Splenocyte transfer from hypertensive donors eliminates premenopausal female protection from Ang II-induced hypertension

Premenopausal females are protected from Angiotensin II (Ang II)-induced hypertension following the adoptive transfer of T cells from normotensive donors. For the present study, we hypothesized that the transfer of hypertensive T cells (HT) or splenocytes (HS) from hypertensive donors would eliminate premenopausal protection from hypertension. Premenopausal Rag-1-/- females received either normotensive (NT) or hypertensive cells, three weeks prior to Ang II infusion (14 days, 490 ng/kg/min). Contrary to our hypothesis, no increase in Ang II-induced blood pressure was observed in the NT/Ang or HT/Ang groups. Flow cytometry demonstrated that renal FoxP3+ T regulatory cells were significantly decreased and IHC showed an increase in renal F4/80+ macrophages in HT/Ang, suggesting a shift in the renal inflammatory environment despite no change in blood pressure. Renal mRNA expression of MCP-1, Endothelin-1, GPER-1 were significantly decreased in HT/Ang. The adoptive transfer of hypertensive splenocytes prior to Ang II infusion (HS/Ang) eliminated premenopausal protection from hypertension and significantly decreased splenic FoxP3+ T regulatory cells compared to females receiving normotensive splenocytes (NS/Ang). Expression of MIP-1a/CCL3, a potent macrophage chemokine was elevated in HS/Ang, however no increase in renal macrophage infiltration occurred. Together, these data show that in premenopausal females T cells from hypertensive donors are not sufficient to induce a robust Ang II mediated hypertension, in contrast, transfer of hypertensive splenocytes (consisting of T/B lymphocytes, dendritic cells, macrophages) is sufficient. Further work is needed to understand how innate and adaptive immune cells and estrogen signaling coordinate to cause differential hypertensive outcomes in premenopausal females.

Acid-Base Effects of Combined Renal Deletion of NBCe1-A and NBCe1-B

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deleting the A splice variant of the Na⁺-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex, but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The current studies determined the effects of combined renal deletion of NBCe1-A and NBCe1-B on systemic and proximal tubule ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. Since renal NBCe1-A and NBCe1-B expression is limited to the proximal tubule, Cre-positive mice had proximal tubule NBCe1-A/B deletion (PT-NBCe1-A/B KO). While on basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased expression of phosphate-dependent glutaminase (PDG) and phospho­enol­pyruvate carboxy­kinase (PEPCK) and increased expression of glutamine synthetase (GS), an ammonia recycling enzyme, in PT in both the cortex and OM. Exogenous acid-loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in PDG, PEPCK, and GS expression in the proximal tubule in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to proximal tubule ammonia metabolism in the OM and thereby to systemic acid-base regulation.

KV7.1 channel blockade inhibits neonatal renal autoregulation triggered by a step decrease in arterial pressure

KV7, the voltage-gated potassium channels encoded by KCNQ genes, mediate heterogeneous vascular responses in adult rodents. Postnatal changes in the functional expression of KV7 channels have been reported in rodent saphenous arteries, but their physiological function in the neonatal renal vascular bed is unclear. Here, we report that, unlike adult pigs, only KCNQ1 (KV7.1) out of the five members of KCNQ genes was detected in neonatal pig renal microvessels. KCNQ1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21. Activation of the renal vascular smooth muscle cell (SMC) KV7.1 stimulated whole-cell currents, inhibited by HMR1556 (HMR), a selective KV7.1 blocker. HMR did not change the steady-state diameter of isolated renal microvessels. Similarly, intrarenal artery infusion of HMR did not alter the mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) in the pigs. An approximately 20 mmHg reduction in the MAP evoked effective autoregulation of the RBF, which HMR inhibited. We conclude that 1) The expression of KCNQ isoforms in porcine renal microvessels is dependent on kidney maturation, 2) KV7.1 is functionally expressed in neonatal pig renal vascular SMCs, 3) a decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs, and 4) SMC KV7.1 does not control basal renal vascular tone but contributes to neonatal renal autoregulation triggered by a step decrease in arterial pressure.

A mathematical model of the rat kidney IV: whole-kidney response to hyperkalemia

The renal response to acute hyperkalemia is mediated by increased K secretion within connecting tubule (CNT), flux that is modulated by tubular effects (e.g. aldosterone) in conjunction with increased luminal flow. There is ample evidence that peritubular K blunts Na reabsorption in proximal tubule, thick ascending Henle limb, and distal convoluted tubule (DCT). While any such reduction may augment CNT delivery, the relative contribution of each is uncertain. The kidney model of this lab was recently advanced with representation of cortical labyrinth and medullary ray. Model tubules capture the impact of hyperkalemia to blunt Na reabsorption within each upstream segment. However, this forces the question of the extent to which increased Na delivery is transmitted past macula densa and its tubuloglomerular feedback (TGF) signal. Beyond increasing macula densa Na delivery, peritubular K is predicted to raise cytosolic Cl and depolarize macula densa cells, which may also activate TGF. Thus, although upstream reduction in Na transport may be larger, it appears that the DCT effect is critical to increasing CNT delivery. Beyond the flow effect, hyperkalemia reduces ammoniagenesis and reduced ammoniagenesis enhances K excretion. What this model provides is a possible mechanism. When cortical NH4 is taken up via peritubular Na,K(NH4)-ATPase, it acidifies principal cells. Consequently, reduced ammoniagenesis increases principal cell pH, thereby increasing conductance of both ENaC and ROMK, enhancing K excretion. In this model, aldosterone's effect on principal cells, diminished DCT Na reabsorption, and reduced ammoniagenesis, all provide relatively equal and additive contributions to renal K excretion.

Multiomic identification of factors associated with progression to cystic kidney disease in mice with nephron Ift88 disruption

Ift88 gene mutations cause primary cilia loss and polycystic kidney disease (PKD) in mice. Nephron Ift88 knockout (KO) at 2 months postnatal does not affect renal histology at 4 months postnatal and causes PKD only in males by 11 months postnatal. To identify factors associated with PKD development, kidneys from 4-month-old male and female control and Ift88 KO mice underwent transcriptomic, proteomic, western, metabolomic and lipidomic analysis. mRNAs involved in extracellular matrix (ECM) synthesis and degradation were selectively upregulated in male KO mice. Proteomic analysis was insufficiently sensitive to detect most ECM components, while western analysis paradoxically revealed reduced fibronectin and collagen I in male KO mice. Only male KO mice upregulated mRNAs encoding fibrinogen subunits and receptors for VEGF and PDGF; Per2, Per3 and Nrld2 clock mRNAs were selectively decreased in male KO mice. Proteomic, metabolomic and lipidomic analysis detected a relative (vs same sex control) decrease in factors involved in fatty acid ß-oxidation in female KO, while increased or unchanged levels in male KO, mice including medium chain acyl-CoA dehydrogenase, 3-hydroxybutyrate, and acylcarnitine. Three putative mRNA biomarkers of cystogenesis in male Ift88 KO mice (similar control levels between sexes and uniquely altered by KO in males) were identified, including high levels (Fga and Sdf2l1) and low levels (Banp) in male KO mice. These findings suggest that relative alterations in renal ECM metabolism, fatty acid ß-oxidation, and other pathways precede cystogenesis in Ift88 KO mice. In addition, potential novel biomarkers of cystogenesis in Ift88 KO mice have been identified.

Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.

Mineralocorticoid receptor blockade with spironolactone has no direct effect on plasma IL-17A and injury markers in urine from kidney transplant patients

Kidney transplantation is associated with increased risk of cardiovascular morbidity. Interleukin-17A (IL-17A) mediates kidney injury. Aldosterone promotes T-helper-17 (Th-17) lymphocyte differentiation and IL-17A production through the mineralocorticoid receptor (MR). In this exploratory, post-hoc substudy, it was hypothesized that 1-year intervention with the MR antagonist spironolactone lowers IL-17A and related cytokines and reduces epithelial injury in kidney transplant recipients. Plasma and urine samples were obtained from kidney transplant recipients from a double-blind randomized clinical trial testing spironolactone (n=39) versus placebo (n=41). Plasma concentrations of cytokines IFN-γ, IL-17A, TNF-α, IL-6, IL-1β, and IL-10 were determined before and after 1-year treatment. Urine calbindin, clusterin, KIM-1, osteoactivin, TFF3, and VEGF/creatinine ratios were analyzed. Blood pressure and plasma aldosterone concentration at inclusion did not relate to plasma cytokines and injury markers. None of the cytokines changed in plasma after spironolactone intervention. Plasma IL-17A increased in the placebo group. Spironolactone induced an increase in plasma K+ (0.4 ± 0.4 mmol/L). This increase did not correlate with plasma IL-17A or urine calbindin and TFF3 changes. Ongoing treatment at inclusion with angiotensin-converting-enzyme inhibitor and/or angiotensin II receptor blockers was not associated with changed levels of IL-17A and injury markers and had no effect on the response to spironolactone. Urinary calbindin and TFF3 decreased in the spironolactone group with no difference in between-group analyses. In conclusion, irrespective of ongoing ANGII inhibition, spironolactone has no effect on plasma IL-17A and related cytokines or urinary injury markers in kidney transplant recipients.

Adaptive Changes in single-nephron GFR, Tubular Morphology, and Transport in a Pregnant Rat Nephron: Modeling and Analysis

Normal pregnancy is characterized by massive increases in plasma volume and electrolyte retention. Given that the kidneys regulate homeostasis of electrolytes and volume, the organ undergoes major adaptations in morphology, hemodynamics, and transport to achieve the volume and electrolyte retention required in pregnancy. These adaptations are complex, sometimes counterintuitive, and not fully understood. In addition, the demands of the developing fetus and placenta change throughout the pregnancy. For example, during late pregnancy, K+ retention and thus enhanced renal K+ reabsorption is required despite many kaliuretic factors. The goal of this study is to unravel how known adaptive changes along the nephrons contribute to the ability of the kidney to meet volume and electrolyte requirements in mid- and late pregnancy. We developed computational models of solute and water transport in the superficial nephron of the kidney of a rat in mid- and late pregnancy. The mid-pregnant and late-pregnant rat superficial nephron models predict that morphological adaptations and increased activity of the sodium hydrogen exchanger 3 (NHE3) and epithelial sodium channel (ENaC) are essential for enhanced Na+ reabsorption observed during pregnancy. Model simulations showed that for sufficient K+ reabsorption, increased H +-K +-ATPase activity and decreased K+ secretion along the distal segments is required in both mid- and late-pregnancy. Furthermore, certain known sex differences in renal transporter pattern (e.g., the higher NHE3 protein abundance but lower activity in the proximal tubules of virgin female rats compared to male) may serve to better prepare the female for the increased transport demand in pregnancy.

IL-1 receptor signaling in podocytes limits susceptibility to glomerular damage

IL-1 receptor (IL-1R1) activation triggers a proinflammatory signaling cascade that can exacerbate kidney injury. However, the functions of the podocyte IL-1R1 in glomerular disease remain unclear. To study the role of IL-1R1 signaling in podocytes, we selectively ablated the podocyte IL-1R1 in mice (PKO). We then subjected PKO mice and wild-type (WT) controls to 2 glomerular injury models: nephrotoxic serum (NTS)- and adriamycin (ADR)-induced nephropathy. Surprisingly, we found IL-1R1 activation in podocytes limited albuminuria and podocyte injury during NTS- and ADR-induced nephropathy. Moreover, deletion of IL-1R1 in podocytes drove podocyte apoptosis and glomerular injury through diminishing Akt activation. Activation of Akt signaling abrogated the differences in albuminuria and podocyte injury between WT and PKO mice during NTS. Thus, IL-1R1 signaling in podocytes limits susceptibility to glomerular injury via an Akt-dependent signaling pathway. These data identify an unexpected protective role for IL-1R1 signaling in podocytes in the pathogenesis of glomerular disease.