Osteoporosis after bariatric surgery is an increasing health concern as the rate of bariatric surgery has risen. In animal studies mimicking bariatric procedures, bone disease, together with decreased serum levels of Ca2+, Mg2+ and the gastric hormone Ghrelin were described. Ghrelin regulates metabolism by binding to and activating the growth hormone secretagogue receptor (GHSR) which is also expressed in the kidney. As calcium and magnesium are key components of bone, we tested the hypothesis that Ghrelin-deficiency contributes to osteoporosis via reduced upregulation of the renal calcium channel TRPV5 and the heteromeric magnesium channel TRPM6/7. We expressed GHSR with TRPV5 or TRPM6/7 channel in HEK293 cells and treated them with purified Ghrelin. Whole-cell current density was analyzed by patch-clamp recording. Nephron-specific gene expression was performed by tubular microdissection followed by qPCR in wild-type (WT) mice, and immunofluorescent imaging of GHSR-eGFP mice. Tubular magnesium homeostasis was analyzed in GHSR-null and WT mice at baseline and after caloric restriction. After Ghrelin exposure, whole-cell current density did not change for TRPV5 but increased for TRPM6/7 in a dose-dependent fashion. Applying the Ghrelin-mimetic (D-Trp7, Ala8,D-Phe10)-α-MSH (6–11) amide without and with the GHSR antagonist (D-Lys3)-GHRP6, we confirmed the stimulatory role of Ghrelin towards TRPM6/7. As GHSR initiates downstream signaling via protein kinase A (PKA), we found that the PKA inhibitor H89 abrogated TRPM6/7 stimulation by Ghrelin. Similarly, transfected Gαs, but not the Gαs mutant Q227L, nor Gαi2, Gαq, or Gα13 upregulated TRPM6/7 current density. In microdissected TALs and DCTs similar levels of GHSR mRNA were detected. In contrast, TRPM6 mRNA was expressed in the DCT and also detected in the TAL at 25% expression compared to DCT. Immunofluorescent studies using reporter GHSR-eGFP mice showed a strong eGFP signal in the TAL but surprisingly displayed no eGFP signal in the DCT. In 3-, 6-, and 9-month-old GHSR-null and WT mice, baseline serum magnesium was not significantly different, but 24-h urinary magnesium excretion was elevated in 9-month-old GHSR-null mice. In calorically restricted GHSR-null mice, we detected excess urinary magnesium excretion and reduced serum magnesium levels compared to WT mice. The kidneys from calorically restricted WT mice showed upregulated gene expression of magnesiotropic genes Hnf1b, Cldn-16, Cldn-19, Fxyd-2b, and Parvalbumin compared to GHSR-null mice. Our in vitro studies show that Ghrelin stimulates TRPM6/7 via GHSR and Gαs-PKA signaling. The murine studies are consistent with Ghrelin-GHSR signaling inducing reduced urinary magnesium excretion, particularly in calorically restricted mice when Ghrelin levels are elevated. This effect may be mediated by Ghrelin-upregulation of TRPM6 in the TAL and/or upregulation of other magnesiotropic genes. We postulate that rising Ghrelin levels with hunger contribute to increased renal Mg2+ reabsorption to compensate for lack of enteral Mg2+ uptake.

ABSTRACT The kidney is the key regulator of magnesium (Mg 2+ ) homeostasis in the human body. In the distal convoluted tubule (DCT), the apical epithelial magnesium (Mg 2+ ) channel TRPM6, determines how much Mg 2+ is excreted in the urine. To better understand the regulation of human renal Mg 2+ absorption we identified novel, potential interaction partners of TRPM6 by pursuing a liquid chromatography – tandem mass spectrometry (LC-MS/MS) proteomics approach. We found insulin receptor substrate 4 (IRS4) enriched with TRPM6 tagged to glutathione S-transferase (TRPM6-GST) but not GST control. Physical interaction between IRS4 and TRPM6 was confirmed by co-immunoprecipitation. Applying microdissection of mouse tubules, we detected Irs4 mRNA expression mostly in the DCT and to a lower degree in the proximal tubule and thick ascending limb of Henle. Given the overall low abundance of Irs4 mRNA along the tubule we investigated the phenotype of Irs4 knockout mice ( Irs4 -/- ). These Irs4 -/- mice displayed significantly higher urinary and fecal Mg 2+ losses and lower blood Mg 2+ levels than wild-type (WT) mice. Claudin-16, claudin-19, and Hnf1b mRNA and Claudin-16 and Trpm6 protein expression was significantly higher in kidneys of 3 month old Irs4 -/- mice consistent with a compensatory mechanism to conserve Mg 2+ . Applying whole-cell patch-clamp recording we confirmed the stimulatory role of insulin on TRPM6 channel activity and showed that IRS4 targets the two TRPM6 phosphorylation sites T1391 and S1583 to enhance TRPM6 current density. To test the effect of Mg 2+ deficiency on metabolism, we performed glucose and insulin tolerance studies, which were mildly abnormal in Irs4 -/- mice. SIGNIFICANCE STATEMENT Magnesium (Mg 2+ ) is the second most abundant intracellular cation but the regulation of Mg 2+ homeostasis is not well understood. The kidney is the key organ for regulating Mg 2+ homeostasis. Insulin is a known stimulator of the apical epithelial Mg 2+ channel TRPM6. We present a novel modifier of Mg 2+ absorption with insulin receptor substrate 4 (IRS4) which illuminates further, how insulin activates the TRPM6 channel and modifies Mg 2+ homeostasis. Applying protein biochemistry, tubular microdissection, whole mouse physiology, and patch-clamp recording, we demonstrate that IRS4 mediates the stimulatory effect of insulin by enhancing phosphorylation of two specific TRPM6 residues. Irs4 -/- mice develop increased urinary and stool Mg 2+ losses, lower serum Mg 2+ concentration, and display mild impairment in glucose and insulin tolerance.

Prenatal dexamethasone has been shown to increase blood pressure in male offspring but the mechanism for the increase in blood pressure is unclear. The present study examined if prenatal programming by maternal injection of dexamethasone on days 15 and 16 of gestation affected the blood pressure comparably in female and male offspring. Our hypothesis was that males would be affected by prenatal dexamethasone to a greater extent than females and that either an increase in renal tubular transporter abundance or an increase in renin or aldosterone system would be associated with hypertension with prenatal programming. Prenatal dexamethasone increased blood pressure at two months and six months of age and resulted in proteinuria and albuminuria at six months in male but not female rat offspring. There was no effect of prenatal dexamethasone on blood pressure and proteinuria at one month in male and in female offspring. While prenatal dexamethasone increased male renal thick ascending limb sodium potassium two chloride cotransporter protein abundance at two months, prenatal dexamethasone on days 15 and 16 of gestation did not affect transporter abundance in males at other ages, nor did it affect proximal tubule sodium/hydrogen exchanger or distal convoluted tubule sodium chloride cotransporter protein abundance at any age. There was no difference in systemic renin or aldosterone in the prenatal dexamethasone group compared to same sex controls. In conclusion, male but not female offspring have an increase in blood pressure and urinary protein excretion with prenatal dexamethasone. The increase in blood pressure with prenatal programming was not associated with a consistent increase in renal tubular transporter protein abundance, nor plasma renin activity and serum aldosterone.

Department of Pediatrics and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas. USA Correspondence to Michel Baum, Department of Pediatrics, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9063, USA. Tel: +1 214 648 3528; fax: +1 214 648 2034; e-mail: [email protected]

Objective Determine prevalence of hyperfiltration (high estimated glomerular filtration rate "eGFR" >95th percentile for age/sex) among youth and association with BMI classification. Methods With the use of 1999 to 2016 National Health and Nutrition Examination Survey data from 12- to 29-year-olds, data for serum creatinine and thresholds for high eGFR were normed using a metabolically healthy subsample (no albuminuria, healthy weights, normal blood pressures, blood glucoses, lipids, and liver enzymes). Logistic regression examined the association of BMI classification (healthy weight, overweight, and obesity classes 1-3) with hyperfiltration (eGFR > 95th percentile for age/sex), adjusted for diabetes and other covariates. Results Of 12- to 29-year-olds (N = 18 698), 27.4% (n = 5493) met criteria for entry into the "healthy subsample" and contributed data to derive normative values for serum creatinine/hyperfiltration thresholds. In the full sample, hyperfiltration prevalence in 12- to 29-year-olds classified as healthy-weight, overweight, and obesity classes 1 to 3 was 4.9%, 4.7%, 6.5%, 8.7%, and 11.8%, respectively (P < .001). In multivariable analysis, obesity classes 2 and 3 were associated with greater likelihood of hyperfiltration (adjusted ORs for class 2: 1.5, 95% CI, 1.1-2.1; and for class 3, 2.1, 95% CI, 1.5-2.9). Diabetes also was associated with hyperfiltration (AOR, 4.0; 95% CI, 2.2-7.4). Conclusion Obesity classes 2 to 3 are associated with hyperfiltration in youth. Age/sex-specific norms for creatinine and hyperfiltration thresholds may aid recognition of kidney dysfunction early.

Epidemiologic studies have shown that small-for-gestational-age infants are at risk for hypertension, cardiovascular disease, chronic kidney disease, and premature death. This may result from intrauterine epigenetic adaptations that are beneficial to the fetus but result in phenotypic changes that are maladaptive in later life. The epidemiologic association between small-for-gestational-age infants and adverse outcomes in later life have been validated in controlled animal studies where maternal insults that result in small-for-gestational-age offspring result in the same adverse outcomes as found in humans. Of importance, recent studies find that premature infants are also at risk for hypertension and chronic kidney disease in later life.

We have previously demonstrated that dexamethasone administered to pregnant rats during specific times during gestation results in a reduction in glomerular number and hypertension in offspring at 2 and 6 months of age. In this study, we examined the effect of prenatal dexamethasone administered daily on days 15 and 16 of gestation in male and female offspring after 1 year of age on glomerular filtration rate. The prenatal dexamethasone male group had a higher systolic blood pressure than the vehicle male group. Females had lower systolic blood pressures than the males and prenatal dexamethasone did not affect blood pressure in female offspring. Prenatal dexamethasone resulted in a reduction in glomerular filtration rate in male but not in female rats. When corrected for body weight, the control male rats had a lower glomerular filtration rate than the control female rats. Males had greater protein excretion than females and prenatal dexamethasone increased the protein excretion only in male rats. Glomerulosclerosis was also greater in male rats than females but was not affected by prenatal dexamethasone. In summary, male rats appear to have evidence of a decline in glomerular filtration rate after 1 year of age and prenatal dexamethasone programs an accelerated decline in glomerular filtration rate in male but not in female offspring.

ConclusionFinish the discussion with a conclusion.This can be as short as two or three sentences.Do not make claims that are not justified by your results or are not part of the study.Many papers finish with 'further research is needed'.We usually know this and it is not helpful.

The diagnosis of end-stage renal disease was once a death sentence. Without dialysis and transplantation, there was nothing to be done and patients died from uremia, electrolyte disturbances, or volume overload. Dialysis was invented by Willem Kolff in 1943, but this form of therapy was initially available to very few patients as the number requiring renal replacement was far greater than the number of available dialysis machines. In the United States and in most developed countries, dialysis is now available to all children and adults with end-stage renal disease. Dialysis is often called renal replacement therapy and this term is a misnomer. The 5-year survival for patients with ESRD is worse than that of many types of cancer [1]. The mortality of children with end-stage renal disease is 30 times greater than children without renal disease and this number is even greater for infants [2–4]. Death in children, as in adults, is predominantly from cardiovascular disease and infection [5]. Thus, dialysis does not replace a normal kidney and dialysis is not adequate long-term therapy. In addition to the high mortality rate, dialysis impacts the quality of life for children who often need to spend 4 h hooked up to a dialysis machine three or four times a week. At this point, only a well functioning kidney can provide true renal replacement therapy. The first successful renal transplant was performed on 23 December 1954 by the eminent team of Joseph Murray, John Merrill and Hartwell Harrison [6]. It had long been known that transplantation of tissue from one individual to another resulted in rejection of the graft in short order. This team overcame this problem by transplanting the kidney of Ronald Herrick to Richard, his identical twin, who was dying of nephritis. Richard lived another 8 years thanks to the gift from his brother and the skill of his team of physicians. Murray, a plastic surgeon, continued to work on transplantation throughout his career and received the Nobel Prize in Physiology or Medicine in 1990 for his efforts. Although this was a major step forward, most patients with end-stage renal disease do not have a healthy identical twin. It had long been known from experiments with skin grafts that transplantation of skin from an unrelated individual resulted in an immunologic response with loss of the graft. It is not surprising that kidney transplantation between unrelated individuals resulted in rejection of the organ. Hume et al.[7] reported nine cases of individuals with renal failure who received renal transplants and four patients made urine for 37–180 days. The functioning transplants were lost to rejection. The problem of immune suppression continues to this day. One has to suppress the immune system enough to prevent graft rejection but not so much as to cause opportunistic infections, malignancy, or toxicity from the immunosuppressive agent itself. The first nonrelated transplants were treated with total body irradiation with or without bone marrow infusion. This was clearly too toxic and nonspecific. This was followed by use of 6-mercaptopurine and azathioprine (Imuran), which inhibit purine synthesis, and thus DNA synthesis in lymphocytes. Imuran was soon administered along with prednisone [8]. The development and success of immunosuppressive agents to suppress the immune system led to transplants from nonrelated donors and cadaveric donors [9]. However, by today's standards, the results were not very good with a 1-year graft survival of 40–50% for cadaveric donors in the 1970s. With the introduction of cyclosporine, a calcineurin inhibitor, in the mid-1980s, the 1-year graft survival increased to 75% [10]. There continues to be an increase in the therapeutic armamentarium to prevent renal transplant rejection with more potent and specific drugs. Induction therapy now includes antilymphocyte or antithymocyte globulin or drugs, such as basiliximab (Simulect) that block the IL-2 receptor. Chronic therapy now includes tacrolimus, a calcineurin inhibitor with fewer side effects than cyclosporine, mycophenolate mofetil, which like Imuran inhibits purine synthesis but is much more potent, and sirolimus, an mammalian target of rapamycin inhibitor. Currently the 1-year graft survival for both adult and pediatric renal transplants is over 90% [11,12]. There has been and continues to be a learning curve as more specific and newer immunosuppressive agents come into clinical use. Transplants can be lost to acute cellular rejection, which is also known as T-cell-mediated rejection. This is characterized by infiltration of T cells into the graft causing tubulitis and interstitial inflammation. With the use of calcineurin inhibitors, the incidence of acute cellular rejection has decreased. However, what has become more and more prevalent is antibody-mediated rejection. If there are preformed antibodies to the allograft, a hyperacute rejection can occur at the time of vascular anastomosis resulting in vascular thrombosis and graft loss. The presence of preformed antibodies is now determined before the transplant and this catastrophe is now a rare event. However, in antibody-mediated rejection that occurs months or years after transplant, acquired antibodies to human leukocyte antigen (HLA) antigens injure peritubular and glomerular capillaries, the latter resulting in transplant glomerulopathy. Antibody-mediated rejection is diagnosed by a reduction in graft function with peritubular (capillary) deposition of C4d, a marker of antibody-mediated endothelial injury and high titers of donor-specific anti-HLA antibodies. Antibody-mediated rejection has proven much more difficult to treat than cellular rejection and can cause acute and chronic allograft rejection and a reduction in graft function. The use of more potent immunosuppressive agents has resulted in a new set of problems. Although there are fewer rejections than previously, over suppression of the immune system has led to an increase in opportunistic infections and cancers including posttransplant lymphoproliferative disorder. In addition to an increase in viral, bacterial, and fungal infections, patients with transplants can reactivate viruses, such as cytomegalovirus, Epstein–Barr virus, and BK virus, a polyomavirus, which may have been dormant for years. Treatment often requires lowering the dose of immunosuppression, which places the patient at risk for rejection of the renal transplant. Although short-term graft survival has improved markedly over the last two decades, long-term graft survival is still problematic. Chronic allograft rejection and calcineurin toxicity as well as exposure to nephrotoxic mediations continues to be a cause of graft loss over time. In pediatrics, nonadherence to medications is a major risk factor for graft loss especially among teenagers whose parents teach them responsibility by putting them in charge of their own medications. There is a myriad of causes for end-stage renal disease. Most causes of end-stage renal disease in children are because of obstructive uropathy. Although the bladder of these patients may not be the perfect reservoir for the transplant, there is no recurrence of the original disease. That is also the case of most of the causes for end-stage renal disease requiring transplant in pediatrics, though there are clearly diseases caused by circulatory factors that are still present in the recipient. This is most dramatic in cases of nephrotic syndrome where the recipient has nephrotic range proteinuria within hours of receiving the transplant. Indeed, the first successful renal transplant to Richard Herrick donated by his identical twin brother was lost after 8 years because of recurrence of disease and not rejection. As patients develop progressive chronic kidney disease, renal elimination of phosphate becomes limiting and the patients develop hyperphosphatemia. The elevated serum phosphorus results in secretion of parathyroid hormone and FGF23. FGF23 is a phosphaturic hormone made in bones that inhibits the proximal tubule phosphate transporter and 1,25 vitamin D synthesis. As renal function declines, the elevated levels of FGF23 and parathyroid hormone cannot cause enough phosphate secretion to maintain normal phosphate levels. At the time of transplant, the patients are usually hyperphosphatemic and have very high levels of FGF23 and parathyroid hormone. After transplant, we have the opposite problem. The patient's renal function may be near normal but the elevated levels of FGF23 and parathyroid hormone do not normalize immediately and the patients become very hypophosphatemic. Patients with end-stage renal disease have metabolic bone disease and osteopenia. The bone disease and risk for fractures does not reverse immediately after transplant and can be potentiated in the immediate posttransplant period because of the continued dysregulation of 1,25 vitamin D, PTH, and FGF-23. In addition, medications, such as calcineurin inhibitors cause magnesium wasting. The miracle of transplantation has changed medicine and the outlook of thousands of children and adults who suffer from end-stage renal disease. However, there are approximately 100 000 patients on the UNOS renal transplant waiting list in the United States. Of those on the waiting list for kidneys in the United States, over 1000 are children 17 years of age and younger. Patients can wait for years before receiving a renal transplant. The mortality of patients awaiting renal transplants is very high and a renal transplant not only extends life but improves the quality of life. This has prompted expansion of both acceptable living and diseased donor transplant donors. The prognosis for long-term graft and patient survival has changed dramatically over the past 2 decades. With more selective and potent immunosuppressants, graft survival will likely continue to improve. The challenge of mitigating the side effects of immunosuppression, especially opportunistic infections and malignancies will need to be addressed. Acknowledgements None. Financial support and sponsorship None. Conflicts of interest There are no conflicts of interest.

microRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. The physiologic function of these noncoding RNAs in postnatal renal tubules still remains unclear. Surprisingly, they appear to be dispensable for mammalian proximal tubule (PT) function. Here, we examined the effects of miRNA suppression in collecting ducts (CDs). To conclusively evaluate the role of miRNAs, we generated three mouse models with CD-specific inactivation of key miRNA pathway genes Dicer, Dgcr8, and the entire Argonaute gene family (Ago1, 2, 3, and 4). Characterization of these three mouse models revealed that inhibition of miRNAs in CDs spontaneously evokes a renal tubule injury-like response, which culminates in progressive tubulointerstitial fibrosis (TIF) and renal failure. Global miRNA profiling of microdissected renal tubules showed that miRNAs exhibit segmental distribution along the nephron and CDs. In particular, the expression of miR-200c is nearly 70-fold higher in CDs compared with PTs. Accordingly, miR-200s are downregulated in Dicer-KO CDs, its direct target genes Zeb1, Zeb2, and Snail2 are upregulated, and miRNA-depleted CDs undergo partial epithelial-to-mesenchymal transition (EMT). Thus, miRNAs are essential for CD homeostasis. Downregulation of CD-enriched miRNAs and the subsequent induction of partial EMT may be a new mechanism for TIF progression.

A maternal low‐protein diet has been shown to program hypertension and a reduction in glomerular filtration rate in adult offspring. This study examined the effect of continuous administration of enalapril in the drinking water and transient administration of enalapril administered from 21 to 42 days of age on blood pressure and glomerular filtration rate (GFR) in male rats whose mothers were fed a 20% protein diet (control) or a 6% protein diet (programmed) during the last half of pregnancy. After birth all rats were fed a 20% protein diet. Programmed rats (maternal 6% protein diet) were hypertensive at 15 months of age compared to control rats and both continuous and transient administration of enalapril had no effect on blood pressure on control offspring, but normalized the blood pressure of programmed offspring. GFR was 3.2 ± 0.1 mL/min in the control group and 1.7 ± 0.1 mL/min in the programmed rats at 17 months of age (P < 0.001). The GFR was 3.0 ± 0.1 mL/min in the control and 2.7 ± 0.1 mL/min in the programmed group that received continuous enalapril in their drinking water showing that enalapril can prevent the decrease in GFR in programmed rats. Transient administration of enalapril had no effect on GFR in the control group (3.2 ± 0.1 mL/min) and prevented the decrease in GFR in the programmed group (2.9 ± 0.1 mL/min). In conclusion, transient exposure to enalapril for 3 weeks after weaning can prevent the hypertension and decrease in GFR in prenatal programmed rats.