Accelerated cystogenesis by dietary protein load is dependent on, but not initiated by kidney macrophages.

Background: Disease severity of autosomal dominant polycystic kidney disease (ADPKD) is influenced by diet. Dietary protein, a recognized cyst-accelerating factor, is catabolized into amino acids (AA) and delivered to the kidney leading to renal hypertrophy. Injury-induced hypertrophic signaling in ADPKD results in increased macrophage (MФ) activation and inflammation followed by cyst growth. We hypothesize that the cystogenesis-prompting effects of HP diet are caused by increased delivery of specific AA to the kidney, ultimately stimulating MФs to promote cyst progression. Methods: Pkd1flox/flox mice with and without Cre (CAGG-ER) were given tamoxifen to induce global gene deletion (Pkd1KO). Pkd1KO mice were fed either a low (LP; 6%), normal (NP; 18%), or high (HP; 60%) protein diet for 1 week (early) or 6 weeks (chronic). Mice were then euthanized and tissues were used for histology, immunofluorescence and various biochemical assays. One week fed kidney tissue was cell sorted to isolate tubular epithelial cells for RNA sequencing. Results: Chronic dietary protein load in Pkd1KO mice increased kidney weight, number of kidney infiltrating and resident MФs, chemokines, cytokines and cystic index compared to LP diet fed mice. Accelerated cyst growth induced by chronic HP were attenuated by liposomal clodronate-mediated MФ depletion. Early HP diet fed Pkd1KO mice had larger cystic kidneys compared to NP or LP fed counterparts, but without increases in the number of kidney MФs, cytokines, or markers of tubular injury. RNA sequencing of tubular epithelial cells in HP compared to NP or LP diet group revealed increased expression of sodium-glutamine transporter Snat3, chloride channel Clcnka, and gluconeogenesis marker Pepck1, accompanied by increased excretion of urinary ammonia, a byproduct of glutamine. Early glutamine supplementation in Pkd1KO mice lead to kidney hypertrophy. Conclusion: Chronic dietary protein load-induced renal hypertrophy and accelerated cyst growth in Pkd1KO mice is dependent on both infiltrating and resident MФ recruitment and subsequent inflammatory response. Early cyst expansion by HP diet, however, is relient on increased delivery of glutamine to kidney epithelial cells, driving downstream metabolic changes prior to inflammatory provocation.

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Endothelin A receptor antagonist attenuated renal iron accumulation in iron overload heme oxygenase-1 knockout mice.

Progressive iron accumulation and renal impairment are prominent in both patients and mouse models of sickle cell disease (SCD). Endothelin A receptor (ETA) antagonism prevents this iron accumulation phenotype and reduces renal iron deposition in the proximal tubules of SCD mice. To better understand the mechanisms of iron metabolism in the kidney and the role of the ETA receptor in iron chelation and transport, we studied renal iron handling in a nonsickle cell iron overload model, heme oxygenase-1 (Hmox-1-/-) knockout mice. We found that Hmox-1-/- mice had elevated plasma endothelin-1 (ET-1), cortical ET-1 mRNA expression, and renal iron content compared with Hmox-1+/+ controls. The ETA receptor antagonist, ambrisentan, attenuated renal iron deposition, without any changes to anemia status in Hmox-1-/- mice. This was accompanied by reduced urinary iron excretion. Finally, ambrisentan had an important iron recycling effect by increasing the expression of the cellular iron exporter, ferroportin-1 (FPN-1), and circulating total iron levels in Hmox-1-/- mice. These findings suggest that the ET-1/ETA signaling pathway contributes to renal iron trafficking in a murine model of iron overload.

Role for ovarian hormones in purinoceptor-dependent natriuresis.

BACKGROUND: Premenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats. METHODS: To test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750 µg/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1δ and ENaCα) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000 nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors. RESULTS: Acute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3. CONCLUSION: These data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Dynamic changes in histone deacetylases following kidney ischemia-reperfusion injury are critical for promoting proximal tubule proliferation.

Deranged histone deacetylase (HDAC) activity causes uncontrolled proliferation, inflammation, fibrosis, and organ damage. It is unclear whether deranged HDAC activity results in acute kidney injury in the renal hypoperfusion model of bilateral ischemia-reperfusion injury (IRI) and whether in vivo inhibition is an appropriate therapeutic approach to limit injury. Male mice were implanted with intraperitoneal osmotic minipumps containing vehicle, the class I HDAC inhibitor, MS275, or the pan-HDAC inhibitor, trichostatin A (TSA), 3 days before sham/bilateral IRI surgery. Kidney cortical samples were analyzed using histological, immunohistochemical, and Western blotting techniques. HDAC-dependent proliferation rate was measured in immortalized rat epithelial cells and primary mouse or human proximal tubule (PT) cells. There were dynamic changes in cortical HDAC localization and abundance following IRI including a fourfold increase in HDAC4 in the PT. HDAC inhibition resulted in a significantly higher plasma creatinine, increased kidney damage, but reduced interstitial fibrosis compared with vehicle-treated IRI mice. HDAC-inhibited mice had reduced interstitial α-smooth muscle actin, fibronectin expression, and Sirius red-positive area, suggesting that IRI activates HDAC-mediated fibrotic pathways. In vivo proliferation of the kidney epithelium was significantly reduced in TSA-treated, but not MS275-treated, IRI mice, suggesting class II HDACs mediate proliferation. Furthermore, HDAC4 activation increased proliferation of human and mouse PTs. Kidney HDACs are activated during IRI with isoform-specific expression patterns. Our data point to mechanisms whereby IRI activates HDACs resulting in fibrotic pathways but also activation of PT proliferation and repair pathways. This study demonstrates the need to develop isoform-selective HDAC inhibitors for the treatment of renal hypoperfusion-induced injury.

Hyperfiltration during early childhood precedes albuminuria in pediatric sickle cell nephropathy.

BACKGROUND: In patients with diabetes mellitus, hyperfiltration precedes the development of albuminuria. Pediatric sickle cell anemia (SCA) patients have a high prevalence of hyperfiltration and albuminuria during early childhood and adolescence. We tested the hypothesis that hyperfiltration precedes the development of albuminuria in a longitudinal pediatric SCA cohort. METHODS: We identified 91 participants with HbSS or SB0 thalassemia 5-21 years of age enrolled in a longitudinal sickle cell nephropathy cohort study who had a cystatin C measured during early childhood (4-10 years of age). Early hyperfiltration was defined as a mean eGFR >180 mL/min/1.73m2 using cystatin C obtained from 4 to 10 years of age. Persistent albuminuria was defined as an albumin to creatinine ratio > 30 mg/g on two of three untimed urine specimens. Time to event analysis estimated survival curves for participants with and without hyperfiltration using Kaplan-Meier curves and used logrank test for categorical variables to assess the association with time to development of the first episode persistent albuminuria. RESULTS: Persistent albuminuria occurred more often and at an earlier age in participants with early hyperfiltration compared to those without early hyperfiltration (log-rank, P = .004). Participants who developed albuminuria have a significant increase in their eGFR during childhood (P = .003) as compared to participants who have not yet progressed to albuminuria (P = .26). For every 1 g/dL increase in hemoglobin, the hazard ratio for developing persistent proteinuria decreased by 0.56 (95% CI: 0.3, 1.06, P = .07). CONCLUSION: Hyperfiltration precedes the development of persistent proteinuria in pediatric SCA patients. Intervention strategies should target lowering eGFR during early childhood.

Interplay between renal endothelin and purinergic signaling systems.

Alterations in extracellular fluid volume regulation and sodium balance may result in the development and maintenance of salt-dependent hypertension, a major risk factor for cardiovascular disease. Numerous pathways contribute to the regulation of sodium excretion and blood pressure, including endothelin and purinergic signaling. Increasing evidence suggests a link between purinergic receptor activation and endothelin production within the renal collecting duct as a means of promoting natriuresis. A better understanding of the relationship between these two systems, especially in regard to sodium homeostasis, will fill a significant knowledge gap and may provide novel antihypertensive treatment options. Therefore, this review focuses on the cross talk between endothelin and purinergic signaling as it relates to the renal regulation of sodium and blood pressure homeostasis.

Intracellular calcium signaling regulates glomerular filtration barrier permeability: the role of the PKGIα-dependent pathway.

Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. Insulin provoked a sustained, approximately 70%, increase in intracellular calcium concentration in podocytes. RT-PCR revealed the presence of mRNA encoding sarco/endoplasmic reticulum calcium ATPase isoforms 1-3, and plasma membrane Ca(2+) pump (PMCA) isoforms 1,3,4; mRNA levels were depressed by the addition of insulin. Inhibitors of PMCA, and the Na(+) -Ca(2+) exchanger, increased podocyte permeability to albumin, induced dimerization of protein kinase G type I alpha (PKGIα), and activation of PKGIα-dependent signaling. These data suggest the involvement of calcium and PKGIα signaling in insulin-enhanced filtration barrier permeability in podocytes.

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.

Endothelin-1 and the kidney: new perspectives and recent findings.

PURPOSE OF REVIEW: The role of endothelin-1 (ET-1) in the kidney has been under study for many years; however, the complex mechanisms by which endothelin controls the physiology/pathophysiology of this organ are not fully resolved. This review aims to summarize recent findings in the field, especially regarding glomerular and tubular damage, Na/water homeostasis and sex differences in ET-1 function. RECENT FINDINGS: Podocytes have been recently identified as a target of ET-1 in the glomerular filtration barrier via ETA receptor activation. Activation of the ETA receptor by ET-1 leads to renal tubular damage by promoting endoplasmic reticulum stress and apoptosis in these cells. In addition, high flow rates in the nephron in response to high salt intake induce ET-1 production by the collecting ducts and promote nitric oxide-dependent natriuresis through epithelial sodium channel inhibition. Recent evidence also indicates that sex hormones regulate the renal ET-1 system differently in men and women, with estrogen suppressing renal ET-1 production and testosterone upregulating that production. SUMMARY: Based on the reports reviewed in here, targeting of the renal endothelin system is a possible therapeutic approach against the development of glomerular injury. More animal and clinical studies are needed to better understand the dimorphic control of this system by sex hormones.

Insulin increases glomerular filtration barrier permeability through PKGIα-dependent mobilization of BKCa channels in cultured rat podocytes.

Podocytes are highly specialized cells that wrap around glomerular capillaries and comprise a key component of the glomerular filtration barrier. They are uniquely sensitive to insulin; like skeletal muscle and fat cells, they exhibit insulin-stimulated glucose uptake and express glucose transporters. Podocyte insulin signaling is mediated by protein kinase G type I (PKGI), and it leads to changes in glomerular permeability to albumin. Here, we investigated whether large-conductance Ca²âº-activated K⁺ channels (BKCa) were involved in insulin-mediated, PKGIα-dependent filtration barrier permeability. Insulin-induced glomerular permeability was measured in glomeruli isolated from Wistar rats. Transepithelial albumin flux was measured in cultured rat podocyte monolayers. Expression of BKCa subunits was detected by RT-PCR. BKCa, PKGIα, and upstream protein expression were examined in podocytes with Western blotting and immunofluorescence. The BKCa-PKGIα interaction was assessed with co-immunoprecipitation. RT-PCR showed that primary cultured rat podocytes expressed mRNAs that encoded the pore-forming α subunit and four accessory ß subunits of BKCa. The BKCa inhibitor, iberiotoxin (ibTX), abolished insulin-dependent glomerular albumin permeability and PKGI-dependent transepithelial albumin flux. Insulin-evoked albumin permeability across podocyte monolayers was also blocked with BKCa siRNA. Moreover, ibTX blocked insulin-induced disruption of the actin cytoskeleton and changes in the phosphorylation of PKG target proteins, MYPT1 and RhoA. These results indicated that insulin increased filtration barrier permeability through mobilization of BKCa channels via PKGI in cultured rat podocytes. This molecular mechanism may explain podocyte injury and proteinuria in diabetes.

Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits.

The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300nM, 5min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.

Chronic renal denervation increases renal tubular response to P2X receptor agonists in rats: implication for renal sympathetic nerve ablation.

BACKGROUND: Kidney noradrenergic innervation regulates tubular function. Adenosine triphosphate (ATP)-a co-transmitter of norepinephrine-acts on purinoceptors, including ion channel receptor, P2X. P2X receptor agonists, α,ß-methylene ATP (α,ß-meATP) and ß,γ-methylene ATP (ß,γ-meATP), induce natriuresis. Regarding the functional co-localization of adrenoceptors and P2X receptors, we evaluated rat renal tubular system sensitivity to natriuretic action of P2X receptor agonists in chronically denervated kidney. METHODS: Clearance studies with α,ß-meATP and ß,γ-meATP (intravenous infusion rate, 2 µmol/kg + 20 nmol/kg/min) were performed after bilateral surgical kidney denervation (DNx) and sham-operation (Sham). Na/K-ATPase activity was measured in isolated rat renal proximal tubules. RESULTS: In DNx compared with Sham, saline infusion significantly increased renal sodium and urine excretion and P2X receptor agonist infusion was significantly more natriuretic and diuretic. In DNx and Sham, respectively, α,ß-meATP increased fractional excretion of sodium (FE(Na)) by 2 ± 0.3 and 0.6 ± 0.1% and urine (FE(V)) by 1.6 ± 0.3 and 0.9 ± 0.2%; ß,γ-meATP had similar effects. In both groups of rats, natriuretic and diuretic actions were abolished by P2 receptor blocker (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate, PPADS), mean arterial blood pressure and glomerular filtration rate remained unchanged during infusion of P2X receptor agonists and antagonist and basal Na/K-ATPase activities in isolated proximal tubules were similar. Both α,ß-meATP and ß,γ-me-ATP decreased the Na/K-ATPase activity, with 20% inhibition (P < 0.05) in denervated and innervated rats; these inhibitory effects were abolished in the presence of PPADS. CONCLUSIONS: Decreased renal sympathetic activity enhances the natriuretic effect of P2X receptor stimulation. This effect is probably not related to altered Na/K-ATPase activity in renal proximal tubules.