN activity and specifically induces β-isoform expression in the kidney. Since NKCC2 is a known target of the CNA b isoform, these findings suggest that a CN-dependent signaling pathway involving this isoform contributes to the mechanism by which TNF inhibits pNKCC2 expression.

Aquaporin-3 (AQP3) mediates basolateral water transport in the kidney principal cells contributing to urine concentration. We previously identified the acetylation of lysine 282 (K282) in the C-terminus of AQP3, which we hypothesized as a positive regulator of AQP3 water permeability. AQP3 acetylation (K282Q or Q), or deacetylation (K282R or R) mimetic mutant mice models were created using CRISPR/Cas9. Male and female wild-type (WT) and mutant mice were assigned to hydrating diets and water deprivation protocols. Urine and plasma osmolality in response to acute vasopressin receptor-2 activation with desmopressin (dDAVP) or inhibition by tolvaptan were determined. In vitro water permeability of murine principal kidney cortical collecting duct (mpkCCD) cells stably expressing AQP3 WT, Q, or R was measured. Acetylated AQP3 was prominent in the cortical to inner medullary collecting ducts of dehydrated versus hydrated mice. At baseline the mutations did not affect the kidney transcriptome, AQP3 abundance, nor subcellular localization. Urine osmolality of the mutant mice was within the normal range. With dehydration, all mice excreted a concentrated urine; however, the female Q mutants exhibited significantly greater 24 h urine osmolality than WT, suggesting greater water reabsorption. In response to acute dDAVP, all mice produced a concentrated urine; however, female Q mutants had a more dilute plasma than WT, further suggesting greater water retention. mpkCCD Q mutant cells exhibited greater water permeability than WT and R cells. We conclude that AQP3 K282 acetylation promotes principal cell water permeability in a sex-dependent manner, however, it is not essential for urine concentration.

Focal Segmental Glomerulosclerosis (FSGS) is a histologic lesion caused by a variety of injurious stimuli that lead to dysfunction/loss of glomerular visceral epithelial cells (i.e. podocytes). Pathogenic mutations in CRB2, encoding the type 1 transmembrane protein crumbs homolog-2, have been shown to cause early-onset corticosteroid-resistant nephrotic syndrome (SRNS)/FSGS. Here, we identified a 2-generation Indian kindred (DUK40595) with biopsy-proven SRNS/FSGS caused by a compound heterozygous mutation in CRB2 comprised of the previously described truncating mutation p.Gly1036_Alafs*43 and a rare 9-bp deletion mutation p.Leu1074_Asp1076del. Because compound heterozygous mutations involving the truncating p.Gly1036_Alafs*43 variant have been associated with reduced CRB2 expression in podocytes and autosomal recessive SRNS/FSGS, we sought to define the pathogenic effects of CRB2 deficiency in podocytes. We show that CRB2 knockdown induces YAP activity and target gene expression in podocytes. It upregulates YAP-mediated mechanosignaling and increases the density of focal adhesion and F-actin. Using elastic resonator interference stress microscopy (ERISM), we demonstrate that CRB2 knockdown also enhances podocyte contractility in a substrate stiffness-dependent manner. The knockdown effect decreases with increasing substrate stiffness, indicating impaired mechanosensing in CRB2 knockdown cells at low substrate stiffness. While the mechanical activation of CRB2 knockdown cells is associated with increased YAP activity, the enhanced cell contractility is not significantly reduced by the selective YAP inhibitors K-975 and verteporfin, suggesting that multiple pathways may be involved in mechanosignaling downstream of CRB2. Taken together, these studies provide the first evidence that CRB2 deficiency may impair podocyte mechanotransduction via disruption of YAP signaling in podocytes.

Sex differences in renal tubular salt and water transporters, channels, claudins and regulatory factors are evident all along the nephron. The influence of sex hormones on physiologic dimorphisms has been established in studies removing, inhibiting or restoring sex hormones and their receptors. The influence of the sex chromosome complement (SCC, XY vs. XX) on renal transporter abundance and activity is an open question. We employed the Four Core Genotypes (FCG) mouse model (in which the testis determining SRY gene is deleted from the Y chromosome and inserted onto an autosomal chromosome) to compare abundance of more than fifty renal transporters and regulators in: FXX gonadal females, FXY gonadal females, MXX Sry males, and MXY XYSry males using semi-quantitative immunoblots. In addition to establishing the significant influence of gonadal hormones, we show, for the first time, that SCC contributes to sexual dimorphisms in abundance of renal transporters including: NHE3, SGLT1 and 2, AQP1, mNKAα1 and β1, NCC, and ENaC β and γ subunits. The findings in this FCG model analysis provide the foundation for future studies of the role of sex hormones vs. chromosomes on physiologic parameters including filtration and flow, on transporter covalent modifications, and trafficking in both heath and disease.

Podocytes are key components of the glomerular filtration barrier, and their injury leads to proteinuria, chronic kidney disease (CKD), and nephrotic syndrome. Effective treatments for these conditions are not well established, and prevention of podocyte injury is a crucial challenge. Nicotinamide (NAM), a form of vitamin B3, has been reported to exert beneficial effects in various renal disease models due to its antioxidant and anti-inflammatory properties and its ability to replenish nicotinamide adenine dinucleotide (NAD⁺). However, its impact on adriamycin (ADR)-induced nephropathy, a model of nephrotic syndrome caused by podocyte injury, remains unclear. We investigated the effects of NAM administration in a mouse model of ADR nephropathy. BALB/c mice were intravenously administered ADR to induce nephropathy. In the NAM-treated group, mice received 0.6% NAM in drinking water ad libitum starting 7 days before ADR administration. After 14 days, NAM treatment decreased albuminuria, glomerular sclerosis, and podocyte injury, and reduced inflammation and oxidative stress markers in the kidneys. NAM and NAD⁺ levels were decreased in ADR-treated kidneys, and the expression of the NAD⁺-consuming enzymes SIRT1 and PARP-1 was decreased and increased, respectively. Nicotinamide N-methyltransferase expression was increased. NAM canceled these abnormalities. In cultured rat podocytes, NAD⁺ alleviated ADR-induced cytotoxicity, apoptosis, and inflammation. These findings suggest that NAM prevents ADR nephropathy and podocyte injury, likely through NAD⁺ replenishment.

Due to the growing obesity epidemic in the United States, it is now estimated that approximately one third of all children are born to obese moms. These data, coupled with data indicating that obesity is associated with accelerated cyst growth in patients with autosomal dominant polycystic kidney disease (ADPKD), led us to hypothesize that maternal obesity may influence the rate of disease progression in offspring. To test this hypothesis, we induced maternal obesity by high-fat diet (HFD) feeding in the orthologous Pkd1^RC/RC mouse model of ADPKD and followed polycystic kidney disease (PKD) progression in offspring for up to 1 year. Surprisingly, and in contrast to our initial hypothesis, exposure to maternal obesity during pregnancy and lactation did not significantly impact PKD severity in offspring at 3 mo or 1 yr of age. In contrast, reexposure to HFD for ∼3 m beginning at 12 wk of age worsened PKD severity in female, but not male, offspring born to obese dams as measured by cystic index, cyst number, and cyst area. Despite worsened cystic parameters, fibrosis and blood urea nitrogen were not altered in these animals. Collectively, these findings indicate that maternal obesity may accelerate PKD severity in female offspring exposed to an obesogenic diet.NEW & NOTEWORTHY Due to the growing obesity pandemic, almost one third of all children are born to mothers with obesity; however, the impact of maternal obesity on polycystic kidney disease (PKD) is unknown. In this manuscript, we found that maternal obesity did not worsen PKD severity in Pkd1^RC/RC mice at 3 mo or 1 yr of age when weaned onto normal chow diet. However, rechallenging pups born to obese mothers worsened PKD severity in female but not male mice.

In previously published work, we elucidated the role of cutaneous arsenical exposure in promoting acute kidney injury (AKI) in adult healthy mice. Here, we determine whether preexisting chronic kidney disease (CKD) increases the severity of AKI. Following exposure to aristolochic acid (AA) (a nephrotoxic phytochemical in humans), mice manifested classical markers of CKD, including robust interstitial fibrosis and loss in kidney function. Skin challenge with phenylarsine oxide (PAO), a surrogate for warfare arsenicals, led to significantly worse kidney injury, as evidenced by tubulointerstitial fibrosis, glomerulosclerosis, a persistent loss of estimated glomerular filtration rate, and mortality in AA-induced CKD mice compared with mice without CKD. These PAO-challenged CKD mice exhibited enhanced production of serum/urine neutrophil gelatinase-associated lipocalin and a significant rise in serum creatinine along with histological markers of kidney injury, including brush border loss, tubular atrophy, cast formation, glomerular injury, and interstitial inflammatory cell infiltration. Serum cytokines IL-4, IL-6, IFN-γ, IL-12p70, TNF-α, and IL-18 significantly elevated in CKD mice following PAO exposure when compared with animals exposed to PAO alone. Furthermore, we found increased TUNEL-positive tubular cells in the kidneys of CKD mice following PAO exposure, suggesting enhanced PAO-mediated cell death in CKD mice. Mechanistically, we determined that DNA damage-regulated p53 signaling was a major mediator of cellular responses to PAO in CKD mice. In summary, our data demonstrate that CKD significantly increased the severity of AKI following exposure to arsenicals and suggest that human populations with preexisting CKD could be highly susceptible to arsenical-mediated kidney injury and associated morbidity and mortality.NEW & NOTEWORTHY Preexisting chronic kidney disease (CKD) predisposes experimental animals to augmented morbidity and mortality following cutaneous vesicant exposure. The mechanism underlying increased susceptibility to renal injury and associated morbidity involves the DNA damage-regulated p53 signaling pathway.

Renal function can be perturbed by a range of stimuli that cause cellular injury and inflammation in the kidney. These injurious and inflammatory processes are typically dynamic and progressive, involving the actions of highly migratory cells such as leukocytes and cellular responses that occur over time spans ranging from seconds to weeks. Understanding these dynamic responses has entailed the use of imaging technologies that allow visualization and capture of events over different time spans, ideally in intact organs in live, experimental animals. The technique that allows this is intravital imaging. Intravital imaging, particularly multiphoton intravital microscopy, has been crucial to the investigation of dynamic physiological and pathophysiological processes in the kidney for many years, driving key developments in our understanding of renal (patho)physiology. This includes the mechanisms of ultrafiltrate generation, the response to acute kidney injury, and how inflammatory leukocytes are recruited to and cause injury in the kidney. This review describes the key studies that have applied intravital imaging to the investigation of models of inflammatory renal disease. The responses examined include those restricted to the glomerulus and the effects of acute kidney injury on the tubulointerstitium. Future innovations and directions in this field of research are also discussed.