Background Every year thousands of hypertensive patients reduce salt consumption in the efforts to control blood pressure. However, most of the studies report a cohort of patients who does not significantly respond to sodium restriction. Generally, most of the studies agreed that about one‐third of the patients had an excellent response, one‐third had only a modest response, and one‐third had little or no response. Irreversibility of high blood pressure is associated with various mechanisms underlying self‐sustaining character of hypertension. Unfortunately, hypertension causes multiple abnormalities in the body which stays even when the trigger, caused the initial blood pressure raise, is gone. We hypothesize that chronic hypertension leads to a significant renal damage and abnormally high sodium reabsorption. Methods We used Dahl salt‐sensitive rats for chronic continuous observation of blood pressure with radiotelemetry in conscious free moving animals. Rats were fed a 4% NaCl diet for 3 weeks to induce hypertension and then diet was switched back to normal (0.4% NaCl). Patch‐clamp analysis was performed on freshly isolated split‐open cortical collecting ducts and CHO cells to characterize activity of the epithelial sodium channel (ENaC), responsible for the sodium reabsorption in the aldosterone‐sensitive distal nephron. Results We found that 4% sodium diet significantly increases blood pressure within 3 weeks (from 111±0.9 to 138±3.5 mmHg) and switching back to 0.4% NaCl diet slightly reduces blood pressure (to 133.6±7.6). Patch clamp analysis reveals that development of hypertension was accompanied with elevated ENaC activity which also stayed high (NPo=1.22±0.23, n=8) after the salt challenge. Subsequent blockage of ENaC with a selective inhibitor, benzamil, caused a dramatic drop in blood pressure indicating that ENaC activity contributes to maintain sustaining hypertension in absence of the high salt consumption. We also found significant renal damage caused by hypertension. As earlier studies report that inflammation and reactive oxygen species production are involved in improper renal function and setting high blood pressure, we performed a subset of whole‐cell patch clamp experiments on CHO cells overexpressing mENaC to study effect of hydrogen peroxide on ENaC activity. We found that treatment with 100μM H2O2 within a few seconds to 2 minutes stimulated the ENaC current of the cells (−144±12 pA/pF vs −190±22 pA/pF before and after treatment respectively, n=8, p=0.0078). Based on our earlier publications and the current data we conclude that ENaC activity contributes to both development of salt‐sensitive hypertension and its continuation in the absence of high salt challenge and reactive oxygen species production can keep ENaC active. Support or Funding Information Acknowledgement ASN Carl W. Gottschalk award; R00 HL116603
Kir5.1 is an inwardly rectifying potassium (Kir) channel subunit abundantly expressed in the kidney and brain. We previously established the physiologic consequences of a Kcnj16 (gene encoding Kir5.1) knockout in the Dahl salt-sensitive rat (SSKcnj16–/–), which caused electrolyte/pH dysregulation and high-salt diet–induced mortality. Since Kir channel gene mutations may alter neuronal excitability and are linked to human seizure disorders, we hypothesized that SSKcnj16–/– rats would exhibit neurological phenotypes, including increased susceptibility to seizures. SSKcnj16–/– rats exhibited increased light sensitivity (fMRI) and reproducible sound-induced tonic-clonic audiogenic seizures confirmed by electroencephalography. Repeated seizure induction altered behavior, exacerbated hypokalemia, and led to approximately 38% mortality in male SSKcnj16–/– rats. Dietary potassium supplementation did not prevent audiogenic seizures but mitigated hypokalemia and prevented mortality induced by repeated seizures. These results reveal a distinct, nonredundant role for Kir5.1 channels in the brain, introduce a rat model of audiogenic seizures, and suggest that yet-to-be identified mutations in Kcnj16 may cause or contribute to seizure disorders.
Histamine is a nitrogenous compound crucial for the inflammatory response. The knowledge regarding the renal effects of histamine is very limited. We showed that renal epithelia exhibit expression of the components of the histaminergic system. Furthermore, we revealed that there was a shift in the histaminergic tone in salt-sensitive rats when they were challenged with a high-salt diet. These data support the notion that histamine plays a role in renal epithelial physiological and pathophysiological functions.
Amiloride‐sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate‐limiting step for Na + absorption. Epidermal growth factor (EGF) is involved in the regulation of Na + transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na + absorption. The aim of the present study was to characterize the EGF regulation of Na + transport in mpkCCD c14 cells. EGF dose dependently regulates basal transepithelial Na + transport in two phases: an acute phase (< 4 hrs) and a chronic phase (> 8 hrs). Similar effects were observed with TGF‐α, HB‐EGF and amphiregulin. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na + reabsorption. Inhibition of PI3‐kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD c14 cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in rat kidneys. Both acute and long term effects of EGF were abolished when cells were treated with tyrphostin AG‐825, a selective inhibitor of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na + transport and that EGF biphasically modulates sodium transport in mpkCCD c14 cells via the ErbB2 receptor. Supported by AHA and ASN.
Epithelial Na + Channel (ENaC) is critical for maintenance of sodium balance. Cortactin is a key signaling protein for many cellular processes, including direct interaction with F‐actin. In this study we have addressed the role of cortactin in mediating regulation of ENaC. As assessed by Western blotting, cortactin is highly expressed in mpkCCD c14 , M‐1 and MDCK cells. Immunohistochemistry analysis showed localization of cortactin in the cortical collecting duct in Sprague‐Dawley rat kidneys. Co‐immunoprecipitation analysis revealed that cortactin directly interacts with ENaC subunits when co‐expressed in CHO cells. We have supported these data with fluorescent microscopy co‐localization approach and fluorescence correlation spectroscopy analysis. Coexpression of cortactin with ENaC decreased channel's activity as measured by patch‐clamp. Surprisingly, cortactin's dynamin‐binding Src homology 3 domain is not required for ENaC regulation. Similarly cortactin mutants with deleted phosphorylation domain, or lacking ability to bind F‐actin decrease ENaC current. However cortactin mutant unable to bind Arp2/3 complex does not influence ENaC activity. Thus, we conclude that cortactin decreases ENaC activity via Arp2/3 complex. Supported by AHA and ASN.
Background ADPKD cysts contain high levels of ATP that contribute to cyst enlargement. Among other effects, ATP excess leads to a reduced reabsorption in cyst-lining cells and cyst fluid accumulation. We demonstrated that Pkd1RC/RC mice, a model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. Probenecid, a uricosuric agent, is also used as a pannexin-1 blocker reducing ATP release. We studied therapeutic potential of probenecid in Pkd1RC/RC mice and its effect on sodium reabsorption. Methods : Pkd1RC/RC mice, a hypomorphic model of ADPKD, were aged till 10.5 months and osmotic minipumps were implanted to deliver probenecid for 42 days. After treatment, 1 year old conscious mice were subjected to a measurement of glomerular filtration rate (GFR) and kidneys were collected for histomorphological studies. Effect of probenecid on Na reabsorption was tested on mpkCCD cells seeded onto permeable supports with open-circuit current measurements. Results In vivo inulin clearance study demonstrates that Pkd1RC/RC mice have normal GFR 1.05±0.09 ml/min/100g at the 6 months old age (n=15) whereas GFR in C57BL/6 mice - 0.94±0.1 ml/min/100g (n=8). With disease progression, GFR in Pkd1RC/RC mice reduces to 0.36±0.08 ml/min/100g at 12 months age. 42 days long probenecid treatment (15.9 mg/kg/day) significantly improves GFR to 1.43±0.11 ml/min/100g (p<0.001). Probenecid treatment also reduced kidney hypertrophy: kidney/TBW ratio in vehicle group was 2.54±0.17% vs 1.76±0.05% probenecid (p<0.05). Histological study on sectioned kidneys revealed that probenecid significantly reduces cyst size. Cyst to total slice area ratio was 13.9±2.7% (vehicle) vs 3.4±0.8% (probenecid) (n=5 each group). Earlier we have shown that probenecid decreases luminal ATP release in immortalized CD cell culture. As ATP is capable of downregulating Na reabsorption via the epithelial sodium channel we tested if probenecid increases ENaC activity. We applied probenecid to mpkCCD cell monolayer and found that the drug causes a bell-shaped dose-dependent increase of amiloride-sensitive transepithelial flux with maximal effect at 50 μM. Conclusion : Probenecid demonstrates therapeutic potential against ADPKD cyst progression in a Pkd1RC/RC mouse model by reducing cyst size, renal hypertrophy and supporting GFR and reabsorption from the cyst space.
Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1 V/V mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1 +/+ and Gpsm1 +/− mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via Gβγ subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease.
Podocytes play a key role in the development of nephropathy: podocyte depletion results in reduced GFR and progressive glomerulosclerosis. Circulating factors can activate calcium influx in these cells triggering processes that lead to podocyte loss. This study was aimed at developing a proper model of diabetic nephropathy (DN) on the basis of the Dahl Salt‐Sensitive (SS) rat that could be further used for assessing calcium signaling in podocytes. Type I diabetes was induced in 6 week old SS rats by an injection of 75 mg/kg streptozotocin (STZ) which caused an elevation of blood glucose up to 700 mg/dL; this was then decreased to 350 mg/dL by an insulin pellet implant. Rats were monitored throughout the next 6 or 12 weeks; urine analyses revealed progressive proteinuria, whereas sodium and potassium fractional excretion levels were normal in both control and diabetic groups (0.3 and 14 for fractional excretion of Na and K, respectively). However, 6 weeks after the STZ injection was not sufficient to develop histological features characteristic in DN, whereas the 12 week treatment resulted in typical DN kidney damage. Urinary ELISA showed a 50‐fold increase in nephrin excretion in the 12 week hyperglycemic animals, which is indicative of podocyte depletion and foot processes effacement. At the end of the experiment rat glomeruli were isolated and successfully used for ratiometric confocal measurements with fluorescent dyes or patch‐clamp measurements. In conclusion, this rat model shows the features of diabetic kidney injury and is a good basis for the assessment of calcium signaling and glomerular filtration barrier function during type 1 DN.
