The angiotensin AT2 receptor (AT2R) has been shown to lower inflammation in the kidney. However the role of the anti-inflammatory cytokine IL-10 in AT2R mediated attenuation of inflammation has not been elucidated. We hypothesized that AT2R activation is renoprotective by directly increasing the levels of anti-inflammatory cytokine IL-10 in the kidney via nitric oxide (NO) signaling. For in vitro studies, the human proximal tubule epithelial cell-line (HK-2) was activated with lipopolysaccharide (LPS, 10 μg/ml) and/or AT2R agonist C21 (1μmol/L) for 24 hours and media cytokine levels were assessed. LPS modestly downregulated AT2R expression. Treatment with C21 lowered LPS-induced levels of both, TNF-α and IL-6 but increased IL-10 levels. Treatment with neutralizing IL-10 antibody (1 μg/ml) or NO synthase inhibitor L-NAME (1 mmol/L) abolished this effect. For in vivo studies, pre-hypertensive obese Zucker rats (OZR) and age-matched lean Zucker rats were treated for 2 weeks with C21 (300 μg/kg/day, i.p) and/or AT2R antagonist (PD123319, 50 μg/kg/min, s.c. infusion). Compared to LZR, OZR had higher levels of renal AT2R expression, TNF-α and IL-6. C21 treatment decreased levels of TNF-α by 75% and IL-6 by 60%. Conversely, PD treatment lowered the renal IL-10 levels in OZR by ~60%. Renal morphometry revealed increased mesangial matrix expansion and glomerular macrophage infiltration which was improved by C21 treatment in OZR. Our findings suggest that proximal tubule AT2R activation is anti-inflammatory by increasing IL-10 production which is largely NO-dependent and thus offers renoprotection by preventing early inflammation-induced renal injury in obesity.

Macrophages, via activation of the Toll-like receptors (TLR4), play an important role in the pathogenesis of hypertension and associated end-organ damage. There is accumulating evidence to suggest a protective role of the angiotensin AT2 receptor (AT2R) in pathological conditions involving inflammation and tissue injury. We have recently shown that AT2R stimulation is renoprotective, in part, via increased anti-inflammatory interleukin-10 (IL-10) production in renal epithelial cells, however the role of AT2R in macrophage inflammatory behavior is not known. The present study was designed to investigate whether AT2R activation exerts an anti-inflammatory response in TLR4-induced inflammation. The anti-inflammatory mechanisms of AT2R agonist C21 (1 µmol/ml) pre-treatment on the cytokine profile of THP-1 macrophages after activation by LPS (1 µg/ml) was studied. The AT2R agonist dose-dependently attenuated LPS-induced TNF-α and IL-6 production but increased IL-10 production. IL-10 was critical for the anti-inflammatory effect of AT2R stimulation, since IL-10 neutralizing antibody dose-dependently abolished the AT2R–mediated decrease in TNF-α level. Further, the enhanced IL-10 levels were associated with a sustained, selective increase in phosphorylation of extracellular signal-regulated kinase (ERK1/2), but not p38 MAPK. Blocking the activation of ERK1/2 prior to C21 pre-treatment completely abrogated this increased IL-10 production in response to AT2R agonist C21, while there was a partial reduction in IL-10 levels on inhibition of p38. We conclude that AT2R stimulation exerts a novel anti-inflammatory response in THP-1 macrophages via enhanced IL-10 production as a result of sustained, selective ERK1/2 phosphorylation, and thus may have protective role in hypertension and associated tissue injury.

Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.

High blood pressure exerts its deleterious effects on health largely through acceleration of end organ diseases. Among these, progressive loss of renal function is particularly important, not only for the direct consequences of kidney damage, but also because loss of renal function is associated with amplification of other adverse cardiovascular outcomes. Genetic susceptibility to hypertension and associated end organ disease is non-Mendelian in both humans and in a rodent model, the spontaneously hypertensive rat (SHR). Here we report that hypertensive end organ disease in the inbred SHR-A3 line is attributable to genetic variation in the immunoglobulin heavy chain on chromosome 6. This variation coexists with variation in a 10 Mbase block on chromosome 17 that contains genetic variation in two genes involved in immunoglobulin Fc receptor signaling. Substitution of these genomic regions into the SHR-A3 genome from the closely-related, but injury resistant, SHR-B2 line normalizes both biomarker and histological measures of renal injury. Our findings indicate that genetic variation leads to a contribution by immune mechanisms hypertensive end organ injury and that, in this rat model, disease is influenced by differences in germ-line antibody repertoire.

Immune cell infiltration plays a central role in mediating endotoxemic acute kidney injury (AKI). Recently, we have reported the anti-inflammatory and reno-protective role of angiotensin-II type-2 receptor (AT2R) activation under chronic low-grade inflammatory condition in the obese Zucker rat model. However, the role of AT2R activation in preventing lipopolysaccharide (LPS)-induced early infiltration of immune cells, inflammation and AKI is not known. Mice were treated with AT2R agonist C21 (0.3 mg/kg), with and without AT2R antagonist PD123319 (5 mg/kg) prior to or concurrently with LPS (5 mg/kg) challenge. Prior-treatment with C21, but not concurrent treatment, significantly prevented the LPS-induced renal infiltration of CD11b+ immune cells, increase in the levels of circulating and/or renal chemotactic cytokines, particularly interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) and markers of renal dysfunction (blood urea nitrogen and albuminuria), while preserving anti-inflammatory interleukin-10 (IL-10) production. Moreover, C21 treatment in the absence of LPS increased renal and circulating IL-10 levels. To investigate the role of IL-10 in a cross-talk between epithelial cells and monocytes, we performed in vitro conditioned media (CM) studies in human kidney proximal tubular epithelial (HK-2) cells and macrophages (differentiated human monocytes, THP-1 cells). These studies revealed that the conditioned-media derived from the C21-treated HK-2 cells reduced LPS-induced THP-1 tumor necrosis factor-α (TNF-α) production via IL-10 originating from HK-2 cells. Our findings suggest that prior activation of AT2R is prophylactic in preventing LPS-induced renal immune cell infiltration and dysfunction, possibly via IL-10 pathway.

Introduction:Renin–angiotensin system (RAS) components exert diverse physiological functions and have been sub-grouped into deleterious angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/angiotensin type 1 receptor (AT1R) and protective ACE2/angiotensin (1-7) (Ang-(1-7))/Mas receptor (MasR) axes. We have reported that chronic activation of angiotensin type 2 receptor (AT2R) alters RAS components and provides protection against obesity-related kidney injury.Materials and methods:We utilized AT2R knockout (AT2KO) mice in this study and evaluated the renal expression of various RAS components and examined the renal injury after placing these mice on high fat diet (HFD) for 16 weeks.Results:The cortical ACE2 activity and MasR expression were significantly decreased in AT2KO mice compared to wild type (WT) mice. LC/MS analysis revealed an increase in renal Ang II levels and a decrease in Ang-(1-7) levels in AT2KO mice. Cortical expression of ACE and AT1R was increased but renin activity remained unchanged in AT2KO compared with WT mice. WT mice fed HFD exhibited increased systolic blood pressure, higher indices of kidney injury, mesangial matrix expansion score, and microalbuminuria, which were further increased in AT2KO mice.Conclusion:This study suggests that deletion of AT2R decreases the expression of the beneficial ACE2/Ang-(1-7)/MasR and increases the deleterious ACE/Ang II/AT1R axis of the renal RAS in mice. Further, AT2KO mice are more susceptible to HFD-induced renal injury.

Background Spontaneously hypertensive rats of the stroke‐prone line ( SHR ‐A3) develop hypertensive renal disease as a result of naturally occurring genetic variation. Our prior work identified a single‐nucleotide polymorphism unique to SHR ‐A3 that results in truncation of the carboxy terminus of STIM 1. The SHR ‐B2 line, which is also hypertensive but resists hypertensive renal injury, expresses the wild‐type STIM 1. STIM 1 plays a central role in lymphocyte calcium signaling that directs immune effector responses. Here we show that major defects in lymphocyte function affecting calcium signaling, nuclear factor of activated T cells activation, cytokine production, proliferation, apoptosis, and regulatory T‐cell development are present in SHR ‐A3 and attributable to STIM1. Methods and Results To assess the role of Stim1 variation in susceptibility to hypertensive renal injury, we created a Stim1 congenic line, SHR ‐A3( Stim1 ‐B2), and STIM1 function was rescued in SHR ‐A3. We found that Stim1 gene rescue restores disturbed lymphocyte function in SHR ‐A3. Hypertensive renal injury was compared in SHR ‐A3 and the SHR ‐A3( Stim1 ‐B2) congenic line. Histologically assessed renal injury was markedly reduced in SHR ‐A3( Stim1 ‐B2), as were renal injury biomarker levels measured in urine. Stim1 deficiency has been linked to the emergence of antibody‐mediated autoimmunity. Renal glomerular immunoglobulin deposition was greater in SHR ‐A3 than SHR ‐B2 and was reduced by Stim1 congenic substitution. Serum anti–double‐stranded DNA antibody titers in SHR ‐A3 were elevated compared with SHR ‐B2 and were reduced in SHR ‐A3( Stim1 ‐B2). Conclusions Stim1 deficiency in lymphocyte function originating from Stim1 truncation in SHR ‐A3 combines with hypertension to create end organ disease and may do so as a result of antibody formation.

Rat genomic tools have been slower to emerge than for those of humans and mice and have remained less thorough and comprehensive. The arrival of a new and improved rat reference genome, mRatBN7.2, in late 2020 is a welcome event. This assembly, like predecessor rat reference assemblies, is derived from an inbred Brown Norway rat. In this "user" survey we hope to provide other users of this assembly some insight into its characteristics and some assessment of its improvements as well as a few caveats that arise from the unique aspects of this assembly. mRatBN7.2 was generated by the Wellcome Sanger Institute as part of the large Vertebrate Genomes Project. This rat assembly has now joined human, mouse, chicken and zebrafish in the NCBI's Genome Reference Consortium, which provides ongoing curation of the assembly. Here we examine the technical procedures by which the assembly was created and assess how this assembly constitutes an improvement over its predecessor. We also indicate the technical limitations affecting the assembly, providing illustrations of how these limitations arise and the impact that results for this reference assembly.