Calcium signalling and transport in the kidney.

The kidney plays a pivotal role in regulating calcium levels within the body. Approximately 98% of the filtered calcium is reabsorbed in the nephron, and this process is tightly controlled to maintain calcium homeostasis, which is required to facilitate optimal bone mineralization, preserve serum calcium levels within a narrow range, and support intracellular signalling mechanisms. The maintenance of these functions is attributed to a delicate balance achieved by various calcium channels, transporters, and calcium-binding proteins in renal cells. Perturbation of this balance due to deficiency or dysfunction of calcium channels and calcium-binding proteins can lead to severe complications. For example, polycystic kidney disease is linked to aberrant calcium transport and signalling. Furthermore, dysregulation of calcium levels can promote the formation of kidney stones. This Review provides an updated description of the key aspects of calcium handling in the kidney, focusing on the function of various calcium channels and the physiological stimuli that control these channels or are communicated through them. A discussion of the role of calcium as an intracellular second messenger and the pathophysiology of renal calcium dysregulation, as well as a summary of gaps in knowledge and future prospects, are also included.

Lysine and salt-sensitive hypertension.

PURPOSE OF REVIEW: Salt-sensitive (SS) hypertension and its associated kidney damage have been extensively studied, yet proper therapeutic strategies are lacking. The interest in altering the metabolome to affect renal and cardiovascular disease has been emerging. Here, we discuss the effect and potential mechanism behind the protective effect of lysine, an essential amino acid, on the progression of SS hypertension. RECENT FINDINGS: We have recently demonstrated that administering lysine in an SS rodent model can control the progression of hypertension. Both the animal and pilot human studies showed that lysine can efficiently inhibit tubular reabsorption of albumin and protect the kidneys from further damage. In addition, we conducted multilevel omics studies that showed increased lysine conjugation and excretion, leading to the depletion of harmful metabolites and an increase in useful ones. SUMMARY: Lysine's twofold action involves both mechanically flushing protein from proximal tubules to shield the kidneys and initiating metabolic adaptations in the kidneys. This results in a net positive impact on SS hypertension. While further research is necessary to apply the current findings in clinical settings, this study offers some evidence suggesting that lysine supplementation holds promise as a therapeutic approach for hypertensive kidney disease.

The presence of xanthine dehydrogenase is crucial for the maturation of the rat kidneys.

The development of the kidney involves essential cellular processes, such as cell proliferation and differentiation, which are led by interactions between multiple signaling pathways. Xanthine dehydrogenase (XDH) catalyzes the reaction producing uric acid in the purine catabolism, which plays a multifaceted role in cellular metabolism. Our previous study revealed that the genetic ablation of the Xdh gene in rats leads to smaller kidneys, kidney damage, decline of renal functions, and failure to thrive. Rats, unlike humans, continue their kidney development postnatally. Therefore, we explored whether XDH plays a critical role in kidney development using SS-/- rats during postnatal development phase. XDH expression was significantly increased from postnatal day 5 to 15 in wild-type but not homozygote rat kidneys. The transcriptomic profile of renal tissue revealed several dysregulated pathways due to the lack of Xdh expression with the remodeling in inflammasome, purinergic signaling, and redox homeostasis. Further analysis suggested that lack of Xdh affects kidney development, likely via dysregulation of epidermal growth factor and its downstream STAT3 signaling. The present study showed that Xdh is essential for kidney maturation. Our data, alongside the previous research, suggests that loss of Xdh function leads to developmental issues, rendering them vulnerable to kidney diseases in adulthood.

Metabolic Communication by SGLT2 Inhibition.

BACKGROUND: SGLT2 (sodium-glucose cotransporter 2) inhibitors (SGLT2i) can protect the kidneys and heart, but the underlying mechanism remains poorly understood. METHODS: To gain insights on primary effects of SGLT2i that are not confounded by pathophysiologic processes or are secondary to improvement by SGLT2i, we performed an in-depth proteomics, phosphoproteomics, and metabolomics analysis by integrating signatures from multiple metabolic organs and body fluids after 1 week of SGLT2i treatment of nondiabetic as well as diabetic mice with early and uncomplicated hyperglycemia. RESULTS: Kidneys of nondiabetic mice reacted most strongly to SGLT2i in terms of proteomic reconfiguration, including evidence for less early proximal tubule glucotoxicity and a broad downregulation of the apical uptake transport machinery (including sodium, glucose, urate, purine bases, and amino acids), supported by mouse and human SGLT2 interactome studies. SGLT2i affected heart and liver signaling, but more reactive organs included the white adipose tissue, showing more lipolysis, and, particularly, the gut microbiome, with a lower relative abundance of bacteria taxa capable of fermenting phenylalanine and tryptophan to cardiovascular uremic toxins, resulting in lower plasma levels of these compounds (including p-cresol sulfate). SGLT2i was detectable in murine stool samples and its addition to human stool microbiota fermentation recapitulated some murine microbiome findings, suggesting direct inhibition of fermentation of aromatic amino acids and tryptophan. In mice lacking SGLT2 and in patients with decompensated heart failure or diabetes, the SGLT2i likewise reduced circulating p-cresol sulfate, and p-cresol impaired contractility and rhythm in human induced pluripotent stem cell-derived engineered heart tissue. CONCLUSIONS: SGLT2i reduced microbiome formation of uremic toxins such as p-cresol sulfate and thereby their body exposure and need for renal detoxification, which, combined with direct kidney effects of SGLT2i, including less proximal tubule glucotoxicity and a broad downregulation of apical transporters (including sodium, amino acid, and urate uptake), provides a metabolic foundation for kidney and cardiovascular protection.

ß-Arrestin pathway activation by selective ATR1 agonism promotes calcium influx in podocytes, leading to glomerular damage.

Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the ß-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated ß-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine ß-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated ß-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of ß-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the ß-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the ß-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated ß-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.

Effect of SGLT2 inhibition on salt-induced hypertension in female Dahl SS rats.

Sodium-glucose co-transporters (SGLTs) in the kidneys play a pivotal role in glucose reabsorption. Several clinical and population-based studies revealed the beneficial effects of SGLT2 inhibition on hypertension. Recent work from our lab provided significant new insight into the role of SGLT2 inhibition in a non-diabetic model of salt-sensitive hypertension, Dahl salt-sensitive (SS) rats. Dapagliflozin (Dapa) blunted the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the Renin-Angiotensin-Aldosterone System. However, our initial study used male SS rats only, and the effect of SGLT2 inhibitors on hypertension in females has not been studied. Therefore, the goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function in female Dahl SS rats. The result showed that administration of Dapa for 3 weeks prevented the progression of salt-induced hypertension in female rats, similar to its effects in male SS rats. Diuresis and glucose excretion were significantly increased in Dapa-treated rats. SGLT2 inhibition also significantly attenuated kidney but not heart fibrosis. Despite significant effects on blood pressure, Dapa treatment caused minor changes to electrolyte balance and no effects on kidney and heart weights were observed. Our data suggest that SGLT2 inhibition in a non-diabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension independent of sex.

Opioids and the Kidney: A Compendium.

Opioids are a class of medications used in pain management. Unfortunately, long-term use, overprescription, and illicit opioid use have led to one of the greatest threats to mankind: the opioid crisis. Accompanying the classical analgesic properties of opioids, opioids produce a myriad of effects including euphoria, immunosuppression, respiratory depression, and organ damage. It is essential to ascertain the physiological role of the opioid/opioid receptor axis to gain an in-depth understanding of the effects of opioid use. This knowledge will aid in the development of novel therapeutic interventions to combat the increasing mortality rate because of opioid misuse. This review describes the current knowledge of opioids, including the opioid epidemic and opioid/opioid receptor physiology. Furthermore, this review intricately relates opioid use to kidney damage, navigates kidney structure and physiology, and proposes potential ways to prevent opioid-induced kidney damage.

Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage. ARTICLE HIGHLIGHTS: Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

Repeated seizures lead to progressive ventilatory dysfunction in SSKcnj16-/- rats.

Patients with uncontrolled epilepsy experience repeated seizures putting them at increased risk for sudden unexpected death in epilepsy (SUDEP). Data from human patients have led to the hypothesis that SUDEP results from severe cardiorespiratory suppression after a seizure, which may involve pathological deficiencies in the brainstem serotonin (5-HT) system. Rats with a genomic Kcnj16 mutation (SSKcnj16-/- rats) are susceptible to sound-induced generalized tonic-clonic seizures (GTCS) which, when repeated once daily for up to 10 days (10-day seizure protocol), increased mortality, particularly in male rats. Here, we test the hypothesis that repeated seizures across the 10-day protocol will cause a progressive ventilatory dysfunction due to time-dependent 5-HT deficiency. Initial severe seizures led to ictal and postictal apneas and transient decreases in breathing frequency, ventilatory drive, breath-to-breath variability, and brief hypoventilation. These seizure-induced effects on ventilation were exacerbated with increasing seizures and ventilatory chemoreflexes became further impaired after repeated seizures. Tissue analyses of key brainstem regions controlling breathing showed time-dependent 5-HT system suppression and increased immunoreactivity for IBA-1 (microglial marker) without changes in overall cell counts at 3, 7, and 10 days of seizures. Fluoxetine treatment in SSKcnj16-/- rats prevented repeated seizure-induced progressive respiratory suppression but failed to prevent seizure-related mortality. We conclude that repeated seizures cause a progressive compromise of ventilatory control in the immediate postictal period largely mediated by serotonin system suppression in brainstem regions of respiratory control. However, other unknown factors contribute to overall survival following repeated seizures in this model.NEW & NOTEWORTHY This study demonstrated that repeated seizures in a novel rat model (SSKcnj16-/- rats) caused a progressively greater ventilatory dysfunction in the immediate postictal period associated with brainstem serotonin (5-HT) suppression. Augmenting brain 5-HT with a selective serotonin reuptake inhibitor prevented the progressive ventilatory dysfunction induced by repeated seizures but failed to prevent seizure-related mortality, suggesting that repeated seizures may lead to cardiorespiratory suppression and failure through multiple mechanisms.

Kir7.1 knockdown and inhibition alter renal electrolyte handling but not the development of hypertension in Dahl salt-sensitive rats.

High K+ supplementation is correlated with a lower risk of the composite of death, major cardiovascular events, and ameliorated blood pressure, but the exact mechanisms have not been established. Inwardly rectifying K+ (Kir) channels expressed in the basolateral membrane of the distal nephron play an essential role in maintaining electrolyte homeostasis. Mutations in this channel family have been shown to result in strong disturbances in electrolyte homeostasis, among other symptoms. Kir7.1 is a member of the ATP-regulated subfamily of Kir channels. However, its role in renal ion transport and its effect on blood pressure have yet to be established. Our results indicate the localization of Kir7.1 to the basolateral membrane of aldosterone-sensitive distal nephron cells. To examine the physiological implications of Kir7.1, we generated a knockout of Kir7.1 (Kcnj13) in Dahl salt-sensitive (SS) rats and deployed chronic infusion of a specific Kir7.1 inhibitor, ML418, in the wild-type Dahl SS strain. Knockout of Kcnj13 (Kcnj13-/-) resulted in embryonic lethality. Heterozygous Kcnj13+/- rats revealed an increase in K+ excretion on a normal-salt diet but did not exhibit a difference in blood pressure development or plasma electrolytes after 3 wk of a high-salt diet. Wild-type Dahl SS rats exhibited increased renal Kir7.1 expression when dietary K+ was increased. K+ supplementation also demonstrated that Kcnj13+/- rats excreted more K+ on normal salt. The development of hypertension was not different when rats were challenged with high salt for 3 wk, although Kcnj13+/- rats excrete less Na+. Interestingly, chronic infusion of ML418 significantly increased Na+ and Cl- excretion after 14 days of high salt but did not alter salt-induced hypertension development. Here, we found that reduction of Kir7.1 function, either through genetic ablation or pharmacological inhibition, can influence renal electrolyte excretion but not to a sufficient degree to impact the development of SS hypertension.NEW & NOTEWORTHY To investigate the role of the Kir7.1 channel in salt-sensitive hypertension, its function was examined using complementary genetic and pharmacological approaches. The results revealed that although reducing Kir7.1 expression had some impact on maintaining K+ and Na+ balance, it did not lead to a significant change in the development or magnitude of salt-induced hypertension. Hence, it is probable that Kir7.1 works in conjunction with other basolateral K+ channels to fine-tune membrane potential.

The small GTPase regulatory protein Rac1 drives podocyte injury independent of cationic channel protein TRPC5.

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.

The circadian clock protein PER1 is important in maintaining endothelin axis regulation in Dahl salt-sensitive rats.

Endothelin-1 (ET-1) is a peptide hormone that acts on its receptors to regulate sodium handling in the kidney's collecting duct. Dysregulation of the endothelin axis is associated with various diseases, including salt-sensitive hypertension and chronic kidney disease. Previously, our lab has shown that the circadian clock gene PER1 regulates ET-1 levels in mice. However, the regulation of ET-1 by PER1 has never been investigated in rats. Therefore, we used a novel model where knockout of Per1 was performed in Dahl salt-sensitive rat background (SS Per1 -/-) to test a hypothesis that PER1 regulates the ET-1 axis in this model. Here, we show increased renal ET-1 peptide levels and altered endothelin axis gene expression in several tissues, including the kidney, adrenal glands, and liver in SS Per1 -/- compared with control SS rats. Edn1 antisense lncRNA Edn1-AS, which has previously been suggested to be regulated by PER1, was also altered in SS Per1 -/- rats compared with control SS rats. These data further support the hypothesis that PER1 is a negative regulator of Edn1 and is important in the regulation of the endothelin axis in a tissue-specific manner.

Ion channels and channelopathies in glomeruli.

An essential step in renal function entails the formation of an ultrafiltrate that is delivered to the renal tubules for subsequent processing. This process, known as glomerular filtration, is controlled by intrinsic regulatory systems and by paracrine, neuronal, and endocrine signals that converge onto glomerular cells. In addition, the characteristics of glomerular fluid flow, such as the glomerular filtration rate and the glomerular filtration fraction, play an important role in determining blood flow to the rest of the kidney. Consequently, disease processes that initially affect glomeruli are the most likely to lead to end-stage kidney failure. The cells that comprise the glomerular filter, especially podocytes and mesangial cells, express many different types of ion channels that regulate intrinsic aspects of cell function and cellular responses to the local environment, such as changes in glomerular capillary pressure. Dysregulation of glomerular ion channels, such as changes in TRPC6, can lead to devastating glomerular diseases, and a number of channels, including TRPC6, TRPC5, and various ionotropic receptors, are promising targets for drug development. This review discusses glomerular structure and glomerular disease processes. It also describes the types of plasma membrane ion channels that have been identified in glomerular cells, the physiological and pathophysiological contexts in which they operate, and the pathways by which they are regulated and dysregulated. The contributions of these channels to glomerular disease processes, such as focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, as well as the development of drugs that target these channels are also discussed.

Modulation of P2X4 receptor activity by ivermectin and 5-BDBD has no effect on the development of ARPKD in PCK rats.

Autosomal recessive polycystic kidney disease (ARPKD) is an inherited pathology caused mainly by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene, which usually leads to end-stage renal disease. Previous studies suggested that the P2X purinoreceptor 4 (P2X4 R) may play an important role in the progression of ARPKD. To test this hypothesis, we assessed the chronic effects of ivermectin (P2X4 R allosteric modulator) and 5-BDBD (P2X4 R antagonist) on the development of ARPKD in PCK/CrljCrl-Pkhd1pck/CRL (PCK) rats. Our data indicated that activation of ATP-mediated P2X4 R signaling with ivermectin for 6 weeks in high dose (50 mg/L; water supplementation) decreased the total body weight of PCK rats while the heart and kidney weight remained unaffected. Smaller doses of ivermectin (0.5 or 5 mg/L, 6 weeks) or the inhibition of P2X4 R signaling with 5-BDBD (18 mg/kg/day, food supplement for 8 weeks) showed no effect on electrolyte balance or the basic physiological parameters. Furthermore, cystic index analysis for kidneys and liver revealed no effect of smaller doses of ivermectin (0.5 or 5 mg/L) and 5-BDBD on the cyst development of PCK rats. We observed a slight increase in the cystic liver index on high ivermectin dose, possibly due to the cytotoxicity of the drug. In conclusion, this study revealed that pharmacological modulation of P2X4 R by ivermectin or 5-BDBD does not affect the development of ARPKD in PCK rats, which may provide insights for future studies on investigating the therapeutic potential of adenosine triphosphate (ATP)-P2 signaling in PKD diseases.

Knockout of the Circadian Clock Protein PER1 (Period1) Exacerbates Hypertension and Increases Kidney Injury in Dahl Salt-Sensitive Rats.

BACKGROUND: Circadian rhythms play an essential role in physiological function. The molecular clock that underlies circadian physiological function consists of a core group of transcription factors, including the protein PER1 (Period1). Studies in mice show that PER1 plays a role in the regulation of blood pressure and renal sodium handling; however, the results are dependent on the strain being studied. Using male Dahl salt-sensitive (SS) rats with global knockout of PER1 (SSPer1-/-), we aim to test the hypothesis that PER1 plays a key role in the regulation of salt-sensitive blood pressure. METHODS: The model was generated using CRISPR/Cas9 and was characterized using radiotelemetry and measures of renal function and circadian rhythm. RESULTS: SSPer1-/- rats had similar mean arterial pressure when fed a normal 0.4% NaCl diet but developed augmented hypertension after three weeks on a high-salt (4% NaCl) diet. Despite being maintained on a normal 12:12 light:dark cycle, SSPer1-/- rats exhibited desynchrony mean arterial pressure rhythms on a high-salt diet, as evidenced by increased variability in the time of peak mean arterial pressure. SSPer1-/- rats excrete less sodium after three weeks on the high-salt diet. Furthermore, SSPer1-/- rats exhibited decreased creatinine clearance, a measurement of renal function, as well as increased signs of kidney tissue damage. SSPer1-/- rats also exhibited higher plasma aldosterone levels. CONCLUSIONS: Altogether, our findings demonstrate that loss of PER1 in Dahl SS rats causes an array of deleterious effects, including exacerbation of the development of salt-sensitive hypertension and renal damage.

Lack of xanthine dehydrogenase leads to a remarkable renal decline in a novel hypouricemic rat model.

Uric acid (UA) is the final metabolite in purine catabolism in humans. Previous studies have shown that the dysregulation of UA homeostasis is detrimental to cardiovascular and kidney health. The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, responsible for producing UA. To explore how hypouricemia can lead to kidney damage, we created a rat model with the genetic ablation of the Xdh gene on the Dahl salt-sensitive rat background (SSXdh-/-). SSXdh-/- rats lacked UA and exhibited impairment in growth and survival. This model showed severe kidney injury with increased interstitial fibrosis, glomerular damage, crystal formation, and an inability to control electrolyte balance. Using a multi-omics approach, we highlighted that lack of Xdh leads to increased oxidative stress, renal cell proliferation, and inflammation. Our data reveal that the absence of Xdh leads to kidney damage and functional decline by the accumulation of purine metabolites in the kidney and increased oxidative stress.

Modulation of blood pressure regulatory genes in the Agtrap-Plod1 locus associated with a deletion in Clcn6.

The AGTRAP-PLOD1 locus is a conserved gene cluster containing several blood pressure regulatory genes, including CLCN6, MTHFR, NPPA, and NPPB. Previous work revealed that knockout of Clcn6 on the Dahl Salt-Sensitive (SS) rat background (SS-Clcn6) resulted in lower diastolic blood pressure compared to SS-WT rats. Additionally, a recent study found sickle cell anemia patients with mutations in CLCN6 had improved survival and reduced stroke risk. We investigated whether loss of Clcn6 would delay the mortality of Dahl SS rats on an 8% NaCl (HS) diet. No significant difference in survival was found. The ability of Clcn6 to affect mRNA expression of nearby Mthfr, Nppa, and Nppb genes was also tested. On normal salt (0.4% NaCl, NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS-Clcn6 rats. MTHFR reduces homocysteine to methionine, but no differences in circulating homocysteine levels were detected. Nppa mRNA levels in cardiac tissue from SS-Clcn6 rat in both normotensive and hypertensive conditions were significantly reduced compared to SS-WT. Nppb mRNA expression in SS-Clcn6 rats on a NS diet was also substantially decreased. Heightened Mthfr expression would be predicted to be protective; however, diminished Nppa and Nppb expression could be deleterious and by preventing or blunting vasodilation, natriuresis, and diuresis that ought to normally occur to offset blood pressure increases. The conserved nature of this genetic locus in humans and rats suggests more studies are warranted to understand how mutations in and around these genes may be influencing the expression of their neighbors.